Sample records for bacterial expression system

  1. Dynamics of Immune System Gene Expression upon Bacterial Challenge and Wounding in a Social Insect (Bombus terrestris)

    PubMed Central

    Erler, Silvio; Popp, Mario; Lattorff, H. Michael G.

    2011-01-01

    The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT). There is a lack of immune genes in social insects (e.g. honeybees) when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals). The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge. Antimicrobial peptides (AMP) (abaecin, defensin 1, hymenoptaecin) were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish) and JNK pathway (basket). Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment. Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

  2. Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems.

    PubMed

    Nishino, Kunihiko

    2018-01-01

    Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

  3. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    PubMed Central

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-01-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  4. Bacterial cell-free expression technology to in vitro systems engineering and optimization.

    PubMed

    Caschera, Filippo

    2017-06-01

    Cell-free expression system is a technology for the synthesis of proteins in vitro . The system is a platform for several bioengineering projects, e.g. cell-free metabolic engineering, evolutionary design of experiments, and synthetic minimal cell construction. Bacterial cell-free protein synthesis system (CFPS) is a robust tool for synthetic biology. The bacteria lysate, the DNA, and the energy module, which are the three optimized sub-systems for in vitro protein synthesis, compose the integrated system. Currently, an optimized E. coli cell-free expression system can produce up to ∼2.3 mg/mL of a fluorescent reporter protein. Herein, I will describe the features of ATP-regeneration systems for in vitro protein synthesis, and I will present a machine-learning experiment for optimizing the protein yield of E. coli cell-free protein synthesis systems. Moreover, I will introduce experiments on the synthesis of a minimal cell using liposomes as dynamic containers, and E. coli cell-free expression system as biochemical platform for metabolism and gene expression. CFPS can be further integrated with other technologies for novel applications in environmental, medical and material science.

  5. [Prokaryotic expression systems].

    PubMed

    Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał

    2013-03-01

    For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

  6. Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

    PubMed Central

    Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

    2014-01-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

  7. Genome engineering and gene expression control for bacterial strain development.

    PubMed

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

    PubMed

    Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

    2014-03-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

    PubMed

    Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

    2018-03-16

    Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

  10. Bacterial expression of self-assembling peptide hydrogelators

    NASA Astrophysics Data System (ADS)

    Sonmez, Cem

    For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to

  11. Distinct antimicrobial peptide expression determines host species-specific bacterial associations

    PubMed Central

    Franzenburg, Sören; Walter, Jonas; Künzel, Sven; Wang, Jun; Baines, John F.; Bosch, Thomas C. G.; Fraune, Sebastian

    2013-01-01

    Animals are colonized by coevolved bacterial communities, which contribute to the host’s health. This commensal microbiota is often highly specific to its host-species, inferring strong selective pressures on the associated microbes. Several factors, including diet, mucus composition, and the immune system have been proposed as putative determinants of host-associated bacterial communities. Here we report that species-specific antimicrobial peptides account for different bacterial communities associated with closely related species of the cnidarian Hydra. Gene family extensions for potent antimicrobial peptides, the arminins, were detected in four Hydra species, with each species possessing a unique composition and expression profile of arminins. For functional analysis, we inoculated arminin-deficient and control polyps with bacterial consortia characteristic for different Hydra species and compared their selective preferences by 454 pyrosequencing of the bacterial microbiota. In contrast to control polyps, arminin-deficient polyps displayed decreased potential to select for bacterial communities resembling their native microbiota. This finding indicates that species-specific antimicrobial peptides shape species-specific bacterial associations. PMID:24003149

  12. Changes in rhizosphere bacterial gene expression following glyphosate treatment.

    PubMed

    Newman, Molli M; Lorenz, Nicola; Hoilett, Nigel; Lee, Nathan R; Dick, Richard P; Liles, Mark R; Ramsier, Cliff; Kloepper, Joseph W

    2016-05-15

    In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria. Copyright © 2016 The Authors. Published by Elsevier B.V. All

  13. Immunomodulators targeting MARCO expression improve resistance to postinfluenza bacterial pneumonia.

    PubMed

    Wu, Muzo; Gibbons, John G; DeLoid, Glen M; Bedugnis, Alice S; Thimmulappa, Rajesh K; Biswal, Shyam; Kobzik, Lester

    2017-07-01

    Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia. Copyright © 2017 the American Physiological Society.

  14. An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

    PubMed

    Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

    2013-04-15

    NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Can dead bacterial cells be defined and are genes expressed after cell death?

    PubMed

    Trevors, J T

    2012-07-01

    There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. RNA-Seq for Bacterial Gene Expression.

    PubMed

    Poulsen, Line Dahl; Vinther, Jeppe

    2018-06-01

    RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

  17. Increased Systemic Cytokine/Chemokine Expression in Asthmatic and Non-asthmatic Patients with Bacterial, Viral or Mixed Lung Infection.

    PubMed

    Giuffrida, M J; Valero, N; Mosquera, J; Duran, A; Arocha, F; Chacín, B; Espina, L M; Gotera, J; Bermudez, J; Mavarez, A; Alvarez-Mon, M

    2017-04-01

    This study was aimed to determine the profiles of serum cytokines (IL-1β, TNF-α, IL-4, IL-5) and chemokines (MCP-1: monocyte chemoattract protein-1 and RANTES: regulated on activation normal T cell expressed and secreted) in individuals with an asthmatic versus a non-asthmatic background with bacterial, viral or mixed acute respiratory infection. Asthmatic (n = 14) and non-asthmatic (n = 29) patients with acute viral, bacterial or mixed (bacterial and viruses) respiratory infection were studied. Patients were also analysed as individuals with pneumonia or bronchitis. Healthy individuals with similar age and sex (n = 10) were used as controls. Cytokine/chemokine content in serum was determined by ELISA. Increased cytokine/chemokine concentration in asthmatic and non-asthmatic patients was observed. However, higher concentrations of chemokines (MCP-1 and RANTES) in asthmatic patients infected by viruses, bacteria or bacteria and viruses (mixed) than in non-asthmatic patients were observed. In general, viral and mixed infections were better cytokine/chemokine inducers than bacterial infection. Cytokine/chemokine expression was similarly increased in both asthmatic and non-asthmatic patients with pneumonia or bronchitis, except that RANTES remained at normal levels in bronchitis. Circulating cytokine profiles induced by acute viral, bacterial or mixed lung infection were not related to asthmatic background, except for chemokines that were increased in asthmatic status. © 2017 The Foundation for the Scandinavian Journal of Immunology.

  18. The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.

    PubMed

    Gawin, Agnieszka; Valla, Svein; Brautaset, Trygve

    2017-07-01

    The XylS/Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW0 is widely used for regulated low- and high-level recombinant expression of genes and gene clusters in Escherichia coli and other bacteria. Induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose-dependent manner and are typically not metabolized by the host cells. Combinatorial mutagenesis and selection using the bla gene encoding β-lactamase as a reporter have demonstrated that the Pm promoter, the DNA sequence corresponding to the 5' untranslated end of its cognate mRNA and the xylS coding region can be modified and improved relative to various types of applications. By combining such mutant genetic elements, altered and extended expression profiles were achieved. Due to their unique properties, obtained systems serve as a genetic toolbox valuable for heterologous protein production and metabolic engineering, as well as for basic studies aiming at understanding fundamental parameters affecting bacterial gene expression. The approaches used to modify XylS/Pm should be adaptable for similar improvements also of other microbial expression systems. In this review, we summarize constructions, characteristics, refinements and applications of expression tools using the XylS/Pm system. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  19. Increase methylmercury accumulation in Arabidopsis thaliana expressing bacterial broad-spectrum mercury transporter MerE

    PubMed Central

    2013-01-01

    The bacterial merE gene derived from the Tn21 mer operon encodes a broad-spectrum mercury transporter that governs the transport of methylmercury and mercuric ions across bacterial cytoplasmic membranes, and this gene is a potential molecular tool for improving the efficiency of methylmercury phytoremediation. A transgenic Arabidopsis engineered to express MerE was constructed and the impact of expression of MerE on methylmercury accumulation was evaluated. The subcellular localization of transiently expressed GFP-tagged MerE was examined in Arabidopsis suspension-cultured cells. The GFP-MerE was found to localize to the plasma membrane and cytosol. The transgenic Arabidopsis expressing MerE accumulated significantly more methymercury and mercuric ions into plants than the wild-type Arabidopsis did. The transgenic plants expressing MerE was significantly more resistant to mercuric ions, but only showed more resistant to methylmercury compared with the wild type Arabidopsis. These results demonstrated that expression of the bacterial mercury transporter MerE promoted the transport and accumulation of methylmercury in transgenic Arabidopsis, which may be a useful method for improving plants to facilitate the phytoremediation of methylmercury pollution. PMID:24004544

  20. Understanding the impact of water distribution system conditions on the biodegradation of haloacetic acids and expression of bacterial dehalogenase genes.

    PubMed

    Behbahani, Mohsen; Lin, Boren; Phares, Tamara L; Seo, Youngwoo

    2018-06-05

    The objective of this study is to evaluate the influence of water distribution system conditions (pH, total organic carbon, residual chlorine, and phosphate) on haloacetic acids (HAAs) biodegradation. A series of batch microcosm tests were conducted to determine biodegradation kinetics and collected biomass was used for real time quantitative reverse transcription polymerase chain reaction analyses to monitor how these drinking water distribution system conditions affect the relative expression of bacterial dehalogenase genes. It was observed that tested water distribution system conditions affected HAA biodegradation with different removal efficiencies (0-100%). HAA biodegradation was improved in tested samples with TOC (3 mg/L) and pH 8.5 compared to those of TOC (0 mg/L) and pH 7, respectively. However, slight improvement was observed with the increased PO 4 concentration (3.5 mg/L), and the presence of residual chlorine even at low concentration prohibited biodegradation of HAAs. The observed trend in the relative expression of dehII genes was compatible with the HAA biodegradation trend. Overall relative expression ratio of dehII genes was lower at pH 7, phosphate (0.5 mg/L), and TOC (0 mg/L) in comparison with pH 8.5, phosphate (3.5 mg/L), and TOC (3 mg/L) in the same experimental conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Targeting bacterial secretion systems: benefits of disarmament in the microcosm.

    PubMed

    Baron, Christian; Coombes, Brian

    2007-03-01

    Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections.

  2. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    PubMed Central

    2010-01-01

    Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this

  3. CRISPR technologies for bacterial systems: Current achievements and future directions.

    PubMed

    Choi, Kyeong Rok; Lee, Sang Yup

    2016-11-15

    Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR technologies. Next, we describe CRISPR/Cas-derived technologies for bacterial strain development, including genome editing and gene expression regulation applications. Then, other CRISPR technologies possessing great potential for industrial applications are described, including typing and tracking of bacterial strains, virome identification, vaccination of bacteria, and advanced antimicrobial approaches. For each application, we note our suggestions for additional improvements as well. In the same context, replication of CRISPR/Cas-based chromosome imaging technologies developed originally in eukaryotic systems is introduced with its potential impact on studying bacterial chromosomal dynamics. Also, the current patent status of CRISPR technologies is reviewed. Finally, we provide some insights to the future of CRISPR technologies for bacterial systems by proposing complementary techniques to be developed for the use of CRISPR technologies in even wider range of applications. Copyright © 2016. Published by Elsevier Inc.

  4. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians.

    PubMed

    Newmark, Phillip A; Reddien, Peter W; Cebrià, Francesc; Sánchez Alvarado, Alejandro

    2003-09-30

    Freshwater planarian flatworms are capable of regenerating complete organisms from tiny fragments of their bodies; the basis for this regenerative prowess is an experimentally accessible stem cell population that is present in the adult planarian. The study of these organisms, classic experimental models for investigating metazoan regeneration, has been revitalized by the application of modern molecular biological approaches. The identification of thousands of unique planarian ESTs, coupled with large-scale whole-mount in situ hybridization screens, and the ability to inhibit planarian gene expression through double-stranded RNA-mediated genetic interference, provide a wealth of tools for studying the molecular mechanisms that regulate tissue regeneration and stem cell biology in these organisms. Here we show that, as in Caenorhabditis elegans, ingestion of bacterially expressed double-stranded RNA can inhibit gene expression in planarians. This inhibition persists throughout the process of regeneration, allowing phenotypes with disrupted regenerative patterning to be identified. These results pave the way for large-scale screens for genes involved in regenerative processes.

  5. Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors.

    PubMed

    Li, Ning; Ren, Aihui; Wang, Xiaoshuang; Fan, Xin; Zhao, Yong; Gao, George F; Cleary, Patrick; Wang, Beinan

    2015-01-06

    Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

  6. Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

    PubMed Central

    Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

    2003-01-01

    Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

  7. The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

    PubMed

    Burbank, Lindsey P; Stenger, Drake C

    2017-04-01

    Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

  8. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

    PubMed Central

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-01-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

  9. Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

    PubMed

    Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

    2015-10-01

    Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

  10. CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation.

    PubMed

    Bruna, Roberto E; Molino, María Victoria; Lazzaro, Martina; Mariscotti, Javier F; García Véscovi, Eleonora

    2018-04-15

    PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescen s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescens IMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is

  11. Drosophila Embryos as Model Systems for Monitoring Bacterial Infection in Real Time

    PubMed Central

    Evans, Iwan R.; Waterfield, Nicholas; ffrench-Constant, Richard H.; Wood, Will

    2009-01-01

    Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors. PMID:19609447

  12. Two poplar-associated bacterial isolates induce additive favorable responses in a constructed plant-microbiome system

    DOE PAGES

    Jawdy, Sara S.; Gunter, Lee E.; Engle, Nancy L.; ...

    2016-04-26

    Here, the biological function of the plant-microbiome system is the result of contributions from the host plant and microbiome members. In this work we study the function of a simplified community consisting of Pseudomonas and Burkholderia bacterial strains isolated from Populus hosts and inoculated on axenic Populus cutting in controlled laboratory conditions. Inoculation individually with either bacterial isolate increased root growth relative to uninoculated controls. Root area, photosynthetic efficiency, gene expression and metabolite expression data in individual and dual inoculated treatments indicate that the effects of these bacteria are unique and additive, suggesting that the function of a microbiome communitymore » may be predicted from the additive functions of the individual members.« less

  13. Reduced toll-like receptor 4 and substance P gene expression is associated with airway bacterial colonization in children.

    PubMed

    Grissell, Terry V; Chang, Anne B; Gibson, Peter G

    2007-04-01

    Neuro-immune interactions are increasingly relevant to human health and disease. The neuropeptide Substance P also has antibacterial activity and bears similarities to the innate immune antibacterial defensins. This suggests possible co-regulation of neuropeptide and innate immune mediators. In this study, non-bronchoscopic bronchoalveolar lavage (BAL) was performed on 69 children. BAL was examined for cellular profile, microbiology (bacteria, virus) and gene expression for TLRs 2, 3, 4; chemokine receptors (CCR3, CCR5, CXCR1); neurotrophins and neurokinin genes (TAC1, TAC3, CGRP, NGF). In children with bacterial colonization (n=10) there was an airway inflammatory response with increased BAL neutrophils, IL-8 protein, and CXCR1 expression. Substance P (TAC1) and TLR4 RNA expression were reduced in children with bacterial colonization. TLR3 mRNA was increased in 7.2% (n=5) children with rhinovirus, and there was a non-significant trend to increased TLR2. There is evidence for co-regulation of neurokinin (TAC1) and TLR4 gene expression in airway cells from children with airway bacterial colonization and their reduced expression may be associated with an impaired bacterial clearance. (c) 2007 Wiley-Liss, Inc.

  14. Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

    PubMed

    Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

    2018-04-15

    Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    PubMed

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  16. Regulation of Bacterial Virulence by Csr (Rsm) Systems

    PubMed Central

    Vakulskas, Christopher A.; Potts, Anastasia H.; Babitzke, Paul; Ahmer, Brian M. M.

    2015-01-01

    SUMMARY Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5′ untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens. PMID:25833324

  17. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution.

    PubMed

    Erickson, Keesha E; Otoupal, Peter B; Chatterjee, Anushree

    2017-01-01

    Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress

  18. Messing with Bacterial Quorum Sensing

    PubMed Central

    González, Juan E.; Keshavan, Neela D.

    2006-01-01

    Quorum sensing is widely recognized as an efficient mechanism to regulate expression of specific genes responsible for communal behavior in bacteria. Several bacterial phenotypes essential for the successful establishment of symbiotic, pathogenic, or commensal relationships with eukaryotic hosts, including motility, exopolysaccharide production, biofilm formation, and toxin production, are often regulated by quorum sensing. Interestingly, eukaryotes produce quorum-sensing-interfering (QSI) compounds that have a positive or negative influence on the bacterial signaling network. This eukaryotic interference could result in further fine-tuning of bacterial quorum sensing. Furthermore, recent work involving the synthesis of structural homologs to the various quorum-sensing signal molecules has resulted in the development of additional QSI compounds that could be used to control pathogenic bacteria. The creation of transgenic plants that express bacterial quorum-sensing genes is yet another strategy to interfere with bacterial behavior. Further investigation on the manipulation of quorum-sensing systems could provide us with powerful tools against harmful bacteria. PMID:17158701

  19. Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

    PubMed

    Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

    2016-01-01

    Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

  20. Computational design of a Zn2+ receptor that controls bacterial gene expression

    NASA Astrophysics Data System (ADS)

    Dwyer, M. A.; Looger, L. L.; Hellinga, H. W.

    2003-09-01

    The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a -galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.

  1. Phytodetoxification of TNT by transplastomic tobacco (Nicotiana tabacum) expressing a bacterial nitroreductase.

    PubMed

    Zhang, Long; Rylott, Elizabeth L; Bruce, Neil C; Strand, Stuart E

    2017-09-01

    Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT. The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.

  2. Determinants of bacterial communities in Canadian agroforestry systems.

    PubMed

    Banerjee, Samiran; Baah-Acheamfour, Mark; Carlyle, Cameron N; Bissett, Andrew; Richardson, Alan E; Siddique, Tariq; Bork, Edward W; Chang, Scott X

    2016-06-01

    Land-use change is one of the most important factors influencing soil microbial communities, which play a pivotal role in most biogeochemical and ecological processes. Using agroforestry systems as a model, this study examined the effects of land uses and edaphic properties on bacterial communities in three agroforestry types covering a 270 km soil-climate gradient in Alberta, Canada. Our results demonstrate that land-use patterns exert stronger effects on soil bacterial communities than soil zones in these agroforestry systems. Plots with trees in agroforestry systems promoted greater bacterial abundance and to some extent species richness, which was associated with more nutrient-rich soil resources. While Acidobacteria, Actinobacteria and Alphaproteobacteria were the dominant bacterial phyla and subphyla across land uses, Arthrobacter, Acidobacteria_Gp16, Burkholderia, Rhodanobacter and Rhizobium were the keystone taxa in these agroforestry systems. Soil pH and carbon contents emerged as the major determinants of bacterial community characteristics. We found non-random co-occurrence and modular patterns of soil bacterial communities, and these patterns were controlled by edaphic factors and not their taxonomy. Overall, this study highlights the drivers and co-occurrence patterns of soil microbial communities in agroforestry systems. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  3. Interplay of Noisy Gene Expression and Dynamics Explains Patterns of Bacterial Operon Organization

    NASA Astrophysics Data System (ADS)

    Igoshin, Oleg

    2011-03-01

    Bacterial chromosomes are organized into operons -- sets of genes co-transcribed into polycistronic messenger RNA. Hypotheses explaining the emergence and maintenance of operons include proportional co-regulation, horizontal transfer of intact ``selfish'' operons, emergence via gene duplication, and co-production of physically interacting proteins to speed their association. We hypothesized an alternative: operons can reduce or increase intrinsic gene expression noise in a manner dependent on the post-translational interactions, thereby resulting in selection for or against operons in depending on the network architecture. We devised five classes of two-gene network modules and show that the effects of operons on intrinsic noise depend on class membership. Two classes exhibit decreased noise with co-transcription, two others reveal increased noise, and the remaining one does not show a significant difference. To test our modeling predictions we employed bioinformatic analysis to determine the relationship gene expression noise and operon organization. The results confirm the overrepresentation of noise-minimizing operon architectures and provide evidence against other hypotheses. Our results thereby suggest a central role for gene expression noise in selecting for or maintaining operons in bacterial chromosomes. This demonstrates how post-translational network dynamics may provide selective pressure for organizing bacterial chromosomes, and has practical consequences for designing synthetic gene networks. This work is supported by National Institutes of Health grant 1R01GM096189-01.

  4. UGT-29 protein expression and localization during bacterial infection in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Wong, Rui-Rui; Lee, Song-Hua; Nathan, Sheila

    2014-09-01

    The nematode Caenorhabditis elegans is routinely used as an animal model to delineate complex molecular mechanisms involved in the host response to pathogen infection. Following up on an earlier study on host-pathogen interaction, we constructed a ugt-29::GFP transcriptional fusion transgenic worm strain to examine UGT-29 protein expression and localization upon bacterial infection. UGT-29 orthologs can be found in higher organisms including humans and is proposed as a member of the UDP-Glucoronosyl Transferase family of proteins which are involved in phase II detoxification of compounds detrimental to the host organism. Under uninfected conditions, UGT-29::GFP fusion protein was highly expressed in the C. elegans anterior pharynx and intestine, two major organs involved in detoxification. We further evaluated the localization of the enzyme in worms infected with the bacterial pathogen, Burkholderia pseudomallei. The infected ugt-29::GFP transgenic strain exhibited increased fluorescence in the pharynx and intestine with pronounced fluorescence also extending to body wall muscle. This transcriptional fusion GFP transgenic worm is a convenient and direct tool to provide information on UGT detoxification enzyme gene expression and could be a useful tool for a number of diverse applications.

  5. Analysis of gene expression levels in individual bacterial cells without image segmentation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J., E-mail: sjhagen@ufl.edu

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on amore » segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.« less

  6. Regulation of bacterial virulence by Csr (Rsm) systems.

    PubMed

    Vakulskas, Christopher A; Potts, Anastasia H; Babitzke, Paul; Ahmer, Brian M M; Romeo, Tony

    2015-06-01

    Most bacterial pathogens have the remarkable ability to flourish in the external environment and in specialized host niches. This ability requires their metabolism, physiology, and virulence factors to be responsive to changes in their surroundings. It is no surprise that the underlying genetic circuitry that supports this adaptability is multilayered and exceedingly complex. Studies over the past 2 decades have established that the CsrA/RsmA proteins, global regulators of posttranscriptional gene expression, play important roles in the expression of virulence factors of numerous proteobacterial pathogens. To accomplish these tasks, CsrA binds to the 5' untranslated and/or early coding regions of mRNAs and alters translation, mRNA turnover, and/or transcript elongation. CsrA activity is regulated by noncoding small RNAs (sRNAs) that contain multiple CsrA binding sites, which permit them to sequester multiple CsrA homodimers away from mRNA targets. Environmental cues sensed by two-component signal transduction systems and other regulatory factors govern the expression of the CsrA-binding sRNAs and, ultimately, the effects of CsrA on secretion systems, surface molecules and biofilm formation, quorum sensing, motility, pigmentation, siderophore production, and phagocytic avoidance. This review presents the workings of the Csr system, the paradigm shift that it generated for understanding posttranscriptional regulation, and its roles in virulence networks of animal and plant pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. In Vitro Trypanocidal Activity of Antibodies to Bacterially Expressed Trypanosoma brucei Tubulin

    PubMed Central

    Kateete, DP; Alezuyo, C; Nanteza, A; Asiimwe, C; Lubega, GW

    2012-01-01

    Background There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth. Methods T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450µg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera. Results Low antisera dilutions (25%) from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions. Conclusion Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes. PMID:23109963

  8. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    PubMed Central

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  9. Systemic acquired tolerance to virulent bacterial pathogens in tomato.

    PubMed

    Block, Anna; Schmelz, Eric; O'Donnell, Phillip J; Jones, Jeffrey B; Klee, Harry J

    2005-07-01

    Recent studies on the interactions between plants and pathogenic microorganisms indicate that the processes of disease symptom development and pathogen growth can be uncoupled. Thus, in many instances, the symptoms associated with disease represent an active host response to the presence of a pathogen. These host responses are frequently mediated by phytohormones. For example, ethylene and salicylic acid (SA) mediate symptom development but do not influence bacterial growth in the interaction between tomato (Lycopersicon esculentum) and virulent Xanthomonas campestris pv vesicatoria (Xcv). It is not apparent why extensive tissue death is integral to a defense response if it does not have the effect of limiting pathogen proliferation. One possible function for this hormone-mediated response is to induce a systemic defense response. We therefore assessed the systemic responses of tomato to Xcv. SA- and ethylene-deficient transgenic lines were used to investigate the roles of these phytohormones in systemic signaling. Virulent and avirulent Xcv did induce a systemic response as evidenced by expression of defense-associated pathogenesis-related genes in an ethylene- and SA-dependent manner. This systemic response reduced cell death but not bacterial growth during subsequent challenge with virulent Xcv. This systemic acquired tolerance (SAT) consists of reduced tissue damage in response to secondary challenge with a virulent pathogen with no effect upon pathogen growth. SAT was associated with a rapid ethylene and pathogenesis-related gene induction upon challenge. SAT was also induced by infection with Pseudomonas syringae pv tomato. These data show that SAT resembles systemic acquired resistance without inhibition of pathogen growth.

  10. Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data.

    PubMed

    Tintle, Nathan L; Sitarik, Alexandra; Boerema, Benjamin; Young, Kylie; Best, Aaron A; Dejongh, Matthew

    2012-08-08

    Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

  11. Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ΦX174 in Escherichia coli

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yung, Mimi C.; Bourguet, Feliza A.; Carpenter, Timothy S.

    Recombinant expression of toxic proteins remains a challenging problem. Furthermore, one potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ΦX174 inside recombinant BMCs to enhance its expression and achieve higher yields duringmore » downstream purification.« less

  12. Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ΦX174 in Escherichia coli

    DOE PAGES

    Yung, Mimi C.; Bourguet, Feliza A.; Carpenter, Timothy S.; ...

    2017-04-26

    Recombinant expression of toxic proteins remains a challenging problem. Furthermore, one potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ΦX174 inside recombinant BMCs to enhance its expression and achieve higher yields duringmore » downstream purification.« less

  13. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

    PubMed Central

    Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

    2005-01-01

    Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  14. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

    PubMed

    Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

    2003-08-01

    Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

  15. Bacterial community changes in an industrial algae production system.

    PubMed

    Fulbright, Scott P; Robbins-Pianka, Adam; Berg-Lyons, Donna; Knight, Rob; Reardon, Kenneth F; Chisholm, Stephen T

    2018-04-01

    While microalgae are a promising feedstock for production of fuels and other chemicals, a challenge for the algal bioproducts industry is obtaining consistent, robust algae growth. Algal cultures include complex bacterial communities and can be difficult to manage because specific bacteria can promote or reduce algae growth. To overcome bacterial contamination, algae growers may use closed photobioreactors designed to reduce the number of contaminant organisms. Even with closed systems, bacteria are known to enter and cohabitate, but little is known about these communities. Therefore, the richness, structure, and composition of bacterial communities were characterized in closed photobioreactor cultivations of Nannochloropsis salina in F/2 medium at different scales, across nine months spanning late summer-early spring, and during a sequence of serially inoculated cultivations. Using 16S rRNA sequence data from 275 samples, bacterial communities in small, medium, and large cultures were shown to be significantly different. Larger systems contained richer bacterial communities compared to smaller systems. Relationships between bacterial communities and algae growth were complex. On one hand, blooms of a specific bacterial type were observed in three abnormal, poorly performing replicate cultivations, while on the other, notable changes in the bacterial community structures were observed in a series of serial large-scale batch cultivations that had similar growth rates. Bacteria common to the majority of samples were identified, including a single OTU within the class Saprospirae that was found in all samples. This study contributes important information for crop protection in algae systems, and demonstrates the complex ecosystems that need to be understood for consistent, successful industrial algae cultivation. This is the first study to profile bacterial communities during the scale-up process of industrial algae systems.

  16. CRISPR Perturbation of Gene Expression Alters Bacterial Fitness under Stress and Reveals Underlying Epistatic Constraints.

    PubMed

    Otoupal, Peter B; Erickson, Keesha E; Escalas-Bordoy, Antoni; Chatterjee, Anushree

    2017-01-20

    The evolution of antibiotic resistance has engendered an impending global health crisis that necessitates a greater understanding of how resistance emerges. The impact of nongenetic factors and how they influence the evolution of resistance is a largely unexplored area of research. Here we present a novel application of CRISPR-Cas9 technology for investigating how gene expression governs the adaptive pathways available to bacteria during the evolution of resistance. We examine the impact of gene expression changes on bacterial adaptation by constructing a library of deactivated CRISPR-Cas9 synthetic devices to tune the expression of a set of stress-response genes in Escherichia coli. We show that artificially inducing perturbations in gene expression imparts significant synthetic control over fitness and growth during stress exposure. We present evidence that these impacts are reversible; strains with synthetically perturbed gene expression regained wild-type growth phenotypes upon stress removal, while maintaining divergent growth characteristics under stress. Furthermore, we demonstrate a prevailing trend toward negative epistatic interactions when multiple gene perturbations are combined simultaneously, thereby posing an intrinsic constraint on gene expression underlying adaptive trajectories. Together, these results emphasize how CRISPR-Cas9 can be employed to engineer gene expression changes that shape bacterial adaptation, and present a novel approach to synthetically control the evolution of antimicrobial resistance.

  17. Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae.

    PubMed

    Yang, Xiao-Yan; Sun, Bin; Zhang, Liang; Li, Nan; Han, Junlong; Zhang, Jing; Sun, Xuesong; He, Qing-Yu

    2014-01-01

    Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

  18. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis.

    PubMed

    Rocha, Danilo J P; Santos, Carolina S; Pacheco, Luis G C

    2015-09-01

    The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.

  19. Potential risk for bacterial contamination in conventional reused ventilator systems and disposable closed ventilator-suction systems.

    PubMed

    Li, Ya-Chi; Lin, Hui-Ling; Liao, Fang-Chun; Wang, Sing-Siang; Chang, Hsiu-Chu; Hsu, Hung-Fu; Chen, Sue-Hsien; Wan, Gwo-Hwa

    2018-01-01

    Few studies have investigated the difference in bacterial contamination between conventional reused ventilator systems and disposable closed ventilator-suction systems. The aim of this study was to investigate the bacterial contamination rates of the reused and disposable ventilator systems, and the association between system disconnection and bacterial contamination of ventilator systems. The enrolled intubated and mechanically ventilated patients used a conventional reused ventilator system and a disposable closed ventilator-suction system, respectively, for a week; specimens were then collected from the ventilator circuit systems to evaluate human and environmental bacterial contamination. The sputum specimens from patients were also analyzed in this study. The detection rate of bacteria in the conventional reused ventilator system was substantially higher than that in the disposable ventilator system. The inspiratory and expiratory limbs of the disposable closed ventilator-suction system had higher bacterial concentrations than the conventional reused ventilator system. The bacterial concentration in the heated humidifier of the reused ventilator system was significantly higher than that in the disposable ventilator system. Positive associations existed among the bacterial concentrations at different locations in the reused and disposable ventilator systems, respectively. The predominant bacteria identified in the reused and disposable ventilator systems included Acinetobacter spp., Bacillus cereus, Elizabethkingia spp., Pseudomonas spp., and Stenotrophomonas (Xan) maltophilia. Both the reused and disposable ventilator systems had high bacterial contamination rates after one week of use. Disconnection of the ventilator systems should be avoided during system operation to decrease the risks of environmental pollution and human exposure, especially for the disposable ventilator system. ClinicalTrials.gov PRS / NCT03359148.

  20. GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium.

    PubMed

    Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

    2015-01-01

    To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

  1. GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

    PubMed Central

    Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

    2015-01-01

    To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

  2. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

    PubMed

    Visser, M; Stephan, D; Jaynes, J M; Burger, J T

    2012-06-01

    Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started. © 2012 The Authors. Letters in Applied Microbiology © 2012

  4. Potential risk for bacterial contamination in conventional reused ventilator systems and disposable closed ventilator-suction systems

    PubMed Central

    Li, Ya-Chi; Lin, Hui-Ling; Liao, Fang-Chun; Wang, Sing-Siang; Chang, Hsiu-Chu; Hsu, Hung-Fu; Chen, Sue-Hsien

    2018-01-01

    Background Few studies have investigated the difference in bacterial contamination between conventional reused ventilator systems and disposable closed ventilator-suction systems. The aim of this study was to investigate the bacterial contamination rates of the reused and disposable ventilator systems, and the association between system disconnection and bacterial contamination of ventilator systems. Methods The enrolled intubated and mechanically ventilated patients used a conventional reused ventilator system and a disposable closed ventilator-suction system, respectively, for a week; specimens were then collected from the ventilator circuit systems to evaluate human and environmental bacterial contamination. The sputum specimens from patients were also analyzed in this study. Results The detection rate of bacteria in the conventional reused ventilator system was substantially higher than that in the disposable ventilator system. The inspiratory and expiratory limbs of the disposable closed ventilator-suction system had higher bacterial concentrations than the conventional reused ventilator system. The bacterial concentration in the heated humidifier of the reused ventilator system was significantly higher than that in the disposable ventilator system. Positive associations existed among the bacterial concentrations at different locations in the reused and disposable ventilator systems, respectively. The predominant bacteria identified in the reused and disposable ventilator systems included Acinetobacter spp., Bacillus cereus, Elizabethkingia spp., Pseudomonas spp., and Stenotrophomonas (Xan) maltophilia. Conclusions Both the reused and disposable ventilator systems had high bacterial contamination rates after one week of use. Disconnection of the ventilator systems should be avoided during system operation to decrease the risks of environmental pollution and human exposure, especially for the disposable ventilator system. Trial registration Clinical

  5. Role of quorum sensing in bacterial infections

    PubMed Central

    Castillo-Juárez, Israel; Maeda, Toshinari; Mandujano-Tinoco, Edna Ayerim; Tomás, María; Pérez-Eretza, Berenice; García-Contreras, Silvia Julieta; Wood, Thomas K; García-Contreras, Rodolfo

    2015-01-01

    Quorum sensing (QS) is cell communication that is widely used by bacterial pathogens to coordinate the expression of several collective traits, including the production of multiple virulence factors, biofilm formation, and swarming motility once a population threshold is reached. Several lines of evidence indicate that QS enhances virulence of bacterial pathogens in animal models as well as in human infections; however, its relative importance for bacterial pathogenesis is still incomplete. In this review, we discuss the present evidence from in vitro and in vivo experiments in animal models, as well as from clinical studies, that link QS systems with human infections. We focus on two major QS bacterial models, the opportunistic Gram negative bacteria Pseudomonas aeruginosa and the Gram positive Staphylococcus aureus, which are also two of the main agents responsible of nosocomial and wound infections. In addition, QS communication systems in other bacterial, eukaryotic pathogens, and even immune and cancer cells are also reviewed, and finally, the new approaches proposed to combat bacterial infections by the attenuation of their QS communication systems and virulence are also discussed. PMID:26244150

  6. Bacterial toxin-antitoxin systems: more than selfish entities?

    PubMed

    Van Melderen, Laurence; Saavedra De Bast, Manuel

    2009-03-01

    Bacterial toxin-antitoxin (TA) systems are diverse and widespread in the prokaryotic kingdom. They are composed of closely linked genes encoding a stable toxin that can harm the host cell and its cognate labile antitoxin, which protects the host from the toxin's deleterious effect. TA systems are thought to invade bacterial genomes through horizontal gene transfer. Some TA systems might behave as selfish elements and favour their own maintenance at the expense of their host. As a consequence, they may contribute to the maintenance of plasmids or genomic islands, such as super-integrons, by post-segregational killing of the cell that loses these genes and so suffers the stable toxin's destructive effect. The function of the chromosomally encoded TA systems is less clear and still open to debate. This Review discusses current hypotheses regarding the biological roles of these evolutionarily successful small operons. We consider the various selective forces that could drive the maintenance of TA systems in bacterial genomes.

  7. Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities?

    PubMed Central

    Van Melderen, Laurence; Saavedra De Bast, Manuel

    2009-01-01

    Bacterial toxin–antitoxin (TA) systems are diverse and widespread in the prokaryotic kingdom. They are composed of closely linked genes encoding a stable toxin that can harm the host cell and its cognate labile antitoxin, which protects the host from the toxin's deleterious effect. TA systems are thought to invade bacterial genomes through horizontal gene transfer. Some TA systems might behave as selfish elements and favour their own maintenance at the expense of their host. As a consequence, they may contribute to the maintenance of plasmids or genomic islands, such as super-integrons, by post-segregational killing of the cell that loses these genes and so suffers the stable toxin's destructive effect. The function of the chromosomally encoded TA systems is less clear and still open to debate. This Review discusses current hypotheses regarding the biological roles of these evolutionarily successful small operons. We consider the various selective forces that could drive the maintenance of TA systems in bacterial genomes. PMID:19325885

  8. Application of Whole Genome Expression Analysis to Assess Bacterial Responses to Environmental Conditions

    NASA Astrophysics Data System (ADS)

    Vukanti, R. V.; Mintz, E. M.; Leff, L. G.

    2005-05-01

    Bacterial responses to environmental signals are multifactorial and are coupled to changes in gene expression. An understanding of bacterial responses to environmental conditions is possible using microarray expression analysis. In this study, the utility of microarrays for examining changes in gene expression in Escherichia coli under different environmental conditions was assessed. RNA was isolated, hybridized to Affymetrix E. coli Genome 2.0 chips and analyzed using Affymetrix GCOS and Genespring software. Major limiting factors were obtaining enough quality RNA (107-108 cells to get 10μg RNA)and accounting for differences in growth rates under different conditions. Stabilization of RNA prior to isolation and taking extreme precautions while handling RNA were crucial. In addition, use of this method in ecological studies is limited by availability and cost of commercial arrays; choice of primers for cDNA synthesis, reproducibility, complexity of results generated and need to validate findings. This method may be more widely applicable with the development of better approaches for RNA recovery from environmental samples and increased number of available strain-specific arrays. Diligent experimental design and verification of results with real-time PCR or northern blots is needed. Overall, there is a great potential for use of this technology to discover mechanisms underlying organisms' responses to environmental conditions.

  9. The effect of dietary bacterial organic selenium on growth performance, antioxidant capacity, and Selenoproteins gene expression in broiler chickens.

    PubMed

    Dalia, A M; Loh, T C; Sazili, A Q; Jahromi, M F; Samsudin, A A

    2017-08-18

    Selenium (Se) is an essential trace mineral in broilers, which has several important roles in biological processes. Organic forms of Se are more efficient than inorganic forms and can be produced biologically via Se microbial reduction. Hence, the possibility of using Se-enriched bacteria as feed supplement may provide an interesting source of organic Se, and benefit broiler antioxidant system and other biological processes. The objective of this study was to examine the impacts of inorganic Se and different bacterial organic Se sources on the performance, serum and tissues Se status, antioxidant capacity, and liver mRNA expression of selenoproteins in broilers. Results indicated that different Se sources did not significantly (P ≤ 0.05) affect broiler growth performance. However, bacterial organic Se of T5 (basal diet +0.3 mg /kg feed ADS18 Se), T4 (basal diet +0.3 mg /kg feed ADS2 Se), and T3 (basal diet +0.3 mg /kg feed ADS1 Se) exhibited significantly (P ≤ 0.05) highest Se concentration in serum, liver, and kidney respectively. Dietary inorganic Se and bacterial organic Se were observed to significantly affect broiler serum ALT, AST, LDH activities and serum creatinine level. ADS18 supplemented Se of (Stenotrophomonas maltophilia) bacterial strain showed the highest GSH-Px activity with the lowest MDA content in serum, and the highest GSH-Px and catalase activity in the kidney, while bacterial Se of ADS2 (Klebsiella pneumoniae) resulted in a higher level of GSH-Px1 and catalase in liver. Moreover, our study showed that in comparison with sodium selenite, only ADS18 bacterial Se showed a significantly higher mRNA level in GSH-Px1, GSH-Px4, DIO1, and TXNDR1, while both ADS18 and ADS2 showed high level of mRNA of DIO2 compared to sodium selenite. The supplementation of bacterial organic Se in broiler chicken, improved tissue Se deposition, antioxidant status, and selenoproteins gene expression, and can be considered as an effective alternative source of

  10. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    PubMed Central

    Branco, Luis M; Matschiner, Alex; Fair, Joseph N; Goba, Augustine; Sampey, Darryl B; Ferro, Philip J; Cashman, Kathleen A; Schoepp, Randal J; Tesh, Robert B; Bausch, Daniel G; Garry, Robert F; Guttieri, Mary C

    2008-01-01

    Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination. PMID:18538016

  11. Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells.

    PubMed

    Inouye, Satoshi; Suzuki, Takahiro

    2016-12-01

    The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc, EpGLuc, and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc. The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Rock bream (Oplegnathus fasciatus) IL-12p40: identification, expression, and effect on bacterial infection.

    PubMed

    Zhang, Lu; Zhang, Bao-Cun; Hu, Yong-Hua

    2014-08-01

    IL-12p40, also called IL-12β, is a subunit of the proinflammatory cytokines interleukin (IL)-12 and IL-23. In teleost, IL-12p40 homologues have been identified in several species, however, the biological function of fish IL-12p40 is essentially unknown. In this work, we reported the identification and analysis of an IL-12p40, OfIL-12p40, from rock bream (Oplegnathus fasciatus). OfIL-12p40 is composed of 361 amino acids and possesses a conserved IL-12p40 domain and a WSxWS signature motif characteristic of known IL-12p40. Constitutive expression of OfIL-12p40 occurred in multiple tissues and was highest in kidney. Experimental infection with bacterial pathogen upregulated the expression of OfIL-12p40 in kidney and spleen in a time-dependent manner. Purified recombinant OfIL-12p40 (rOfIL-12p40) stimulated the respiratory burst activity of peripheral blood leukocytes in a dose-dependent manner. rOfIL-12p40 also enhanced the resistance of rock bream against bacterial infection and upregulated the expression of innate immune genes in kidney. Taken together, these results indicate that OfIL-12p40 possesses cytokine-like property and plays a role in immune defense against bacterial infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Insight into the effects of different cropping systems on soil bacterial community and tobacco bacterial wilt rate.

    PubMed

    Niu, Jiaojiao; Chao, Jin; Xiao, Yunhua; Chen, Wu; Zhang, Chao; Liu, Xueduan; Rang, Zhongwen; Yin, Huaqun; Dai, Linjian

    2017-01-01

    Rotation is an effective strategy to control crop disease and improve plant health. However, the effects of crop rotation on soil bacterial community composition and structure, and crop health remain unclear. In this study, using 16S rRNA gene sequencing, we explored the soil bacterial communities under four different cropping systems, continuous tobacco cropping (control group), tobacco-maize rotation, tobacco-lily rotation, and tobacco-turnip rotation. Results of detrended correspondence analysis and dissimilarity tests showed that soil bacterial community composition and structure changed significantly among the four groups, such that Acidobacteria and Actinobacteria were more abundant in the maize rotation group (16.6 and 11.5%, respectively) than in the control (8.5 and 7.1%, respectively). Compared with the control group (57.78%), maize and lily were effective rotation crops in controlling tobacco bacterial wilt (about 23.54 and 48.67%). On the other hand, tobacco bacterial wilt rate was increased in the turnip rotation (59.62%) relative to the control. Further study revealed that the abundances of several bacterial populations were directly correlated with tobacco bacterial wilt. For example, Acidobacteria and Actinobacteria were significantly negatively correlated to the tobacco bacterial wilt rate, so they may be probiotic bacteria. Canonical correspondence analysis showed that soil pH and calcium content were key factors in determining soil bacterial communities. In conclusion, our study revealed the composition and structure of bacterial communities under four different cropping systems and may unveil molecular mechanisms for the interactions between soil microorganisms and crop health. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

    PubMed

    Steinmetz, Eric J; Auldridge, Michele E

    2017-11-01

    The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  15. Increased bacterial cell density and recombinant protein yield using a commercial microbial cultivation system.

    PubMed

    Peck, Grantley R; Bowden, Timothy R; Shiell, Brian J; Michalski, Wojtek P

    2014-01-01

    EnBase (BioSilta, Finland) is a microbial cultivation system that replicates fed-batch systems through sustained release of glucose by enzymatic degradation of a polymeric substrate. Achievable bacterial cell densities and recombinant capripoxvirus protein expression levels, solubility, and antigenicity using the EnBase system were assessed. BL21-AI Escherichia coli expressing capripoxvirus proteins achieved up to eightfold higher cell densities when grown in EnBase media compared with standard media. Greater yields of capripoxvirus proteins were attained using EnBase media, either through increases in the amount of expressed protein per cell in conjunction with higher cell density or through the increase in cell density alone. Addition of EnBase booster enhanced protein yield for one of the proteins tested but reduced yield for the other. However, the amount of soluble forms of the capripoxvirus proteins tested was not different from that observed from cultures grown under standard conditions. Purified capripoxvirus proteins expressed using EnBase or standard media were assessed for their performance by enzyme-linked immunosorbent assay (ELISA) and were shown to be equally capable of specifically binding capripoxvirus antibodies.

  16. Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+ -pumping pyrophosphatase in pepper plants.

    PubMed

    Vigani, Gianpiero; Rolli, Eleonora; Marasco, Ramona; Dell'Orto, Marta; Michoud, Grégoire; Soussi, Asma; Raddadi, Noura; Borin, Sara; Sorlini, Claudia; Zocchi, Graziano; Daffonchio, Daniele

    2018-05-22

    It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H + -ATPase (V-ATPase) and H + -PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H + -ATPase (V-ATPase) and H + -PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  17. Evaluation of zraP gene expression characteristics and construction of a lead (Pb) sensing and removal system in a recombinant Escherichia coli.

    PubMed

    Maruthamuthu, Murali Kannan; Ganesh, Irisappan; Ravikumar, Sambandam; Hong, Soon Ho

    2015-03-01

    A ZraP-based lead sensing and removal system was constructed in E. coli. It was regulated by the ZraS/ZraR two-component system. The expression profile of the zraP gene towards extracellular lead was studied via real-time PCR. A dual-function bacterial system was also designed to express GFP and OmpC-lead binding peptide under the control of zraP for the simultaneous sensing and adsorption of environmental lead without additional manipulation. The constructed bacterial system can emit fluorescence and it adsorbed a maximum of 487 µmol lead/g cell DCW. From a study of artificial wastewater, the constructed bacteria adsorbed lead highly selectively (427 µmol lead/g cell DCW) among other metal ions. The newly-constructed dual function bacterial system can be applied for the development of an efficient process for the removal of lead from polluted wastes.

  18. Dynamic network reconstruction from gene expression data applied to immune response during bacterial infection.

    PubMed

    Guthke, Reinhard; Möller, Ulrich; Hoffmann, Martin; Thies, Frank; Töpfer, Susanne

    2005-04-15

    The immune response to bacterial infection represents a complex network of dynamic gene and protein interactions. We present an optimized reverse engineering strategy aimed at a reconstruction of this kind of interaction networks. The proposed approach is based on both microarray data and available biological knowledge. The main kinetics of the immune response were identified by fuzzy clustering of gene expression profiles (time series). The number of clusters was optimized using various evaluation criteria. For each cluster a representative gene with a high fuzzy-membership was chosen in accordance with available physiological knowledge. Then hypothetical network structures were identified by seeking systems of ordinary differential equations, whose simulated kinetics could fit the gene expression profiles of the cluster-representative genes. For the construction of hypothetical network structures singular value decomposition (SVD) based methods and a newly introduced heuristic Network Generation Method here were compared. It turned out that the proposed novel method could find sparser networks and gave better fits to the experimental data. Reinhard.Guthke@hki-jena.de.

  19. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

    PubMed

    Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

    2016-07-21

    Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

    PubMed

    Yan, Kelly; Merritt, Hanne; Crawford, Kenneth; Pardee, Gwynn; Cheng, Jan Marie; Widger, Stephania; Hekmat-Nejad, Mohammad; Zaror, Isabel; Sim, Janet

    2015-06-01

    Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins.

    PubMed

    Angius, Federica; Ilioaia, Oana; Amrani, Amira; Suisse, Annabelle; Rosset, Lindsay; Legrand, Amélie; Abou-Hamdan, Abbas; Uzan, Marc; Zito, Francesca; Miroux, Bruno

    2018-06-05

    Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.

  2. A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

    PubMed Central

    Tian, Tian; Salis, Howard M.

    2015-01-01

    Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

  3. Evolvable social agents for bacterial systems modeling.

    PubMed

    Paton, Ray; Gregory, Richard; Vlachos, Costas; Saunders, Jon; Wu, Henry

    2004-09-01

    We present two approaches to the individual-based modeling (IbM) of bacterial ecologies and evolution using computational tools. The IbM approach is introduced, and its important complementary role to biosystems modeling is discussed. A fine-grained model of bacterial evolution is then presented that is based on networks of interactivity between computational objects representing genes and proteins. This is followed by a coarser grained agent-based model, which is designed to explore the evolvability of adaptive behavioral strategies in artificial bacteria represented by learning classifier systems. The structure and implementation of the two proposed individual-based bacterial models are discussed, and some results from simulation experiments are presented, illustrating their adaptive properties.

  4. Comparative Genomic Analyses of the Bacterial Phosphotransferase System

    PubMed Central

    Barabote, Ravi D.; Saier, Milton H.

    2005-01-01

    We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer. PMID:16339738

  5. Putative bacterial volatile-mediated growth in soybean (Glycine max L. Merrill) and expression of induced proteins under salt stress.

    PubMed

    Vaishnav, A; Kumari, S; Jain, S; Varma, A; Choudhary, D K

    2015-08-01

    Plant root-associated rhizobacteria elicit plant immunity referred to as induced systemic tolerance (IST) against multiple abiotic stresses. Among multibacterial determinants involved in IST, the induction of IST and promotion of growth by putative bacterial volatile compounds (VOCs) is reported in the present study. To characterize plant proteins induced by putative bacterial VOCs, proteomic analysis was performed by MALDI-MS/MS after exposure of soybean seedlings to a new strain of plant growth promoting rhizobacteria (PGPR) Pseudomonas simiae strain AU. Furthermore, expression analysis by Western blotting confirmed that the vegetative storage protein (VSP), gamma-glutamyl hydrolase (GGH) and RuBisCo large chain proteins were significantly up-regulated by the exposure to AU strain and played a major role in IST. VSP has preponderant roles in N accumulation and mobilization, acid phosphatase activity and Na(+) homeostasis to sustain plant growth under stress condition. More interestingly, plant exposure to the bacterial strain significantly reduced Na(+) and enhanced K(+) and P content in root of soybean seedlings under salt stress. In addition, high accumulation of proline and chlorophyll content also provided evidence of protection against osmotic stress during the elicitation of IST by bacterial exposure. The present study reported for the first time that Ps. simiae produces a putative volatile blend that can enhance soybean seedling growth and elicit IST against 100 mmol l(-1) NaCl stress condition. The identification of such differentially expressed proteins provide new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to stresses. Further work should uncover more about the chemical side of VOC compounds and a detailed study about their molecular mechanism responsible for plant growth. © 2015 The Society for Applied Microbiology.

  6. Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

    PubMed Central

    Aleshin, SE; Timofeev, AV; Khoretonenko, MV; Zakharova, LG; Pashvykina, GV; Stephenson, JR; Shneider, AM; Altstein, AD

    2005-01-01

    Background Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. Results The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. Conclusion Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines. PMID:16076390

  7. Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

    PubMed

    Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

    2018-10-01

    The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

    PubMed

    Jalomo-Khayrova, Ekaterina; Mares, Rosa E; Muñoz, Patricia L A; Meléndez-López, Samuel G; Rivero, Ignacio A; Ramos, Marco A

    2018-04-03

    Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

  9. Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System

    PubMed Central

    Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya

    2018-01-01

    ABSTRACT We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B. pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B. bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant

  10. Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System.

    PubMed

    Ishigaki, Keisuke; Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko

    2018-01-01

    We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica , which is required for O antigen biosynthesis, into the chromosome of B. pertussis , which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti- B. bronchiseptica antibody but not the anti- B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica . O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis , which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant

  11. Interference in Bacterial Quorum Sensing: A Biopharmaceutical Perspective

    PubMed Central

    Rémy, Benjamin; Mion, Sonia; Plener, Laure; Elias, Mikael; Chabrière, Eric; Daudé, David

    2018-01-01

    Numerous bacteria utilize molecular communication systems referred to as quorum sensing (QS) to synchronize the expression of certain genes regulating, among other aspects, the expression of virulence factors and the synthesis of biofilm. To achieve this process, bacteria use signaling molecules, known as autoinducers (AIs), as chemical messengers to share information. Naturally occurring strategies that interfere with bacterial signaling have been extensively studied in recent years, examining their potential to control bacteria. To interfere with QS, bacteria use quorum sensing inhibitors (QSIs) to block the action of AIs and quorum quenching (QQ) enzymes to degrade signaling molecules. Recent studies have shown that these strategies are promising routes to decrease bacterial pathogenicity and decrease biofilms, potentially enhancing bacterial susceptibility to antimicrobial agents including antibiotics and bacteriophages. The efficacy of QSIs and QQ enzymes has been demonstrated in various animal models and are now considered in the development of new medical devices against bacterial infections, including dressings, and catheters for enlarging the therapeutic arsenal against bacteria. PMID:29563876

  12. Macrophage-expressed perforins mpeg1 and mpeg1.2 have an anti-bacterial function in zebrafish.

    PubMed

    Benard, Erica L; Racz, Peter I; Rougeot, Julien; Nezhinsky, Alexander E; Verbeek, Fons J; Spaink, Herman P; Meijer, Annemarie H

    2015-01-01

    Macrophage-expressed gene 1 (MPEG1) encodes an evolutionarily conserved protein with a predicted membrane attack complex/perforin domain associated with host defence against invading pathogens. In vertebrates, MPEG1/perforin-2 is an integral membrane protein of macrophages, suspected to be involved in the killing of intracellular bacteria by pore-forming activity. Zebrafish have 3 copies of MPEG1; 2 are expressed in macrophages, whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is downregulated during infection with both pathogens, mpeg1.2 is infection inducible. Upregulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll-like receptor adaptor molecule MyD88 and the transcription factor NFκB. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in an increased bacterial burden. In Salmonella typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase the bacterial burdens, but mpeg1 morphants show increased survival times. The combined results of these two in vivo infection models support the anti-bacterial function of the MPEG1/perforin-2 family and indicate that the intricate cross-regulation of the two mpeg1 copies aids the zebrafish host in combatting infection of various pathogens. © 2014 S. Karger AG, Basel.

  13. Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

    PubMed

    Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

    2001-04-01

    Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

  14. Phytodegradation of organophosphorus compounds by transgenic plants expressing a bacterial organophosphorus hydrolase.

    PubMed

    Wang, Xiaoxue; Wu, Ningfeng; Guo, Jun; Chu, Xiaoyu; Tian, Jian; Yao, Bin; Fan, Yunliu

    2008-01-18

    Organophosphorus (OP) compounds are widely used as pesticides in agriculture but cause broad-area environmental pollution. In this work, we have expressed a bacterial organophosphorus hydrolase (OPH) gene in tobacco plants. An assay of enzyme activity showed that transgenic plants could secrete OPH into the growth medium. The transgenic plants were resistant to methyl parathion (Mep), an OP pesticide, as evidenced by a toxicity test showing that the transgenic plants produced greater shoot and root biomass than did the wild-type plants. Furthermore, at 0.02% (v/v) Mep, the transgenic plants degraded more than 99% of Mep after 14 days of growth. Our work indicates that transgenic plants expressing an OPH gene may provide a new strategy for decontaminating OP pollutants.

  15. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    PubMed

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  16. Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate.

    PubMed

    Valeeva, Lia R; Nyamsuren, Chuluuntsetseg; Sharipova, Margarita R; Shakirov, Eugene V

    2018-01-01

    Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo -inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2 . The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future

  17. Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate

    PubMed Central

    Valeeva, Lia R.; Nyamsuren, Chuluuntsetseg; Sharipova, Margarita R.; Shakirov, Eugene V.

    2018-01-01

    Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop

  18. Bacterial and cytokine mixtures predict the length of gestation and are associated with miRNA expression in the cervix.

    PubMed

    Sanders, Alison P; Gennings, Chris; Svensson, Katherine; Motta, Valeria; Mercado-Garcia, Adriana; Solano, Maritsa; Baccarelli, Andrea A; Tellez-Rojo, Martha M; Wright, Robert O; Burris, Heather H

    2017-01-01

    Bacterial vaginosis may lead to preterm birth through epigenetic programming of the inflammatory response, specifically via miRNA expression. We quantified bacterial 16S rRNA, cytokine mRNA and 800 miRNA from cervical swabs obtained from 80 women at 16-19 weeks' gestation. We generated bacterial and cytokine indices using weighted quantile sum regression and examined associations with miRNA and gestational age at delivery. Each decile of the bacterial and cytokine indices was associated with shorter gestations (p < 0.005). The bacterial index was associated with miR-494, 371a, 4286, 185, 320e, 888 and 23a (p < 0.05). miR-494 remained significant after false discovery rate correction (q < 0.1). The cytokine index was associated with 27 miRNAs (p < 0.05; q < 0.01). Future investigation into the role of bacterial vaginosis- and inflammation-associated miRNA and preterm birth is warranted.

  19. Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

    PubMed

    Hassan, Karl A; Liu, Qi; Henderson, Peter J F; Paulsen, Ian T

    2015-02-10

    Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug

  20. Elevated gene expression of S100A12 is correlated with the predominant clinical inflammatory factors in patients with bacterial pneumonia.

    PubMed

    Hou, Fei; Wang, Likui; Wang, Hong; Gu, Junchao; Li, Meiling; Zhang, Jingkai; Ling, Xiao; Gao, Xiaofang; Luo, Cheng

    2015-06-01

    Inflammation is the predominant characteristic of pneumonia. The present study aimed to to identify a faster and more reliable novel inflammatory marker for the diagnosis of pneumonia. The expression of the S100A12 gene was analyzed by reverse transcription quantitative polymerase chain reaction in samples obtained from 46 patients with bacterial pneumonia and other infections, compared with samples from 20 healthy individuals, using the 2‑ΔΔCt method. The expression levels of S100A12 were increased in 12 patients with bacterial pneumonia. Compared with clinical inflammatory data, a positive correlation was observed between the expression of the S100A12 gene and levels of white blood cells, C‑reactive protein (CRP), thrombocytocrit, neutrophils, erythrocyte sedimentation and soterocytes, and an inverse correlation was observed with the width of red blood cell volume distribution and platelet distribution, monocytes and hemoglobin, using Pearson's product‑moment correlation method. The P‑value of CRP and erythrocyte sedimentation were revealed to be statistically significant (P<0.05). A sporadic distribution of S100A12 was observed in a heatmap among the patients with different infections and bacterial pneumonia. Furthermore, the expression of S100A12 occurred in parallel to the number of clumps of inflamed tissue observed in chest computed tomography and X‑ray. The value of gene expression of S100A12 (>1.0) determined using the 2‑ΔΔCt method was associated with more severe respiratory diseases in the patients compromised by bacterial pneumonia, sepsis and pancreatitis. These findings suggested that S100A12 is an effective marker for inflammatory diseases.

  1. Autologous tumor cells engineered to express bacterial antigens.

    PubMed

    Ramiya, Vijayakumar K; Jerald, Maya M; Lawman, Patricia D; Lawman, Michael J P

    2014-01-01

    Cancer immunotherapies are emerging as promising treatment modalities in the management of the disease. As a result, cancer vaccines are considered to be immensely crucial in preventing recurrence, a well-known nemesis in cancer patients because they have the potential to activate memory antitumor immunity. Due to poor antigenicity and self-tolerance, most tumor antigens require interventional vaccine therapies to provide an adequate "danger" signal to the immune system in order to activate a robust, clinically meaningful antitumor immunity. It has been postulated that this requirement may be achieved by providing bacterial and/or viral immunogens to prime this type of immune response. Briefly, we provide here a method of transfecting whole tumor cells with plasmid DNA encoding an immunogenic bacterial protein such as Emm55, which was derived from Streptococcus pyogenes (S. pyogenes). Subsequent inactivation of the transfected cells by irradiation (100 Gray) prevents replication. This type of whole-cell vaccine, e.g., ImmuneFx™, has demonstrated activity in a murine neuroblastoma model, in canine lymphoma patients with naturally occurring disease, and in many cancer types in companion animals. The protocols described in this chapter provide the necessary materials and methodologies to manufacture such a vaccine.

  2. Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

    PubMed

    Gorgoglione, Bartolomeo; Carpio, Yamila; Secombes, Christopher J; Taylor, Nick G H; Lugo, Juana María; Estrada, Mario Pablo

    2015-12-01

    Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens. Copyright © 2015

  3. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

    PubMed Central

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

    2016-01-01

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

  4. Increased Bacterial Load and Expression of Antimicrobial Peptides in Skin of Barrier-Deficient Mice with Reduced Cancer Susceptibility.

    PubMed

    Natsuga, Ken; Cipolat, Sara; Watt, Fiona M

    2016-01-01

    Mice lacking three epidermal barrier proteins-envoplakin, periplakin, and involucrin (EPI-/- mice)-have a defective cornified layer, reduced epidermal γδ T cells, and increased dermal CD4(+) T cells. They are also resistant to developing skin tumors. The tumor-protective mechanism involves signaling between Rae-1 expressing keratinocytes and the natural killer group 2D receptor on immune cells, which also plays a role in host defenses against infection. Given the emerging link between bacteria and cancer, we investigated whether EPI-/- mice have an altered skin microbiota. The bacterial phyla were similar in wild-type and EPI-/- skin. However, bacteria were threefold more abundant in EPI-/- skin and penetrated deeper into the epidermis. The major epithelial defense mechanism against bacteria is production of antimicrobial proteins (AMPs). EPI-/- skin exhibited enhanced expression of antimicrobial peptides. However, reducing the bacterial load by antibiotic treatment or breeding mice under specific pathogen-free conditions did not reduce AMP expression or alleviate the abnormalities in T-cell populations. We conclude that the atopic characteristics of EPI-/- skin are a consequence of the defective barrier rather than a response to the increased bacterial load. It is therefore unlikely that the increase in skin microbiota contributes directly to the observed cancer resistance. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    PubMed Central

    Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2016-01-01

    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

  6. Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype.

    PubMed

    Mushtaq, Naseem; Redpath, Maria B; Luzio, J Paul; Taylor, Peter W

    2004-05-01

    Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack. We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model. Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E. coli K1 strains. Intraperitoneal administration of small quantities of recombinant endoE (20 micro g) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection. The enzyme had no effect on the viability of E. coli strains but sensitized strains expressing PSA to killing by the complement system. This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen.

  7. Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

    PubMed Central

    O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

    1994-01-01

    The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

  8. Analysis of gene expression levels in individual bacterial cells without image segmentation.

    PubMed

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J

    2012-05-11

    Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

    PubMed Central

    Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

    2009-01-01

    Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

  10. Bacterial communications in implant infections: a target for an intelligence war.

    PubMed

    Costerton, J W; Montanaro, L; Arciola, C R

    2007-09-01

    The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called "quorum-sensing". Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In grampositive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.

  11. Recognition of bacterial plant pathogens: local, systemic and transgenerational immunity.

    PubMed

    Henry, Elizabeth; Yadeta, Koste A; Coaker, Gitta

    2013-09-01

    Bacterial pathogens can cause multiple plant diseases and plants rely on their innate immune system to recognize and actively respond to these microbes. The plant innate immune system comprises extracellular pattern recognition receptors that recognize conserved microbial patterns and intracellular nucleotide binding leucine-rich repeat (NLR) proteins that recognize specific bacterial effectors delivered into host cells. Plants lack the adaptive immune branch present in animals, but still afford flexibility to pathogen attack through systemic and transgenerational resistance. Here, we focus on current research in plant immune responses against bacterial pathogens. Recent studies shed light onto the activation and inactivation of pattern recognition receptors and systemic acquired resistance. New research has also uncovered additional layers of complexity surrounding NLR immune receptor activation, cooperation and sub-cellular localizations. Taken together, these recent advances bring us closer to understanding the web of molecular interactions responsible for coordinating defense responses and ultimately resistance. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  12. Bacterial uptake of antibiotics in model unsaturated systems

    NASA Astrophysics Data System (ADS)

    Zhang, W.; Chen, Z.; Zhang, Y.; Zhao, Z.; Wang, G.; Gao, Y.; Boyd, S. A.; Zhu, D.; Li, H.

    2016-12-01

    Anthropogenic antibiotics are ubiquitously present in the environment due to large uses in human medicine and animal agriculture, and are causing unintended consequence to human and ecosystem health. Bacterial uptake of antibiotics could exert selection pressure on antibiotic resistance development among bacteria population. Therefore, understanding environmental factors controlling bioavailability of antibiotics to bacteria is critical to better assessing exposure risks and developing mitigation strategies. Nonetheless, conventional bioavailability assays are often performed in water-saturated systems that do not represent unsaturated soils where most bacteria live, therefore neglecting soil water as a controlling factor in determining the extent of antibiotic bacterial uptake. Therefore, we propose to study bacterial uptake of antibiotics in model unsaturated systems using GFP-tagged Escherichia coli bioreporter for tetracyclines. Our preliminary studies demonstrated the important role of water content (or water matric potential) in determining the bioavailability of antibiotics, and complex interactions of water potential, tetracycline diffusion, and E. coli growth. Therefore, unsaturated processes are important for understanding antibiotic resistance development and developing mitigation strategies.

  13. A Transcriptional Regulatory Mechanism Finely Tunes the Firing of Type VI Secretion System in Response to Bacterial Enemies.

    PubMed

    Lazzaro, Martina; Feldman, Mario F; García Véscovi, Eleonora

    2017-08-22

    The ability to detect and measure danger from an environmental signal is paramount for bacteria to respond accordingly, deploying strategies that halt or counteract potential cellular injury and maximize survival chances. Type VI secretion systems (T6SSs) are complex bacterial contractile nanomachines able to target toxic effectors into neighboring bacteria competing for the same colonization niche. Previous studies support the concept that either T6SSs are constitutively active or they fire effectors in response to various stimuli, such as high bacterial density, cell-cell contact, nutrient depletion, or components from dead sibling cells. For Serratia marcescens , it has been proposed that its T6SS is stochastically expressed, with no distinction between harmless or aggressive competitors. In contrast, we demonstrate that the Rcs regulatory system is responsible for finely tuning Serratia T6SS expression levels, behaving as a transcriptional rheostat. When confronted with harmless bacteria, basal T6SS expression levels suffice for Serratia to eliminate the competitor. A moderate T6SS upregulation is triggered when, according to the aggressor-prey ratio, an unbalanced interplay between homologous and heterologous effectors and immunity proteins takes place. Higher T6SS expression levels are achieved when Serratia is challenged by a contender like Acinetobacter , which indiscriminately fires heterologous effectors able to exert lethal cellular harm, threatening the survival of the Serratia population. We also demonstrate that Serratia 's RcsB-dependent T6SS regulatory mechanism responds not to general stress signals but to the action of specific effectors from competitors, displaying an exquisite strategy to weigh risks and keep the balance between energy expenditure and fitness costs. IMPORTANCE Serratia marcescens is among the health-threatening pathogens categorized by the WHO as research priorities to develop alternative antimicrobial strategies, and it was

  14. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

    PubMed

    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis.

    PubMed

    Chen, Yanhong; Oba, Masahito; Guan, Le Luo

    2012-10-12

    In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n=3) and acidosis-resistant (AR, n=3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis. Global R analysis of bacterial profile similarity revealed that bacterial diversity was significantly different between AR and AS groups for both rumen content (P=0.001) and epithelial (P=0.002) communities. The copy number of total bacterial 16S rRNA genes in content of AS steers was 10-fold higher than that of AR steers, and the copy number of total 16S rRNA genes of epimural bacteria in AR steers was positively correlated with ruminal pH (r=0.59, P=0.04), and negatively correlated with total VFA concentration (r=-0.59, P=0.05). The expressions of host TLR2 and 4 genes were significantly higher in AR steers compared to those in AS steers. These findings enhance our understanding about the ruminal microbial ecology and host gene expression changes that may be useful in the prevention of ruminal acidosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced Performance of Microbial Electrochemical Systems Using Nano-Decorated Anodes

    NASA Astrophysics Data System (ADS)

    Xu, Shoutao

    Microbial electrochemical systems (MESs) have attracted much research attention in recent years due to their promising applications in renewable energy generation, bioremediation, and wastewater treatment. In a MES, microorganisms interact with electrodes via electrons, catalyzing oxidation and reduction reactions at the anode and the cathode. The bacterial community of a high power mixed consortium MESs (maximum power density is 6.5W/m2) was analyzed by using denature gradient gel electrophoresis (DGGE) and 16S DNA clone library methods. The bacterial DGGE profiles were relatively complex (more than 10 bands) but only three brightly dominant bands in DGGE results. These results indicated there are three dominant bacterial species in mixed consortium MFCs. The 16S DNA clone library method results revealed that the predominant bacterial species in mixed culture is Geobacter sp (66%), Arcobacter sp and Citrobacter sp. These three bacterial species reached to 88% of total bacterial species. This result is consistent with the DGGE result which showed that three bright bands represented three dominant bacterial species. Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial fuel cell by conventional plating techniques with ferric citrate as electron acceptor under anaerobic conditions. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the most closely related species. The bacterial strain SX-1 produced electricity from citrate, acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density of 205 mA/m2 generated from citrate. Cyclic voltammetry analysis indicated that membrane associated proteins may play an important role in facilitating electron transfer from the bacteria to the electrode. This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors

  17. Bacterial vaginosis-associated microflora isolated from the female genital tract activates HIV-1 expression.

    PubMed

    Al-Harthi, L; Roebuck, K A; Olinger, G G; Landay, A; Sha, B E; Hashemi, F B; Spear, G T

    1999-07-01

    Alteration of cervicovaginal microbial flora can lead to vaginosis, which is associated with an increased risk of HIV-1 transmission. We recently characterized a soluble HIV-inducing factor (HIF) from the cervicovaginal lavage (CVL) samples of women. The goals of this study were to determine the effect of cervicovaginal microflora on HIV-1 expression and to elucidate the relationship between HIF activity and microflora. Physiologically relevant microorganisms, Mycoplasma, diphtheroid-like bacteria, Gardnerella vaginalis, Streptococcus agalactiae, and Streptococcus constellatus, cultured from the CVL of a representative woman with a clinical condition of bacterial vaginosis and possessing HIF activity, induced HIV-1 expression. The magnitude of virus induction varied widely with the greatest stimulation induced by diphtheroid-like bacteria and Mycoplasma. The transcriptional induction by Mycoplasma was mediated by activation of the KB enhancer, an activation mechanism shared with HIF. Also as with HIF, Mycoplasma induced AP-1 dependent transcription. Polymerase chain reaction (PCR)-based speciation showed that the isolate was M. hominis. Our data indicate that bacterial vaginosis-associated microflora can enhance HIV-1 transcription and replication and identify M. hominis as a potential source for HIF activity. The virus-enhancing activities associated with the microflora and HIF may increase genital tract viral load, potentially contributing to HIV transmission.

  18. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  19. Using Bacterial Extract along with Differential Gene Expression in Acropora millepora Larvae to Decouple the Processes of Attachment and Metamorphosis

    PubMed Central

    Siboni, Nachshon; Abrego, David; Seneca, Francois; Motti, Cherie A.; Andreakis, Nikos; Tebben, Jan; Blackall, Linda L.; Harder, Tilmann

    2012-01-01

    Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0–2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p<0.05) in gene expression profiles of 24 GOI, 12 hours post exposure. Fourteen of those GOI also presented differences in expression (p<0.05) following exposure to the threshold concentration of bacterial TBP-containing extract. The specificity of the bacterial TBP-containing extract to induce the metamorphic stage in A. millepora larvae without attachment, using a robust, low cost, accurate, ecologically relevant and highly reproducible RT-qPCR assay, allowed partially decoupling of the transcriptomic processes of attachment and metamorphosis. The bacterial TBP-containing extract provided a unique opportunity to monitor the regulation of genes exclusively involved in the process of metamorphosis, contrasting previous gene expression studies that utilized cues

  20. α-Intercalated cells defend the urinary system from bacterial infection.

    PubMed

    Paragas, Neal; Kulkarni, Ritwij; Werth, Max; Schmidt-Ott, Kai M; Forster, Catherine; Deng, Rong; Zhang, Qingyin; Singer, Eugenia; Klose, Alexander D; Shen, Tian Huai; Francis, Kevin P; Ray, Sunetra; Vijayakumar, Soundarapandian; Seward, Samuel; Bovino, Mary E; Xu, Katherine; Takabe, Yared; Amaral, Fábio E; Mohan, Sumit; Wax, Rebecca; Corbin, Kaitlyn; Sanna-Cherchi, Simone; Mori, Kiyoshi; Johnson, Lynne; Nickolas, Thomas; D'Agati, Vivette; Lin, Chyuan-Sheng; Qiu, Andong; Al-Awqati, Qais; Ratner, Adam J; Barasch, Jonathan

    2014-07-01

    α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

  1. Double promoter expression systems for recombinant protein production by industrial microorganisms.

    PubMed

    Öztürk, Sibel; Ergün, Burcu Gündüz; Çalık, Pınar

    2017-10-01

    Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more

  2. Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

    PubMed Central

    2017-01-01

    ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that

  3. Comparison of bacterial communities of conventional and A-stage activated sludge systems

    PubMed Central

    Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Lotti, Tommaso; Garcia-Ruiz, Maria-Jesus; Osorio, Francisco; Gonzalez-Lopez, Jesus; van Loosdrecht, Mark C. M.

    2016-01-01

    The bacterial community structure of 10 different wastewater treatment systems and their influents has been investigated through pyrosequencing, yielding a total of 283486 reads. These bioreactors had different technological configurations: conventional activated sludge (CAS) systems and very highly loaded A-stage systems. A-stage processes are proposed as the first step in an energy producing municipal wastewater treatment process. Pyrosequencing analysis indicated that bacterial community structure of all influents was similar. Also the bacterial community of all CAS bioreactors was similar. Bacterial community structure of A-stage bioreactors showed a more case-specific pattern. A core of genera was consistently found for all influents, all CAS bioreactors and all A-stage bioreactors, respectively, showing that different geographical locations in The Netherlands and Spain did not affect the functional bacterial communities in these technologies. The ecological roles of these bacteria were discussed. Influents and A-stage bioreactors shared several core genera, while none of these were shared with CAS bioreactors communities. This difference is thought to reside in the different operational conditions of the two technologies. This study shows that bacterial community structure of CAS and A-stage bioreactors are mostly driven by solids retention time (SRT) and hydraulic retention time (HRT), as suggested by multivariate redundancy analysis. PMID:26728449

  4. Pesticide Side Effects in an Agricultural Soil Ecosystem as Measured by amoA Expression Quantification and Bacterial Diversity Changes

    PubMed Central

    Feld, Louise; Hjelmsø, Mathis Hjort; Nielsen, Morten Schostag; Jacobsen, Anne Dorthe; Rønn, Regin; Ekelund, Flemming; Krogh, Paul Henning; Strobel, Bjarne Westergaard; Jacobsen, Carsten Suhr

    2015-01-01

    Background and Methods Assessing the effects of pesticide hazards on microbiological processes in the soil is currently based on analyses that provide limited insight into the ongoing processes. This study proposes a more comprehensive approach. The side effects of pesticides may appear as changes in the expression of specific microbial genes or as changes in diversity. To assess the impact of pesticides on gene expression, we focused on the amoA gene, which is involved in ammonia oxidation. We prepared soil microcosms and exposed them to dazomet, mancozeb or no pesticide. We hypothesized that the amount of amoA transcript decreases upon pesticide application, and to test this hypothesis, we used reverse-transcription qPCR. We also hypothesized that bacterial diversity is affected by pesticides. This hypothesis was investigated via 454 sequencing and diversity analysis of the 16S ribosomal RNA and RNA genes, representing the active and total soil bacterial communities, respectively. Results and Conclusion Treatment with dazomet reduced both the bacterial and archaeal amoA transcript numbers by more than two log units and produced long-term effects for more than 28 days. Mancozeb also inhibited the numbers of amoA transcripts, but only transiently. The bacterial and archaeal amoA transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial amoA transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit, but the population size was restored after twelve days. The diversity of the active soil bacteria also seemed to be re-established after twelve days. However, the total bacterial diversity as reflected in the 16S ribosomal RNA gene sequences was largely dominated by Firmicutes and Proteobacteria at day twelve, likely reflecting a halt in the growth of early opportunists and the re-establishment of a more diverse population. We observed no

  5. Induction of Systemic Resistance against Aphids by Endophytic Bacillus velezensis YC7010 via Expressing PHYTOALEXIN DEFICIENT4 in Arabidopsis

    PubMed Central

    Rashid, Md. Harun-Or-; Khan, Ajmal; Hossain, Mohammad T.; Chung, Young R.

    2017-01-01

    Aphids are the most destructive insect pests. They suck the sap and transmit plant viruses, causing widespread yield loss of many crops. A multifunctional endophytic bacterial strain Bacillus velezensis YC7010 has been found to induce systemic resistance against bacterial and fungal pathogens of rice. However, its activity against insects attack and underlying cellular and molecular defense mechanisms are not elucidated yet. Here, we show that root drenching of Arabidopsis seedlings with B. velezensis YC7010 can induce systemic resistance against green peach aphid (GPA), Myzus persicae. Treatment of bacterial suspension of B. velezensis YC7010 at 2 × 107 CFU/ml to Arabidopsis rhizosphere induced higher accumulation of hydrogen peroxide, cell death, and callose deposition in leaves compared to untreated plants at 6 days after infestation of GPA. Salicylic acid, jasmonic acid, ethylene, and abscisic acid were not required to confer defense against GPA in Arabidopsis plants treated by B. velezensis YC7010. Bacterial treatment with B. velezensis YC7010 significantly reduced settling, feeding and reproduction of GPA on Arabidopsis leaves via strongly expressing senescence-promoting gene PHYTOALEXIN DEFICIENT4 (PAD4) while suppressing BOTRYTIS-INDUCED KINASE1 (BIK1). These results indicate that B. velezensis YC7010-induced systemic resistance to the GPA is a hypersensitive response mainly dependent on higher expression of PAD4 with suppression of BIK1, resulting in more accumulation of hydrogen peroxide, cell death, and callose deposition in Arabidopsis. PMID:28261260

  6. Identification of Bacterial Factors Involved in Type 1 Fimbria Expression using an Escherichia coli K12 Proteome Chip*

    PubMed Central

    Chen, Yi-Wen; Teng, Ching-Hao; Ho, Yu-Hsuan; Jessica Ho, Tien Yu; Huang, Wen-Chun; Hashimoto, Masayuki; Chiang, I-Yuan; Chen, Chien-Sheng

    2014-01-01

    Type 1 fimbriae are filamentous structures on Escherichia coli. These structures are important adherence factors. Because binding to the host cells is the first step of infection, type 1 fimbria is an important virulence factor of pathogenic E. coli. Expression of type 1 fimbria is regulated by a phase variation in which each individual bacterium can alternate between fimbriated (phase-ON) and nonfimbriated (phase-OFF) states. The phase variation is regulated by the flipping of the 314-bp fimS fragment, which contains the promoter driving the expression of the genes required for the synthesis of type 1 fimbria. Thus, the bacterial proteins able to interact with fimS are likely to be involved in regulating the expression of type 1 fimbria. To identify novel type 1 fimbria-regulating factors, we used an E. coli K12 proteome chip to screen for the bacterial factors able to interact with a 602-bp DNA fragment containing fimS and its adjacent regions. The Spr protein was identified by the proteome chip-based screening and further confirmed to be able to interact with fimS by electrophoretic mobility shift assay. Deletion of spr in the neonatal meningitis E. coli strain RS218 significantly increased the ratio of the bacterial colonies that contained the type 1 fimbria phase-ON cells on agar plates. In addition, Spr interfered with the interactions of fimS with the site-specific recombinases, FimB and FimE, which are responsible for mediating the flipping of fimS. These results suggest that Spr is involved in the regulation of type 1 fimbria expression through direct interaction with the invertible element fimS. These findings facilitate our understanding of the regulation of type 1 fimbria. PMID:24692643

  7. Safety assessment of genetically modified rice expressing human serum albumin from urine metabonomics and fecal bacterial profile.

    PubMed

    Qi, Xiaozhe; Chen, Siyuan; Sheng, Yao; Guo, Mingzhang; Liu, Yifei; He, Xiaoyun; Huang, Kunlun; Xu, Wentao

    2015-02-01

    The genetically modified (GM) rice expressing human serum albumin (HSA) is used for non-food purposes; however, its food safety assessment should be conducted due to the probability of accidental mixture with conventional food. In this research, Sprague Dawley rats were fed diets containing 50% (wt/wt) GM rice expressing HSA or non-GM rice for 90 days. Urine metabolites were detected by (1)H NMR to examine the changes of the metabolites in the dynamic process of metabolism. Fecal bacterial profiles were detected by denaturing gradient gel electrophoresis to reflect intestinal health. Additionally, short chain fatty acids and fecal enzymes were investigated. The results showed that compared with rats fed the non-GM rice, some significant differences were observed in rats fed with the GM rice; however, these changes were not significantly different from the control diet group. Additionally, the gut microbiota was associated with blood indexes and urine metabolites. In conclusion, the GM rice diet is as safe as the traditional daily diet. Furthermore, urine metabonomics and fecal bacterial profiles provide a non-invasive food safety assessment rat model for genetically modified crops that are used for non-food/feed purposes. Fecal bacterial profiles have the potential for predicting the change of blood indexes in future. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Bacterial bioluminescence regulates expression of a host cryptochrome gene in the squid-Vibrio symbiosis.

    PubMed

    Heath-Heckman, Elizabeth A C; Peyer, Suzanne M; Whistler, Cheryl A; Apicella, Michael A; Goldman, William E; McFall-Ngai, Margaret J

    2013-04-02

    The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut.

  9. Bacterial Bioluminescence Regulates Expression of a Host Cryptochrome Gene in the Squid-Vibrio Symbiosis

    PubMed Central

    Heath-Heckman, Elizabeth A. C.; Peyer, Suzanne M.; Whistler, Cheryl A.; Apicella, Michael A.; Goldman, William E.; McFall-Ngai, Margaret J.

    2013-01-01

    ABSTRACT The symbiosis between the squid Euprymna scolopes and its luminous symbiont, Vibrio fischeri, is characterized by daily transcriptional rhythms in both partners and daily fluctuations in symbiont luminescence. In this study, we sought to determine whether symbionts affect host transcriptional rhythms. We identified two transcripts in host tissues (E. scolopes cry1 [escry1] and escry2) that encode cryptochromes, proteins that influence circadian rhythms in other systems. Both genes cycled daily in the head of the squid, with a pattern similar to that of other animals, in which expression of certain cry genes is entrained by environmental light. In contrast, escry1 expression cycled in the symbiont-colonized light organ with 8-fold upregulation coincident with the rhythms of bacterial luminescence, which are offset from the day/night light regime. Colonization of the juvenile light organ by symbionts was required for induction of escry1 cycling. Further, analysis with a mutant strain defective in light production showed that symbiont luminescence is essential for cycling of escry1; this defect could be complemented by presentation of exogenous blue light. However, blue-light exposure alone did not induce cycling in nonsymbiotic animals, but addition of molecules of the symbiont cell envelope to light-exposed animals did recover significant cycling activity, showing that light acts in synergy with other symbiont features to induce cycling. While symbiont luminescence may be a character specific to rhythms of the squid-vibrio association, resident microbial partners could similarly influence well-documented daily rhythms in other systems, such as the mammalian gut. PMID:23549919

  10. Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein.

    PubMed

    Müller, Ina; Gernold, Marina; Schneider, Bernd; Geider, Klaus

    2012-01-01

    Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme. Copyright © 2012 S. Karger AG, Basel.

  11. A bacterial reporter system for the evaluation of antisense oligodeoxynucleotides directed against human papillomavirus type 16 (HPV-16).

    PubMed

    Guapillo, Mario R; Márquez, Miguel A; Benítez-Hess, María L; Alvarez-Salas, Luis M

    2006-07-01

    Antisense oligodeoxynucleotides (AS-ODNs) are a promising alternative for the cure of many diseases because of their in vivo specificity and stability. However, AS-ODNs have a strong dependence on the target mRNA structure making necessary extensive in vivo testing. There is, therefore, a need to develop assays to rapidly evaluate in vivo ODN performance. We report a simple and inexpensive bacterial reporter system for the rapid in vivo evaluation of AS-ODNs directed against human papillomavirus type 16 (HPV-16) based on the destruction of a chimeric CFP mRNA using the reported HPV-16 nt 410-445 target. In vitro RNaseH assays confirmed target RNA accessibility after AS-ODN treatment. Expression of CFP in Escherichia coli BL21(DE3) with pGST-TSd2-CFP plasmid containing HPV-16 nt 410-445 target linked to CFP was blocked by transformed antisense PS-ODNs but not by two different scrambled ODN controls. A correlation was observed between bacterial CFP downregulation with the HPV-16 E6/E7 mRNA downregulation and the inhibition of anchorage-independent growth of HPV-16 containing cells suggesting that inhibition of HPV-16 E6/E7 expression by AS-ODNs directed against 410-445 target in cervical tumor cells can be tested in bacterial models.

  12. A Transcriptional Regulatory Mechanism Finely Tunes the Firing of Type VI Secretion System in Response to Bacterial Enemies

    PubMed Central

    Lazzaro, Martina; Feldman, Mario F.

    2017-01-01

    ABSTRACT The ability to detect and measure danger from an environmental signal is paramount for bacteria to respond accordingly, deploying strategies that halt or counteract potential cellular injury and maximize survival chances. Type VI secretion systems (T6SSs) are complex bacterial contractile nanomachines able to target toxic effectors into neighboring bacteria competing for the same colonization niche. Previous studies support the concept that either T6SSs are constitutively active or they fire effectors in response to various stimuli, such as high bacterial density, cell-cell contact, nutrient depletion, or components from dead sibling cells. For Serratia marcescens, it has been proposed that its T6SS is stochastically expressed, with no distinction between harmless or aggressive competitors. In contrast, we demonstrate that the Rcs regulatory system is responsible for finely tuning Serratia T6SS expression levels, behaving as a transcriptional rheostat. When confronted with harmless bacteria, basal T6SS expression levels suffice for Serratia to eliminate the competitor. A moderate T6SS upregulation is triggered when, according to the aggressor-prey ratio, an unbalanced interplay between homologous and heterologous effectors and immunity proteins takes place. Higher T6SS expression levels are achieved when Serratia is challenged by a contender like Acinetobacter, which indiscriminately fires heterologous effectors able to exert lethal cellular harm, threatening the survival of the Serratia population. We also demonstrate that Serratia’s RcsB-dependent T6SS regulatory mechanism responds not to general stress signals but to the action of specific effectors from competitors, displaying an exquisite strategy to weigh risks and keep the balance between energy expenditure and fitness costs. PMID:28830939

  13. Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons.

    PubMed Central

    Cheetham, M E; Brion, J P; Anderton, B H

    1992-01-01

    The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation. The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK. Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described. We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues. The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease. The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70. Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal. These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1599432

  14. High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer.

    PubMed

    Bhoir, Siddhant; Shaik, Althaf; Thiruvenkatam, Vijay; Kirubakaran, Sivapriya

    2018-03-19

    Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein-drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

  15. Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System.

    PubMed

    Schwarz-Schilling, Matthaeus; Dupin, Aurore; Chizzolini, Fabio; Krishnan, Swati; Mansy, Sheref S; Simmel, Friedrich C

    2018-04-11

    Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

  16. Newer systems for bacterial resistances to toxic heavy metals.

    PubMed Central

    Silver, S; Ji, G

    1994-01-01

    Bacterial plasmids contain specific genes for resistances to toxic heavy metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, and Zn2+. Recent progress with plasmid copper-resistance systems in Escherichia coli and Pseudomonas syringae show a system of four gene products, an inner membrane protein (PcoD), an outer membrane protein (PcoB), and two periplasmic Cu(2+)-binding proteins (PcoA and PcoC). Synthesis of this system is governed by two regulatory proteins (the membrane sensor PcoS and the soluble responder PcoR, probably a DNA-binding protein), homologous to other bacterial two-component regulatory systems. Chromosomally encoded Cu2+ P-type ATPases have recently been recognized in Enterococcus hirae and these are closely homologous to the bacterial cadmium efflux ATPase and the human copper-deficiency disease Menkes gene product. The Cd(2+)-efflux ATPase of gram-positive bacteria is a large P-type ATPase, homologous to the muscle Ca2+ ATPase and the Na+/K+ ATPases of animals. The arsenic-resistance system of gram-negative bacteria functions as an oxyanion efflux ATPase for arsenite and presumably antimonite. However, the structure of the arsenic ATPase is fundamentally different from that of P-type ATPases. The absence of the arsA gene (for the ATPase subunit) in gram-positive bacteria raises questions of energy-coupling for arsenite efflux. The ArsC protein product of the arsenic-resistance operons of both gram-positive and gram-negative bacteria is an intracellular enzyme that reduces arsenate [As(V)] to arsenite [As(III)], the substrate for the transport pump. Newly studied cation efflux systems for Cd2+, Zn2+, and Co2+ (Czc) or Co2+ and Ni2+ resistance (Cnr) lack ATPase motifs in their predicted polypeptide sequences. Therefore, not all plasmid-resistance systems that function through toxic ion efflux are ATPases. The first well-defined bacterial metallothionein was found in the cyanobacterium Synechococcus

  17. Intra-species bacterial quorum sensing studied at single cell level in a double droplet trapping system.

    PubMed

    Bai, Yunpeng; Patil, Santoshkumar N; Bowden, Steven D; Poulter, Simon; Pan, Jie; Salmond, George P C; Welch, Martin; Huck, Wilhelm T S; Abell, Chris

    2013-05-21

    In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

  18. Harnessing CRISPR-Cas systems for bacterial genome editing.

    PubMed

    Selle, Kurt; Barrangou, Rodolphe

    2015-04-01

    Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Effect of bacterial endotoxin LPS on expression of INF-gamma and IL-5 in T-lymphocytes from asthmatics.

    PubMed

    Koch, Andrea; Knobloch, Jürgen; Dammhayn, Cathrin; Raidl, Maria; Ruppert, Andrea; Hag, Haitham; Rottlaender, Dennis; Müller, Katja; Erdmann, Erland

    2007-11-01

    Epidemiological evidence, in vitro studies and animal models suggest that exposure to the bacterial endotoxin lipopolysaccharide (LPS) can influence the development and severity of asthma. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 and 2 responses, it is unclear whether the LPS ligand TLR 4 is expressed on CD4(+) and CD8(+) T-lymphocytes and if so, whether LPS could modulate the T(H)1 or T(H)2 response in this context. The present authors have, therefore, examined the expression of TLR 4 on peripheral blood CD4(+) and CD8(+) T-lymphocytes using RT-PCR method and FACS analyses. Furthermore, the authors have studied the IL-12-induced expression of the T(H)1-associated cytokine INF-gamma and the IL-4-induced expression of the T(H)2-specific cytokine IL-5 in the presence of LPS using ELISA and compared nine atopic asthmatic subjects and eleven nonatopic normal volunteers. There was an increased anti-CD3/anti-CD28-induced IL-5 expression in T cells of asthmatics compared with normals (p<0.01). In the presence of IL-4 (10 ng/ml), there was an additional increase in IL-5 expression and this additional increase was greater in T cells of normals compared with asthmatics (p<0.05). There was an expression of INF-gamma in anti-CD3/anti-CD28-induced T-lymphocytes without differences between both groups (NS). In the presence of IL-12 (10 ng/ml), there was an increase in INF-gamma release without differences between normals and asthmatics (NS). In the presence of different concentrations of LPS (10 ng/ml, 1 mug/ml), there was a decrease in IL-4-induced IL-5 expression without differences in both groups, indicating an intact T(H)2 response to bacterial endotoxin LPS in asthma. Interestingly, LPS increased the IL-12-induced INF-gamma release in a concentration-dependent manner in T-lymphocytes of normals but this could not be found in T cells of asthmatics, indicating an impaired T(H)1 response to bacterial

  20. Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

    PubMed

    Woods, J P; Heinecke, E L; Goldman, W E

    1998-04-01

    We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption

  1. α–Intercalated cells defend the urinary system from bacterial infection

    PubMed Central

    Paragas, Neal; Kulkarni, Ritwij; Werth, Max; Schmidt-Ott, Kai M.; Forster, Catherine; Deng, Rong; Zhang, Qingyin; Singer, Eugenia; Klose, Alexander D.; Shen, Tian Huai; Francis, Kevin P.; Ray, Sunetra; Vijayakumar, Soundarapandian; Seward, Samuel; Bovino, Mary E.; Xu, Katherine; Takabe, Yared; Amaral, Fábio E.; Mohan, Sumit; Wax, Rebecca; Corbin, Kaitlyn; Sanna-Cherchi, Simone; Mori, Kiyoshi; Johnson, Lynne; Nickolas, Thomas; D’Agati, Vivette; Lin, Chyuan-Sheng; Qiu, Andong; Al-Awqati, Qais; Ratner, Adam J.; Barasch, Jonathan

    2014-01-01

    α–Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC–dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system. PMID:24937428

  2. Interspecies Systems Biology Uncovers Metabolites Affecting C. elegans Gene Expression and Life History Traits

    PubMed Central

    Watson, Emma; MacNeil, Lesley T.; Ritter, Ashlyn D.; Yilmaz, L. Safak; Rosebrock, Adam P.; Caudy, Amy A.; Walhout, Albertha J. M.

    2014-01-01

    SUMMARY Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its diet. Here we used an interspecies systems biology approach with Caenorhabditis elegans and two if its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal’s gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development and reduces fertility, but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology. PMID:24529378

  3. Bacterial diversity in water injection systems of Brazilian offshore oil platforms.

    PubMed

    Korenblum, Elisa; Valoni, Erika; Penna, Mônica; Seldin, Lucy

    2010-01-01

    Biogenic souring and microbial-influenced corrosion is a common scenario in water-flooded petroleum reservoirs. Water injection systems are continuously treated to control bacterial contamination, but some bacteria that cause souring and corrosion can persist even after different treatments have been applied. Our aim was to increase our knowledge of the bacterial communities that persist in the water injection systems of three offshore oil platforms in Brazil. To achieve this goal, we used a culture-independent molecular approach (16S ribosomal RNA gene clone libraries) to analyze seawater samples that had been subjected to different treatments. Phylogenetic analyses revealed that the bacterial communities from the different platforms were taxonomically different. A predominance of bacterial clones affiliated with Gammaproteobacteria, mostly belonging to the genus Marinobacter (60.7%), were observed in the platform A samples. Clones from platform B were mainly related to the genera Colwellia (37.9%) and Achromobacter (24.6%), whereas clones obtained from platform C were all related to unclassified bacteria. Canonical correspondence analyses showed that different treatments such as chlorination, deoxygenation, and biocide addition did not significantly influence the bacterial diversity in the platforms studied. Our results demonstrated that the injection water used in secondary oil recovery procedures contained potentially hazardous bacteria, which may ultimately cause souring and corrosion.

  4. Low-shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis

    NASA Technical Reports Server (NTRS)

    Nickerson, Cheryl A.; Ott, C. Mark; Wilson, James W.; Ramamurthy, Rajee; LeBlanc, Carly L.; Honer zu Bentrup, Kerstin; Hammond, Timothy; Pierson, Duane L.

    2003-01-01

    Bacteria inhabit an impressive variety of ecological niches and must adapt constantly to changing environmental conditions. While numerous environmental signals have been examined for their effect on bacteria, the effects of mechanical forces such as shear stress and gravity have only been investigated to a limited extent. However, several important studies have demonstrated a key role for the environmental signals of low shear and/or microgravity in the regulation of bacterial gene expression, physiology, and pathogenesis [Chem. Rec. 1 (2001) 333; Appl. Microbiol. Biotechnol. 54 (2000) 33; Appl. Environ. Microbiol. 63 (1997) 4090; J. Ind. Microbiol. 18 (1997) 22; Curr. Microbiol. 34(4) (1997) 199; Appl. Microbiol. Biotechnol. 56(3-4) (2001) 384; Infect Immun. 68(6) (2000) 3147; Cell 109(7) (2002) 913; Appl. Environ. Microbiol. 68(11) (2002) 5408; Proc. Natl. Acad. Sci. U. S. A. 99(21) (2002) 13807]. The response of bacteria to these environmental signals, which are similar to those encountered during prokaryotic life cycles, may provide insight into bacterial adaptations to physiologically relevant conditions. This review focuses on the current and potential future research trends aimed at understanding the effect of the mechanical forces of low shear and microgravity analogues on different bacterial parameters. In addition, this review also discusses the use of microgravity technology to generate physiologically relevant human tissue models for research in bacterial pathogenesis.

  5. Applications of CRISPR/Cas System to Bacterial Metabolic Engineering.

    PubMed

    Cho, Suhyung; Shin, Jongoh; Cho, Byung-Kwan

    2018-04-05

    The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli . Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

  6. Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen

    PubMed Central

    Barry, Kevin C; Ingolia, Nicholas T; Vance, Russell E

    2017-01-01

    The inducible innate immune response to infection requires a concerted process of gene expression that is regulated at multiple levels. Most global analyses of the innate immune response have focused on transcription induced by defined immunostimulatory ligands, such as lipopolysaccharide. However, the response to pathogens involves additional complexity, as pathogens interfere with virtually every step of gene expression. How cells respond to pathogen-mediated disruption of gene expression to nevertheless initiate protective responses remains unclear. We previously discovered that a pathogen-mediated blockade of host protein synthesis provokes the production of specific pro-inflammatory cytokines. It remains unclear how these cytokines are produced despite the global pathogen-induced block of translation. We addressed this question by using parallel RNAseq and ribosome profiling to characterize the response of macrophages to infection with the intracellular bacterial pathogen Legionella pneumophila. Our results reveal that mRNA superinduction is required for the inducible immune response to a bacterial pathogen. DOI: http://dx.doi.org/10.7554/eLife.22707.001 PMID:28383283

  7. Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria.

    PubMed

    Ham, Jong Hyun

    2013-04-01

    Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N-acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL-mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram-negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid-type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194-amino-acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF-mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c-di-GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  8. The Bacterial Cytoskeleton Modulates Motility, Type 3 Secretion, and Colonization in Salmonella

    PubMed Central

    Bulmer, David M.; Kharraz, Lubna; Grant, Andrew J.; Dean, Paul; Morgan, Fiona J. E.; Karavolos, Michail H.; Doble, Anne C.; McGhie, Emma J.; Koronakis, Vassilis; Daniel, Richard A.; Mastroeni, Pietro; Anjam Khan, C. M.

    2012-01-01

    Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase Rcs

  9. Bacterial RNA Biology on a Genome Scale.

    PubMed

    Hör, Jens; Gorski, Stanislaw A; Vogel, Jörg

    2018-06-07

    Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

    PubMed

    Pombo, Marina A; Zheng, Yi; Fernandez-Pozo, Noe; Dunham, Diane M; Fei, Zhangjun; Martin, Gregory B

    2014-01-01

    Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

  11. Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

    PubMed Central

    Li, Yiyan; Yang, Xing; Zhao, Weian

    2018-01-01

    Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies. PMID:28850804

  12. Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing.

    PubMed

    Li, Yiyan; Yang, Xing; Zhao, Weian

    2017-12-01

    Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies.

  13. Tissue- and cell-specific expression of mouse xanthine oxidoreductase gene in vivo: regulation by bacterial lipopolysaccharide.

    PubMed Central

    Kurosaki, M; Li Calzi, M; Scanziani, E; Garattini, E; Terao, M

    1995-01-01

    The expression of the xanthine oxidoreductase gene was studied in various mouse organs and tissues, under basal conditions and on treatment with bacterial lipopolysaccharide. Levels of xanthine oxidoreductase protein and mRNA were compared in order to understand the molecular mechanisms regulating the expression of this enzyme system. The highest amounts of xanthine oxidoreductase and the respective mRNA are observed in the duodenum and jejunum, where the protein is present in an unusual form because of a specific proteolytic cleavage of the primary translation product present in all locations. Under basal conditions, multiple tissue-specific mechanisms of xanthine oxidoreductase regulation are evident. Lipopolysaccharide increases enzyme activity in some, but not all tissues, mainly via modulation of the respective transcript, although translational and post-translational mechanisms are also active. In situ hybridization studies on tissue sections obtained from mice under control conditions or with lipopolysaccharide treatment demonstrate that xanthine oxidoreductase is present in hepatocytes, predominantly in the proximal tubules of the kidney, epithelial layer of the gastrointestinal mucosa, the alveolar compartment of the lung, the pulpar region of the spleen and the vascular component of the heart. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7864814

  14. Bacterial Cysteine-Inducible Cysteine Resistance Systems

    PubMed Central

    Takumi, Kazuhiro

    2016-01-01

    ABSTRACT Cysteine donates sulfur to macromolecules and occurs naturally in many proteins. Because low concentrations of cysteine are cytotoxic, its intracellular concentration is stringently controlled. In bacteria, cysteine biosynthesis is regulated by feedback inhibition of the activities of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) and is also regulated at the transcriptional level by inducing the cysteine regulon using the master regulator CysB. Here, we describe two novel cysteine-inducible systems that regulate the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for biotechnological, medical, and industrial purposes. One locus, designated ccdA (formerly PAJ_0331), encodes a novel cysteine-inducible cysteine desulfhydrase (CD) that degrades cysteine, and its expression is controlled by the transcriptional regulator encoded by ccdR (formerly PAJ_0332 or ybaO), located just upstream of ccdA. The other locus, designated cefA (formerly PAJ_3026), encodes a novel cysteine-inducible cysteine efflux pump that is controlled by the transcriptional regulator cefR (formerly PAJ_3027), located just upstream of cefA. To our knowledge, this is the first example where the expression of CD and an efflux pump is regulated in response to cysteine and is directly involved in imparting resistance to excess levels of cysteine. We propose that ccdA and cefA function as safety valves that maintain homeostasis when the intra- or extracellular cysteine concentration fluctuates. Our findings contribute important insights into optimizing the production of cysteine and related biomaterials by P. ananatis. IMPORTANCE Because of its toxicity, the bacterial intracellular cysteine level is stringently regulated at biosynthesis. This work describes the identification and characterization of two novel cysteine-inducible systems that regulate, through degradation and

  15. A synthetic system for expression of components of a bacterial microcompartment.

    PubMed

    Sargent, Frank; Davidson, Fordyce A; Kelly, Ciarán L; Binny, Rachelle; Christodoulides, Natasha; Gibson, David; Johansson, Emelie; Kozyrska, Katarzyna; Lado, Lucia Licandro; Maccallum, Jane; Montague, Rachel; Ortmann, Brian; Owen, Richard; Coulthurst, Sarah J; Dupuy, Lionel; Prescott, Alan R; Palmer, Tracy

    2013-11-01

    In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilization (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T and -U, and each was shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

  16. Lack of Toll-like receptor 2 results in higher mortality of bacterial meningitis by impaired host resistance.

    PubMed

    Böhland, Martin; Kress, Eugenia; Stope, Matthias B; Pufe, Thomas; Tauber, Simone C; Brandenburg, Lars-Ove

    2016-10-15

    Bacterial meningitis is - despite therapeutical progress during the last decades - still characterized by high mortality and severe permanent neurogical sequelae. The brain is protected from penetrating pathogens by both the blood-brain barrier and the innate immune system. Invading pathogens are recognized by so-called pattern recognition receptors including the Toll-like receptors (TLR) which are expressed by glial immune cells in the central nervous system. Among these, TLR2 is responsible for the detection of Gram-positive bacteria such as the meningitis-causing pathogen Streptococcus pneumoniae. Here, we used TLR2-deficient mice to investigate the effects on mortality, bacterial growth and inflammation in a mouse model of pneumococcal meningitis. Our results revealed a significantly increased mortality rate and higher bacterial burden in TLR2-deficient mice with pneumococcal meningitis. Furthermore, infected TLR2-deficient mice suffered from a significantly increased pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and Chemokine (C-C motif) ligand 2 (CCL2) or CCL3 chemokine expression and decreased expression of anti-inflammatory cytokines and antimicrobial peptides. In contrast, glial cell activation assessed by glial cell marker expression was comparable to wildtype mice. Taken together, the results suggest that TLR2 is essential for an efficient immune response against Streptococcus pneumoniae meningitis since lack of the receptor led to a worse outcome by higher mortality due to increased bacterial burden, weakened innate immune response and reduced expression of antimicrobial peptides. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Changes in bacterial composition of biofilm in a metropolitan drinking water distribution system.

    PubMed

    Revetta, R P; Gomez-Alvarez, V; Gerke, T L; Santo Domingo, J W; Ashbolt, N J

    2016-07-01

    This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e. groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. The biofilm community was characterized using 16S rRNA gene clone libraries and functional potential analysis, generated from total DNA extracted from coupons in biofilm annular reactors fed with onsite drinking water for up to 18 months. Differences in the bacterial community structure were observed between GW and SW. Representatives that explained the dissimilarity were associated with the classes Betaproteobacteria, Alphaproteobacteria, Actinobacteria, Gammaproteobacteria and Firmicutes. After 9 months the biofilm bacterial community from both GW and SW were dominated by Mycobacterium species. The distribution of the dominant operational taxonomic unit (OTU) (Mycobacterium) positively correlated with the drinking water distribution system (DWDS) temperature. In this study, the biofilm community structure observed between GW and SW were dissimilar, while communities from different locations receiving SW did not show significant differences. The results suggest that source water and/or the water quality shaped by their respective treatment processes may play an important role in shaping the bacterial communities in the distribution system. In addition, several bacterial groups were present in all samples, suggesting that they are an integral part of the core microbiota of this DWDS. These results provide an ecological insight into biofilm bacterial structure in chlorine-treated drinking water influenced by different water sources and their respective treatment processes. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  18. Genetic and epigenetic control of gene expression by CRISPR–Cas systems

    PubMed Central

    Lo, Albert; Qi, Lei

    2017-01-01

    The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363

  19. Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

    PubMed Central

    Deng, Meihong; Loughran, Patricia; Gibson, Gregory; Sodhi, Chhinder; Watkins, Simon; Hackam, David

    2013-01-01

    The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type–selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations. PMID:23562812

  20. Bacterial-based Systems for Expression and Purification of Recombinant Lassa Virus Proteins of Immunological Relevance

    DTIC Science & Technology

    2008-06-06

    sites. Abbreviations include: MBP gene (malE), MBP promoter (Ptac), philamentous phage origin of replication ( M13 ori), bacterial origin of replica...notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display

  1. Gene expression in gut symbiotic organ of stinkbug affected by extracellular bacterial symbiont.

    PubMed

    Futahashi, Ryo; Tanaka, Kohjiro; Tanahashi, Masahiko; Nikoh, Naruo; Kikuchi, Yoshitomo; Lee, Bok Luel; Fukatsu, Takema

    2013-01-01

    The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.

  2. Microbial Genomics: The Expanding Universe of Bacterial Defense Systems.

    PubMed

    Forsberg, Kevin J; Malik, Harmit S

    2018-04-23

    Bacteria protect themselves against infection using multiple defensive systems that move by horizontal gene transfer and accumulate in genomic 'defense islands'. A recent study exploited these features to uncover ten novel defense systems, substantially expanding the catalog of bacterial defense systems and predicting the discovery of many more. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Forest floor community metatranscriptomes identify fungal and bacterial responses to N deposition in two maple forests

    DOE PAGES

    Hesse, Cedar N.; Mueller, Rebecca C.; Vuyisich, Momchilo; ...

    2015-04-23

    Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes)more » in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.« less

  4. Role of Honey in Topical and Systemic Bacterial Infections.

    PubMed

    Hussain, Muhammad Barkaat

    2018-01-01

    The development of bacterial resistance to antibiotics has made it more difficult and expensive to treat infections. Honey is getting worldwide attention as a topical therapeutic agent for wound infections and potential future candidate for systemic infections. The purpose of this review was to summarise different antibacterial bio-active compounds in honey, their synergistic interaction and their clinical implications in topical and systemic infections. In addition, contemporary testing methods for evaluating peroxide and non-peroxide antibacterial activity of honey were also critically appraised. MEDLINE, EMBASE, Cochrane Library, Pub Med, reference lists and databases were used to review the literature. Honey contains several unique antibacterial components. These components are believed to act on diverse bacterial targets, are broad spectrum, operate synergistically, prevent biofilm formation, and decrease production of virulence factors. Moreover, honey has the ability to block bacterial communication (quorum sensing), and therefore, it is unlikely that bacteria develop resistance against honey. Bacterial resistance against honey has not been documented so far. Unlike conventional antibiotics, honey only targets pathogenic bacteria without disturbing the growth of normal gastrointestinal flora when taken orally. It also contains prebiotics, probiotics, and zinc and enhances the growth of beneficial gut flora. The presence of such plethora of antibacterial properties in one product makes it a promising candidate not only in wound infections but also in systemic and particularly for gastrointestinal infections. Agar diffusion assay, being used for evaluating antibacterial activity of honey, is not the most appropriate and sensitive assay as it only detects non-peroxide activity when present at a higher level. Therefore, there is a need to develop more sensitive techniques that may be capable of detecting and evaluating different important components in honey as

  5. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    PubMed

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-11-01

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

  6. The combination of decoy receptor 3 and soluble triggering receptor expressed on myeloid cells-1 for the diagnosis of nosocomial bacterial meningitis.

    PubMed

    Liu, Yong-Juan; Shao, Li-Hua; Zhang, Jian; Fu, Shan-Ji; Wang, Gang; Chen, Feng-Zhe; Zheng, Feng; Ma, Rui-Ping; Liu, Hai-Hong; Dong, Xiao-Meng; Ma, Li-Xian

    2015-03-23

    Early diagnosis and appropriate antibiotic treatment can significantly reduce mortality of nosocomial bacterial meningitis. However, it is a challenge for clinicians to make an accurate and rapid diagnosis of bacterial meningitis. This study aimed at determining whether combined biomarkers can provide a useful tool for the diagnosis of bacterial meningitis. A retrospective study was carried out. Cerebrospinal fluid (CSF) levels of decoy receptor 3 (DcR3) and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) were detected by enzyme-linked immunosorbent assay (ELISA). The patients with bacterial meningitis had significantly elevated levels of the above mentioned biomarkers. The two biomarkers were all risk factors with bacterial meningitis. The biomarkers were constructed into a "bioscore". The discriminative performance of the bioscore was better than that of each biomarker, with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.842 (95% confidence intervals (CI) 0.770-0.914; p< 0.001). Combined measurement of CSF DcR3 and sTREM-1 concentrations improved the prediction of nosocomial bacterial meningitis. The combined strategy is of interest and the validation of that improvement needs further studies.

  7. Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

    PubMed

    Merchant, Mark; Kinney, Clint; Sanders, Paige

    2009-12-01

    Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

  8. Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles.

    PubMed

    Kwon, Young Sang; Ryu, Choong-Min; Lee, Soohyun; Park, Hyo Bee; Han, Ki Soo; Lee, Jung Han; Lee, Kyunghee; Chung, Woo Sik; Jeong, Mi-Jeong; Kim, Hee Kyu; Bae, Dong-Won

    2010-11-01

    Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.

  9. A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

    PubMed Central

    Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

    2017-01-01

    We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

  10. A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells.

    PubMed

    Chatelle, Claire; Ochoa-Fernandez, Rocio; Engesser, Raphael; Schneider, Nils; Beyer, Hannes M; Jones, Alex R; Timmer, Jens; Zurbriggen, Matias D; Weber, Wilfried

    2018-05-18

    The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene repression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.

  11. Annual Surveillance Summary: Bacterial Infections in the Military Health System (MHS), 2015

    DTIC Science & Technology

    2017-03-01

    Approved for public release. Distribution is unlimited. The views expressed in this document are those of the authors and do not necessarily reflect... account for low incidence rates. Data Source: NMCPHC HL7-formatted CHCS microbiology and M2 databases. Prepared by the EpiData Center Department, Navy...and Marine Corps Public Health Center, on 24 March 2017. Bacterial Infections in the MHS, 2015 Prepared March 2017 EpiData Center Department

  12. Strategies used for genetically modifying bacterial genome: ite-directed mutagenesis, gene inactivation, and gene over-expression*

    PubMed Central

    Xu, Jian-zhong; Zhang, Wei-guo

    2016-01-01

    With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators. PMID:26834010

  13. Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors

    PubMed Central

    Mo, Irene Fung Ying; Yip, Kevin Hak Kong; Chan, Wing Keung; Law, Helen Ka Wai; Lau, Yu Lung; Chan, Godfrey Chi Fung

    2008-01-01

    Background Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings. Results We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria. Conclusion In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment. PMID:18799018

  14. Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

    PubMed

    Li, Jun; Zhang, Yuehuan; Liu, Ying; Zhang, Yang; Xiao, Shu; Yu, Ziniu

    2016-01-01

    Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Estimate of blow-up and relaxation time for self-gravitating Brownian particles and bacterial populations.

    PubMed

    Chavanis, P-H; Sire, C

    2004-08-01

    We determine an exact asymptotic expression of the blow-up time t(coll) for self-gravitating Brownian particles or bacterial populations (chemotaxis) close to the critical point in d=3. We show that t(coll) = t(*) (eta- eta(c) )(-1/2) with t(*) =0.917 677 02..., where eta represents the inverse temperature (for Brownian particles) or the mass (for bacterial colonies), and eta(c) is the critical value of eta above which the system blows up. This result is in perfect agreement with the numerical solution of the Smoluchowski-Poisson system. We also determine the exact asymptotic expression of the relaxation time close to but above the critical temperature and derive a large time asymptotic expansion for the density profile exactly at the critical point.

  16. Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression

    PubMed Central

    Puthiyaveetil, Sujith; Allen, John F.

    2009-01-01

    Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles—chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems. PMID:19324807

  17. Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression.

    PubMed

    Puthiyaveetil, Sujith; Allen, John F

    2009-06-22

    Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles-chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems.

  18. Pyrosequencing analysis for characterization of bacterial diversity in a soil as affected by integrated livestock-cotton production systems

    USDA-ARS?s Scientific Manuscript database

    Impacts of integrated livestock-crop production systems compared to specialized systems on soil bacterial diversity have not been well documented. We used a bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) method to evaluate bacterial diversity of a clay loam soil (Fine, mixed, thermic To...

  19. Bacterial responses to antibiotics and their combinations.

    PubMed

    Mitosch, Karin; Bollenbach, Tobias

    2014-12-01

    Antibiotics affect bacterial cell physiology at many levels. Rather than just compensating for the direct cellular defects caused by the drug, bacteria respond to antibiotics by changing their morphology, macromolecular composition, metabolism, gene expression and possibly even their mutation rate. Inevitably, these processes affect each other, resulting in a complex response with changes in the expression of numerous genes. Genome-wide approaches can thus help in gaining a comprehensive understanding of bacterial responses to antibiotics. In addition, a combination of experimental and theoretical approaches is needed for identifying general principles that underlie these responses. Here, we review recent progress in our understanding of bacterial responses to antibiotics and their combinations, focusing on effects at the levels of growth rate and gene expression. We concentrate on studies performed in controlled laboratory conditions, which combine promising experimental techniques with quantitative data analysis and mathematical modeling. While these basic research approaches are not immediately applicable in the clinic, uncovering the principles and mechanisms underlying bacterial responses to antibiotics may, in the long term, contribute to the development of new treatment strategies to cope with and prevent the rise of resistant pathogenic bacteria.

  20. Candidate innate immune system gene expression in the ecological model Daphnia

    PubMed Central

    Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E.; Little, Tom J.

    2011-01-01

    The last ten years have witnessed increasing interest in host–pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host–pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia–pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia–Pasteuria system will need to balance a candidate gene approach with more

  1. Candidate innate immune system gene expression in the ecological model Daphnia.

    PubMed

    Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E; Little, Tom J

    2011-10-01

    The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive

  2. Activation of the immune system by bacterial CpG-DNA

    PubMed Central

    Häcker, Georg; Redecke, Vanessa; Häcker, Hans

    2002-01-01

    The past decade has seen a remarkable process of refocusing in immunology. Cells of the innate immune system, especially macrophages and dendritic cells, have been at the centre of this process. These cells had been regarded by some scientists as non-specific, sometimes perhaps even confined to the menial job of serving T cells by scavenging antigen and presenting it to the sophisticated adaptive immune system. Only over the last few years has it become unequivocally clear that cells of the innate immunity hold, by variation of context and mode of antigen presentation, the power of shaping an adaptive immune response. The innate immune response, in turn, is to a significant degree the result of stimulation by so-called pathogen-associated molecular patterns (PAMPs). One compound with high stimulatory potential for the innate immune system is bacterial DNA. Here we will review recent evidence that bacterial DNA should be ranked with other PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid. We will further review our present knowledge of DNA recognition and DNA-dependent signal transduction in cells of the immune system. PMID:11918685

  3. Gene Expression in Gut Symbiotic Organ of Stinkbug Affected by Extracellular Bacterial Symbiont

    PubMed Central

    Futahashi, Ryo; Tanaka, Kohjiro; Tanahashi, Masahiko; Nikoh, Naruo; Kikuchi, Yoshitomo; Lee, Bok Luel; Fukatsu, Takema

    2013-01-01

    The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations. PMID:23691247

  4. Analysis of host response to bacterial infection using error model based gene expression microarray experiments

    PubMed Central

    Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

    2005-01-01

    A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

  5. Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system.

    PubMed

    Douterelo, I; Sharpe, R L; Boxall, J B

    2013-02-01

    Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this

  6. Impact of bacterial biocontrol agents on aflatoxin biosynthetic genes, aflD and aflR expression, and phenotypic aflatoxin B₁ production by Aspergillus flavus under different environmental and nutritional regimes.

    PubMed

    Al-Saad, Labeed A; Al-Badran, Adnan I; Al-Jumayli, Sami A; Magan, Naresh; Rodríguez, Alicia

    2016-01-18

    The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B1 (AFB1) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (aw) on nutrient medium; 0.995, 0.98 aw on maize meal agar medium) at 35°C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway (aflD and aflR respectively) and the production of AFB1. These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 aw on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 aw and some at 0.995 aw. However, the results for AFB1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB1 production on both nutrient and maize-based media regardless of aw. However, some bacterial treatments did inhibit AFB1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB1. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Development of a Plant Transformation Selection System Based on Expression of Genes Encoding Gentamicin Acetyltransferases

    PubMed Central

    Hayford, Maria B.; Medford, June I.; Hoffman, Nancy L.; Rogers, Stephen G.; Klee, Harry J.

    1988-01-01

    The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666057

  8. Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues.

    PubMed

    Peyer, Suzanne M; Pankey, M Sabrina; Oakley, Todd H; McFall-Ngai, Margaret J

    2014-02-01

    The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or 'light organ', which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ∼1/4 into embryogenesis (Naef stage 18) and the light organ ∼3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light

  9. Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues

    PubMed Central

    Peyer, Suzanne M.; Pankey, M. Sabrina; Oakley, Todd H.; McFall-Ngai, Margaret J.

    2014-01-01

    The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or ‘light organ’, which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ~1/4 into embryogenesis (Naef stage 18) and the light organ ~3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light

  10. Vizantin inhibits bacterial adhesion without affecting bacterial growth and causes Streptococcus mutans biofilm to detach by altering its internal architecture.

    PubMed

    Takenaka, Shoji; Oda, Masataka; Domon, Hisanori; Ohsumi, Tatsuya; Suzuki, Yuki; Ohshima, Hayato; Yamamoto, Hirofumi; Terao, Yutaka; Noiri, Yuichiro

    2016-11-11

    An ideal antibiofilm strategy is to control both in the quality and quantity of biofilm while maintaining the benefits derived from resident microflora. Vizantin, a recently developed immunostimulating compound, has also been found to have antibiofilm property. This study evaluated the influence on biofilm formation of Streptococcus mutans in the presence of sulfated vizantin and biofilm development following bacterial adhesion on a hydroxyapatite disc coated with sulfated vizantin. Supplementation with sulfated vizantin up to 50 μM did not affect either bacterial growth or biofilm formation, whereas 50 μM sulfated vizantin caused the biofilm to readily detach from the surface. Sulfated vizantin at the concentration of 50 μM upregulated the expression of the gtfB and gtfC genes, but downregulated the expression of the gtfD gene, suggesting altered architecture in the biofilm. Biofilm development on the surface coated with sulfated vizantin was inhibited depending on the concentration, suggesting prevention from bacterial adhesion. Among eight genes related to bacterial adherence in S. mutans, expression of gtfB and gtfC was significantly upregulated, whereas the expression of gtfD, GbpA and GbpC was downregulated according to the concentration of vizantin, especially with 50 μM vizantin by 0.8-, 0.4-, and 0.4-fold, respectively. These findings suggest that sulfated vizantin may cause structural degradation as a result of changing gene regulation related to bacterial adhesion and glucan production of S. mutans. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Down-regulation of monocarboxylate transporter 1 (MCT1) gene expression in the colon of piglets is linked to bacterial protein fermentation and pro-inflammatory cytokine-mediated signalling.

    PubMed

    Villodre Tudela, Carmen; Boudry, Christelle; Stumpff, Friederike; Aschenbach, Jörg R; Vahjen, Wilfried; Zentek, Jürgen; Pieper, Robert

    2015-02-28

    The present study investigated the influence of bacterial metabolites on monocarboxylate transporter 1 (MCT1) expression in pigs using in vivo, ex vivo and in vitro approaches. Piglets (n 24) were fed high-protein (26 %) or low-protein (18 %) diets with or without fermentable carbohydrates. Colonic digesta samples were analysed for a broad range of bacterial metabolites. The expression of MCT1, TNF-α, interferon γ (IFN-γ) and IL-8 was determined in colonic tissue. The expression of MCT1 was lower and of TNF-α and IL-8 was higher with high-protein diets (P< 0·05). MCT1 expression was positively correlated with l-lactate, whereas negatively correlated with NH₃ and putrescine (P< 0·05). The expression of IL-8 and TNF-α was negatively correlated with l-lactate and positively correlated with NH₃ and putrescine, whereas the expression of IFN-γ was positively correlated with histamine and 4-ethylphenol (P< 0·05). Subsequently, porcine colonic tissue and Caco-2 cells were incubated with Na-butyrate, NH₄Cl or TNF-α as selected bacterial metabolites or mediators of inflammation. Colonic MCT1 expression was higher after incubation with Na-butyrate (P< 0·05) and lower after incubation with NH₄Cl or TNF-α (P< 0·05). Incubation of Caco-2 cells with increasing concentrations of these metabolites confirmed the up-regulation of MCT1 expression by Na-butyrate (linear, P< 0·05) and down-regulation by TNF-α and NH₄Cl (linear, P< 0·05). The high-protein diet decreased the expression of MCT1 in the colon of pigs, which appears to be linked to NH₃- and TNF-α-mediated signalling.

  12. Bacterial collagen-like proteins that form triple-helical structures

    PubMed Central

    Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara

    2014-01-01

    A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612

  13. Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC.

    PubMed Central

    Ellis, R W; Keller, P M; Lowe, R S; Zivin, R A

    1985-01-01

    The genome of varicella-zoster virus (VZV) encodes at least three major glycoprotein genes. Among viral gene products, the gC gene products are the most abundant glycoproteins and induce a substantial humoral immune response (Keller et al., J. Virol. 52:293-297, 1984). We utilized two independent approaches to map the gC gene. Small fragments of randomly digested VZV DNA were inserted into a bacterial expression vector. Bacterial colonies transformed by this vector library were screened serologically for antigen expression with monoclonal antibodies to gC. Hybridization of the plasmid DNA from a gC antigen-positive clone revealed homology to the 3' end of the VZV Us segment. In addition, mRNA from VZV-infected cells was hybrid selected by a set of VZV DNA recombinant plasmids and translated in vitro, and polypeptide products were immunoprecipitated by convalescent zoster serum or by monoclonal antibodies to gC. This analysis revealed that the mRNA encoding a 70,000-dalton polypeptide precipitable by anti-gC antibodies mapped to the HindIII C fragment, which circumscribes the entire Us region. We conclude that the VZV gC glycoprotein gene maps to the 3' end of the Us region and is expressed as a 70,000-dalton primary translational product. These results are consistent with the recently reported DNA sequence of Us (A.J. Davison, EMBO J. 2:2203-2209, 1983). Furthermore, glycosylation appears not to be required for a predominant portion of the antigenicity of gC glycoproteins. We also report the tentative map assignments for eight other VZV primary translational products. Images PMID:2981365

  14. Quantification, Distribution, and Possible Source of Bacterial Biofilm in Mouse Automated Watering Systems

    PubMed Central

    Meier, Thomas R; Maute, Carrie J; Cadillac, Joan M; Lee, Ji Young; Righter, Daniel J; Hugunin, Kelly MS; Deininger, Rolf A; Dysko, Robert C

    2008-01-01

    The use of automated watering systems for providing drinking water to rodents has become commonplace in the research setting. Little is known regarding bacterial biofilm growth within the water piping attached to the racks (manifolds). The purposes of this project were to determine whether the mouse oral flora contributed to the aerobic bacterial component of the rack biofilm, quantify bacterial growth in rack manifolds over 6 mo, assess our rack sanitation practices, and quantify bacterial biofilm development within sections of the manifold. By using standard methods of bacterial identification, the aerobic oral flora of 8 strains and stocks of mice were determined on their arrival at our animal facility. Ten rack manifolds were sampled before, during, and after sanitation and monthly for 6 mo. Manifolds were evaluated for aerobic bacterial growth by culture on R2A and trypticase soy agar, in addition to bacterial ATP quantification by bioluminescence. In addition, 6 racks were sampled at 32 accessible sites for evaluation of biofilm distribution within the watering manifold. The identified aerobic bacteria in the oral flora were inconsistent with the bacteria from the manifold, suggesting that the mice do not contribute to the biofilm bacteria. Bacterial growth in manifolds increased while they were in service, with exponential growth of the biofilm from months 3 to 6 and a significant decrease after sanitization. Bacterial biofilm distribution was not significantly different across location quartiles of the rack manifold, but bacterial levels differed between the shelf pipe and connecting elbow pipes. PMID:18351724

  15. Quantification, distribution, and possible source of bacterial biofilm in mouse automated watering systems.

    PubMed

    Meier, Thomas R; Maute, Carrie J; Cadillac, Joan M; Lee, Ji Young; Righter, Daniel J; Hugunin, Kelly M S; Deininger, Rolf A; Dysko, Robert C

    2008-03-01

    The use of automated watering systems for providing drinking water to rodents has become commonplace in the research setting. Little is known regarding bacterial biofilm growth within the water piping attached to the racks (manifolds). The purposes of this project were to determine whether the mouse oral flora contributed to the aerobic bacterial component of the rack biofilm, quantify bacterial growth in rack manifolds over 6 mo, assess our rack sanitation practices, and quantify bacterial biofilm development within sections of the manifold. By using standard methods of bacterial identification, the aerobic oral flora of 8 strains and stocks of mice were determined on their arrival at our animal facility. Ten rack manifolds were sampled before, during, and after sanitation and monthly for 6 mo. Manifolds were evaluated for aerobic bacterial growth by culture on R2A and trypticase soy agar, in addition to bacterial ATP quantification by bioluminescence. In addition, 6 racks were sampled at 32 accessible sites for evaluation of biofilm distribution within the watering manifold. The identified aerobic bacteria in the oral flora were inconsistent with the bacteria from the manifold, suggesting that the mice do not contribute to the biofilm bacteria. Bacterial growth in manifolds increased while they were in service, with exponential growth of the biofilm from months 3 to 6 and a significant decrease after sanitization. Bacterial biofilm distribution was not significantly different across location quartiles of the rack manifold, but bacterial levels differed between the shelf pipe and connecting elbow pipes.

  16. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

    PubMed Central

    Ihssen, Julian; Reiss, Renate; Luchsinger, Ronny; Thöny-Meyer, Linda; Richter, Michael

    2015-01-01

    Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development. PMID:26068013

  17. Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

    DOEpatents

    Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL

    2007-02-27

    The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

  18. Mechano-sensitization of mammalian neuronal networks through expression of the bacterial large-conductance mechanosensitive ion channel

    PubMed Central

    Contestabile, Andrea; Moroni, Monica; Hallinan, Grace I.; Palazzolo, Gemma; Chad, John; Deinhardt, Katrin; Carugo, Dario

    2018-01-01

    ABSTRACT Development of remote stimulation techniques for neuronal tissues represents a challenging goal. Among the potential methods, mechanical stimuli are the most promising vectors to convey information non-invasively into intact brain tissue. In this context, selective mechano-sensitization of neuronal circuits would pave the way to develop a new cell-type-specific stimulation approach. We report here, for the first time, the development and characterization of mechano-sensitized neuronal networks through the heterologous expression of an engineered bacterial large-conductance mechanosensitive ion channel (MscL). The neuronal functional expression of the MscL was validated through patch-clamp recordings upon application of calibrated suction pressures. Moreover, we verified the effective development of in-vitro neuronal networks expressing the engineered MscL in terms of cell survival, number of synaptic puncta and spontaneous network activity. The pure mechanosensitivity of the engineered MscL, with its wide genetic modification library, may represent a versatile tool to further develop a mechano-genetic approach. This article has an associated First Person interview with the first author of the paper. PMID:29361543

  19. Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens.

    PubMed

    Tanhaeian, Abbas; Shahriari Ahmadi, Farajollah; Sekhavati, Mohammad Hadi; Mamarabadi, Mojtaba

    2018-04-04

    Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 μg/ml for different bacterial isolates.

  20. Expression of a soluble truncated Vargula luciferase in Escherichia coli

    PubMed Central

    Hunt, Eric A.; Moutsiopoulou, Angeliki; Broyles, David; Head, Trajen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K.

    2017-01-01

    Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties. PMID:28108349

  1. Expression of a soluble truncated Vargula luciferase in Escherichia coli.

    PubMed

    Hunt, Eric A; Moutsiopoulou, Angeliki; Broyles, David; Head, Trajen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K

    2017-04-01

    Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Venus Express Chemical Propulsion System - The Mars Express Legacy

    NASA Astrophysics Data System (ADS)

    Hunter, C. J.

    2004-10-01

    ESA's ambition of inter-planetary exploration using a fast-track low cost industrial programme was well achieved with Mars Express. Reusing the platform architecture for the service module and specifically the Propulsion system enabled Venus Express to benefit from several lessons learnt from the Mars Express experience. Using all existing components qualified for previous programmes, many of them commercial telecommunication spacecraft programmes with components available from stock, an industrial organisation familiar from Mars Express was able to compress the schedule to make the November 2005 launch window a realistic target. While initial inspection of the CPS schematic indicates a modified Eurostar type architecture, - a similar system using some Eurostar components - would be a fairer description. The use of many parts of the system on arrival at the destination (Mars or Venus in this case) is a departure from the usual mode of operation, where many components are used during the initial few weeks of GTO or GEO. The system modifications over the basic Eurostar system have catered for this in terms of reliability contingencies by replacing components, or providing different levels of test capability or isolation in flight. This paper aims to provide an introduction to the system, address the evolution from Eurostar, and provide an initial assessment of the success of these modifications using the Mars Express experience, and how measures have been adopted specifically for Venus Express.

  3. Alteration of intracellular protein expressions as a key mechanism of the deterioration of bacterial denitrification caused by copper oxide nanoparticles.

    PubMed

    Su, Yinglong; Zheng, Xiong; Chen, Yinguang; Li, Mu; Liu, Kun

    2015-10-28

    The increasing production and utilization of copper oxide nanoparticles (CuO NPs) result in the releases into the environment. However, the influence of CuO NPs on bacterial denitrification, one of the most important pathways to transform nitrate to dinitrogen in environment, has seldom been studied. Here we reported that CuO NPs caused a significant alteration of key protein expressions of a model denitrifier, Paracoccus denitrificans, leading to severe inhibition to denitrification. Total nitrogen removal efficiency was decreased from 98.3% to 62.1% with the increase of CuO NPs from 0.05 to 0.25 mg/L. Cellular morphology and integrity studies indicated that nanoparticles entered the cells. The proteomic bioinformatics analysis showed that CuO NPs caused regulation of proteins involved in nitrogen metabolism, electron transfer and substance transport. The down-regulation of GtsB protein (responsible for glucose transport) decreased the production of NADH (electron donor for denitrification). Also, the expressions of key electron-transfer proteins (including NADH dehydrogenase and cytochrome) were suppressed by CuO NPs, which adversely affected electrons transfer for denitrification. Further investigation revealed that CuO NPs significantly inhibited the expressions and catalytic activities of nitrate reductase and nitrite reductase. These results provided a fundamental understanding of the negative influences of CuO NPs on bacterial denitrification.

  4. Alteration of intracellular protein expressions as a key mechanism of the deterioration of bacterial denitrification caused by copper oxide nanoparticles

    PubMed Central

    Su, Yinglong; Zheng, Xiong; Chen, Yinguang; Li, Mu; Liu, Kun

    2015-01-01

    The increasing production and utilization of copper oxide nanoparticles (CuO NPs) result in the releases into the environment. However, the influence of CuO NPs on bacterial denitrification, one of the most important pathways to transform nitrate to dinitrogen in environment, has seldom been studied. Here we reported that CuO NPs caused a significant alteration of key protein expressions of a model denitrifier, Paracoccus denitrificans, leading to severe inhibition to denitrification. Total nitrogen removal efficiency was decreased from 98.3% to 62.1% with the increase of CuO NPs from 0.05 to 0.25 mg/L. Cellular morphology and integrity studies indicated that nanoparticles entered the cells. The proteomic bioinformatics analysis showed that CuO NPs caused regulation of proteins involved in nitrogen metabolism, electron transfer and substance transport. The down-regulation of GtsB protein (responsible for glucose transport) decreased the production of NADH (electron donor for denitrification). Also, the expressions of key electron-transfer proteins (including NADH dehydrogenase and cytochrome) were suppressed by CuO NPs, which adversely affected electrons transfer for denitrification. Further investigation revealed that CuO NPs significantly inhibited the expressions and catalytic activities of nitrate reductase and nitrite reductase. These results provided a fundamental understanding of the negative influences of CuO NPs on bacterial denitrification. PMID:26508362

  5. Comparison of bacterial counts in expressed breast milk following standard or strict infection control regimens in neonatal intensive care units: compliance of mothers does matter.

    PubMed

    Haiden, N; Pimpel, B; Assadian, O; Binder, C; Kreissl, A; Repa, A; Thanhäuser, M; Roberts, C D; Berger, A

    2016-03-01

    Bacterial counts in 1466 expressed breast milk (EBM) samples from women following one of two infection control regimens (standard vs strict) were investigated. Overall, 12% of samples yielded Gram-negative bacteria, with no significant differences between the standard [11.9% (94/788)] and strict [12.1% (82/678)] regimens (P = 0.92). Significantly more samples were contaminated when expressed at home (standard regimen home/hospital: 17.9% vs 6.1%; strict regimen home/hospital: 19.6% vs 3.4%; P < 0.001). Bacterial contamination of EBM was not associated with the regimen, but was associated with the location of breast milk expression. Attempts to improve personal hygiene during milk collection seem to be of limited value. Good hygiene of collection and storage equipment is likely to be the most important way to ensure the microbiological quality of EBM. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  6. Role of the cathelicidin-related antimicrobial peptide in inflammation and mortality in a mouse model of bacterial meningitis.

    PubMed

    Merres, Julika; Höss, Jonas; Albrecht, Lea-Jessica; Kress, Eugenia; Soehnlein, Oliver; Jansen, Sandra; Pufe, Thomas; Tauber, Simone C; Brandenburg, Lars-Ove

    2014-01-01

    Antimicrobial peptides (AP) are important components of the innate immune system, yet little is known about their expression and function in the brain. Our previous work revealed upregulated gene expression of cathelicidin-related AP (CRAMP) following bacterial meningitis in primary rat glial cells as well as bactericidal activity against frequent meningitis-causing bacteria. However, the effect of cathelicidin expression on the progression of inflammation and mortality in bacterial meningitis remains unknown. Therefore, we used CRAMP-deficient mice to investigate the effect of CRAMP on bacterial growth, inflammatory responses and mortality in meningitis. Meningitis was induced by intracerebral injection of type 3 Streptococcus pneumoniae. The degree of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time RT-PCR. CRAMP deficiency led to a higher mortality rate that was associated with increased bacterial titers in the cerebellum, blood and spleen as well as decreased meningeal neutrophil infiltration. CRAMP-deficient mice displayed a higher degree of glial cell activation that was accompanied by a more pronounced proinflammatory response. Taken together, this work provides insight into the important role of CRAMP as part of the innate immune defense against pathogens in bacterial CNS infections. Copyright © 2013 S. Karger AG, Basel

  7. Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

    PubMed

    Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

    2016-12-28

    Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19 +FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

  8. Nitrous oxide production and mRNA expression analysis of nitrifying and denitrifying bacterial genes under floodwater disappearance and fertilizer application.

    PubMed

    Riya, Shohei; Takeuchi, Yuki; Zhou, Sheng; Terada, Akihiko; Hosomi, Masaaki

    2017-06-01

    A pulse of nitrous oxide (N 2 O) emission has been observed following the disappearance of floodwater by drainage. However, its mechanism is not well understood. We conducted a column study to clarify the mechanism for N 2 O production during floodwater disappearance by using a microsensor and determining the bacterial gene expression. An increase in N 2 O flux was observed following floodwater disappearance after the addition of NH 4 + , with a corresponding increase in the concentrations of NO 3 - and dissolved N 2 O in the oxic and anoxic soil layers, respectively. The transcription level of the bacterial amoA mRNA did not change, while that of nirK mRNA increased sharply after an hour of floodwater disappearance. An additional anoxic soil slurry experiment demonstrated that the addition of NO 3 - induced the expression of nirK gene and caused a concomitant increase in N 2 O production. These findings suggest that NO 3 - production in the oxic layers is important as it provides a substrate and induces the synthesis of denitrification enzymes in the anoxic layer during N 2 O production.

  9. Encyclopedia of bacterial gene circuits whose presence or absence correlate with pathogenicity--a large-scale system analysis of decoded bacterial genomes.

    PubMed

    Shestov, Maksim; Ontañón, Santiago; Tozeren, Aydin

    2015-10-13

    Bacterial infections comprise a global health challenge as the incidences of antibiotic resistance increase. Pathogenic potential of bacteria has been shown to be context dependent, varying in response to environment and even within the strains of the same genus. We used the KEGG repository and extensive literature searches to identify among the 2527 bacterial genomes in the literature those implicated as pathogenic to the host, including those which show pathogenicity in a context dependent manner. Using data on the gene contents of these genomes, we identified sets of genes highly abundant in pathogenic but relatively absent in commensal strains and vice versa. In addition, we carried out genome comparison within a genus for the seventeen largest genera in our genome collection. We projected the resultant lists of ortholog genes onto KEGG bacterial pathways to identify clusters and circuits, which can be linked to either pathogenicity or synergy. Gene circuits relatively abundant in nonpathogenic bacteria often mediated biosynthesis of antibiotics. Other synergy-linked circuits reduced drug-induced toxicity. Pathogen-abundant gene circuits included modules in one-carbon folate, two-component system, type-3 secretion system, and peptidoglycan biosynthesis. Antibiotics-resistant bacterial strains possessed genes modulating phagocytosis, vesicle trafficking, cytoskeletal reorganization, and regulation of the inflammatory response. Our study also identified bacterial genera containing a circuit, elements of which were previously linked to Alzheimer's disease. Present study produces for the first time, a signature, in the form of a robust list of gene circuitry whose presence or absence could potentially define the pathogenicity of a microbiome. Extensive literature search substantiated a bulk majority of the commensal and pathogenic circuitry in our predicted list. Scanning microbiome libraries for these circuitry motifs will provide further insights into the complex

  10. Reduction of Airborne Bacterial Burden in the OR by Installation of Unidirectional Displacement Airflow (UDF) Systems.

    PubMed

    Fischer, Sebastian; Thieves, Martin; Hirsch, Tobias; Fischer, Klaus-Dieter; Hubert, Helmine; Beppler, Steffen; Seipp, Hans-Martin

    2015-08-13

    Intraoperative bacterial contamination is a major risk factor for postoperative wound infections. This study investigated the influence of type of ventilation system on intraoperative airborne bacterial burden before and after installation of unidirectional displacement air flow systems. We microbiologically monitored 1286 surgeries performed by a single surgical team that moved from operating rooms (ORs) equipped with turbulent mixing ventilation (TMV, according to standard DIN-1946-4 [1999], ORs 1, 2, and 3) to ORs with unidirectional displacement airflow (UDF, according to standard DIN-1946-4, annex D [2008], ORs 7 and 8). The airborne bacteria were collected intraoperatively with sedimentation plates. After incubation for 48 h, we analyzed the average number of bacteria per h, peak values, and correlation to surgery duration. In addition, we compared the last 138 surgeries in ORs 1-3 with the first 138 surgeries in ORs 7 and 8. Intraoperative airborne bacterial burden was 5.4 CFU/h, 5.5 CFU/h, and 6.1 CFU/h in ORs 1, 2, and 3, respectively. Peak values of burden were 10.7 CFU/h, 11.1 CFU/h, and 11.0 CFU/h in ORs 1, 2, and 3, respectively). With the UDF system, the intraoperative airborne bacterial burden was reduced to 0.21 CFU/h (OR 7) and 0.35 CFU/h (OR 8) on average (p<0.01). Accordingly, peak values decreased to 0.9 CFU/h and 1.0 CFU/h in ORs 7 and 8, respectively (p<0.01). Airborne bacterial burden increased linearly with surgery duration in ORs 1-3, but the UDF system in ORs 7 and 8 kept bacterial levels constantly low (<3 CFU/h). A comparison of the last 138 surgeries before with the first 138 surgeries after changing ORs revealed a 94% reduction in average airborne bacterial burden (5 CFU/h vs. 0.29 CFU/h, p<0.01). The unidirectional displacement airflow, which fulfills the requirements of standard DIN-1946-4 annex D of 2008, is an effective ventilation system that reduces airborne bacterial burden under real clinical conditions by more than 90

  11. Identification of gold sensing peptide by integrative proteomics and a bacterial two-component system

    NASA Astrophysics Data System (ADS)

    Ng, I.-Son; Yu, You-Jin; Yi, Ying-Chen; Tan, Shih-I.; Huang, Bo-Chuan; Han, Yin-Lung

    2017-12-01

    The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from Tepidimonas fonticaldi AT-A2, due to its outstanding performance in gold-binding and recovery. The results showed that three small proteins, including histidine biosynthesis protein (HisIE), iron donor protein (CyaY) and hypothetical protein_65aa, have a higher ability to adsorb gold ions because of the negatively charged domains or metal binding sites. On the other hand, the Salmonella PmrA/PmrB two-component system first replaces the iron (III)-binding motif using the peptide sequence from hypothetical protein_65aa, and this is then used to reveal the sensing and responsiveness to gold metal ions, which is totally different from the performance of traditional gold binding peptide (GBP) on the crystals on the surface of gold (111). We have successfully demonstrated an integrative proteomics and bacterial two-component system to explore the novel gold binding peptide. Finally, the heterologous over-expression of gold binding peptide by E. coli and the equilibrium of binding capacity for Au(III) have been conducted.

  12. Bacterial Signaling to the Nervous System through Toxins and Metabolites.

    PubMed

    Yang, Nicole J; Chiu, Isaac M

    2017-03-10

    Mammalian hosts interface intimately with commensal and pathogenic bacteria. It is increasingly clear that molecular interactions between the nervous system and microbes contribute to health and disease. Both commensal and pathogenic bacteria are capable of producing molecules that act on neurons and affect essential aspects of host physiology. Here we highlight several classes of physiologically important molecular interactions that occur between bacteria and the nervous system. First, clostridial neurotoxins block neurotransmission to or from neurons by targeting the SNARE complex, causing the characteristic paralyses of botulism and tetanus during bacterial infection. Second, peripheral sensory neurons-olfactory chemosensory neurons and nociceptor sensory neurons-detect bacterial toxins, formyl peptides, and lipopolysaccharides through distinct molecular mechanisms to elicit smell and pain. Bacteria also damage the central nervous system through toxins that target the brain during infection. Finally, the gut microbiota produces molecules that act on enteric neurons to influence gastrointestinal motility, and metabolites that stimulate the "gut-brain axis" to alter neural circuits, autonomic function, and higher-order brain function and behavior. Furthering the mechanistic and molecular understanding of how bacteria affect the nervous system may uncover potential strategies for modulating neural function and treating neurological diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Complexity of Bacterial Communities in a River-Floodplain System (Danube, Austria)

    PubMed Central

    Besemer, Katharina; Moeseneder, Markus M.; Arrieta, Jesus M.; Herndl, Gerhard J.; Peduzzi, Peter

    2005-01-01

    Natural floodplains play an essential role in the processing and decomposition of organic matter and in the self-purification ability of rivers, largely due to the activity of bacteria. Knowledge about the composition of bacterial communities and its impact on organic-matter cycling is crucial for the understanding of ecological processes in river-floodplain systems. Particle-associated and free-living bacterial assemblages from the Danube River and various floodplain pools with different hydrological characteristics were investigated using terminal restriction fragment length polymorphism analysis. The particle-associated bacterial community exhibited a higher number of operational taxonomic units (OTUs) and was more heterogeneous in time and space than the free-living community. The temporal dynamics of the community structure were generally higher in isolated floodplain pools. The community structures of the river and the various floodplain pools, as well as those of the particle-associated and free-living bacteria, differed significantly. The compositional dynamics of the planktonic bacterial communities were related to changes in the algal biomass, temperature, and concentrations of organic and inorganic nutrients. The OTU richness of the free-living community was correlated with the concentration and origin of organic matter and the concentration of inorganic nutrients, while no correlation with the OTU richness of the particle-associated assemblage was found. Our results demonstrate the importance of the river-floodplain interactions and the influence of damming and regulation on the bacterial-community composition. PMID:15691909

  14. Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks.

    PubMed

    Jers, Carsten; Soufi, Boumediene; Grangeasse, Christophe; Deutscher, Josef; Mijakovic, Ivan

    2008-08-01

    Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.

  15. A real-time control system of gene expression using ligand-bound nucleic acid aptamer for metabolic engineering.

    PubMed

    Wang, Jing; Cui, Xun; Yang, Le; Zhang, Zhe; Lv, Liping; Wang, Haoyuan; Zhao, Zhenmin; Guan, Ningzi; Dong, Lichun; Chen, Rachel

    2017-07-01

    Artificial control of bio-functions through regulating gene expression is one of the most important and attractive technologies to build novel living systems that are useful in the areas of chemical synthesis, nanotechnology, pharmacology, cell biology. Here, we present a novel real-time control system of gene regulation that includes an enhancement element by introducing duplex DNA aptamers upstream promoter and a repression element by introducing a RNA aptamer upstream ribosome binding site. With the presence of ligands corresponding to the DNA aptamers, the expression of the target gene can be potentially enhanced at the transcriptional level by strengthening the recognition capability of RNAP to the recognition region and speeding up the separation efficiency of the unwinding region due to the induced DNA bubble around the thrombin-bound aptamers; while with the presence of RNA aptamer ligand, the gene expression can be repressed at the translational level by weakening the recognition capability of ribosome to RBS due to the shielding of RBS by the formed aptamer-ligand complex upstream RBS. The effectiveness and potential utility of the developed gene regulation system were demonstrated by regulating the expression of ecaA gene in the cell-free systems. The realistic metabolic engineering application of the system has also tested by regulating the expression of mgtC gene and thrombin cDNA in Escherichia coli JD1021 for controlling metabolic flux and improving thrombin production, verifying that the real-time control system of gene regulation is able to realize the dynamic regulation of gene expression with potential applications in bacterial physiology studies and metabolic engineering. Copyright © 2017. Published by Elsevier Inc.

  16. Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

    PubMed

    Úbeda, María; Lario, Margaret; Muñoz, Leticia; Borrero, María-José; Rodríguez-Serrano, Macarena; Sánchez-Díaz, Ana-María; Del Campo, Rosa; Lledó, Lourdes; Pastor, Óscar; García-Bermejo, Laura; Díaz, David; Álvarez-Mon, Melchor; Albillos, Agustín

    2016-05-01

    In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  17. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

    PubMed

    Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

    2011-11-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

  18. Stable long-term indigo production by overexpression of dioxygenase genes using a chromosomal integrated cascade expression circuit.

    PubMed

    Royo, Jose Luis; Moreno-Ruiz, Emilia; Cebolla, Angel; Santero, Eduardo

    2005-03-16

    In our laboratory we have analyzed different factors to maximize the yield in heterologous protein expression for long-term cultivation, by combination of an efficient cascade expression system and stable integration in the bacterial chromosome. In this work, we have explored this system for the production of indigo dye as a model for biotechnological production, by expressing in Escherichia coli the thnA1A2A3A4 genes from Sphingomonas macrogolitabida strain TFA, which encode the components of a tetralin dioxygenase activity. We compared Ptac, and the Pm-based cascade expression circuit in a multicopy plasmid and stably integrated into the bacterial chromosome. Plasmid-based expression systems resulted in instability of indigo production when serially diluted batch experiments were performed without a selective pressure. This problem was solved by integrating the expression module in the chromosome. Despite the gene dosage reduction, the synergic effect of the cascade expression system produced comparable expression to the dioxygenase activity in the plasmid configuration but could be stably maintained for at least 5 days. Here, we show that the cascade amplification circuit integrated in the chromosome could be an excellent system for tight control and stable production of recombinant products.

  19. Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato

    PubMed Central

    Tai, Thomas H.; Dahlbeck, Douglas; Clark, Eszter T.; Gajiwala, Paresh; Pasion, Romela; Whalen, Maureen C.; Stall, Robert E.; Staskawicz, Brian J.

    1999-01-01

    The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species. PMID:10570214

  20. Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato.

    PubMed

    Tai, T H; Dahlbeck, D; Clark, E T; Gajiwala, P; Pasion, R; Whalen, M C; Stall, R E; Staskawicz, B J

    1999-11-23

    The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

  1. The bacterial alarmone (p)ppGpp activates the type III secretion system in Erwinia amylovora.

    PubMed

    Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In; Zhao, Youfu

    2015-04-01

    The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp(0)) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp(0) and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp(0) and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation

  2. The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

    PubMed Central

    Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In

    2015-01-01

    ABSTRACT The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for

  3. Influence of sugarbeet tillage systems on rhizoctonia-bacterial root rot complex

    USDA-ARS?s Scientific Manuscript database

    The Rhizoctonia-bacterial root rot complex on sugarbeet caused by Rhizoctonia solani and Leuconostoc mesenteroides can cause significant yield losses. To investigate the impact of different tillage systems on this complex, field studies were conducted from 2009 to 2011. Split blocks with conventio...

  4. Riboregulation of bacterial and archaeal transposition.

    PubMed

    Ellis, Michael J; Haniford, David B

    2016-05-01

    The coexistence of transposons with their hosts depends largely on transposition levels being tightly regulated to limit the mutagenic burden associated with frequent transposition. For 'DNA-based' (class II) bacterial transposons there is growing evidence that regulation through small noncoding RNAs and/or the RNA-binding protein Hfq are prominent mechanisms of defense against transposition. Recent transcriptomics analyses have identified many new cases of antisense RNAs (asRNA) that potentially could regulate the expression of transposon-encoded genes giving the impression that asRNA regulation of DNA-based transposons is much more frequent than previously thought. Hfq is a highly conserved bacterial protein that plays a central role in posttranscriptional gene regulation and stress response pathways in many bacteria. Three different mechanisms for Hfq-directed control of bacterial transposons have been identified to date highlighting the versatility of this protein as a regulator of bacterial transposons. There is also evidence emerging that some DNA-based transposons encode RNAs that could regulate expression of host genes. In the case of IS200, which appears to have lost its ability to transpose, contributing a regulatory RNA to its host could account for the persistence of this mobile element in a wide range of bacterial species. It remains to be seen how prevalent these transposon-encoded RNA regulators are, but given the relatively large amount of intragenic transcription in bacterial genomes, it would not be surprising if new examples are forthcoming. WIREs RNA 2016, 7:382-398. doi: 10.1002/wrna.1341 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  5. SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

    PubMed

    Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

    2016-02-04

    Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with

  6. Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs

    PubMed Central

    Zhang, Can; Chan, Chio Mui; Wang, Pei; Huang, Raven H.

    2012-01-01

    Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed. PMID:22190744

  7. CHANGES IN BACTERIAL COMPOSITION OF BIOFILM IN A METROPOLITAN DRINKING WATER DISTRIBUTION SYSTEM

    EPA Science Inventory

    This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e., groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. The b...

  8. Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System

    PubMed Central

    Whiteley, Andrew S.; Bailey, Mark J.

    2000-01-01

    The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the α, β and γ subclasses of the class Proteobacteria [α-, β-, and γ-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the γ-Proteobacteria. The α subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the β subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The γ subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical

  9. Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction

    PubMed Central

    Tugaeva, Kristina V.; Tsvetkov, Philipp O.

    2017-01-01

    Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods. PMID:28575131

  10. Connexin 26 facilitates gastrointestinal bacterial infection in vitro.

    PubMed

    Simpson, Charlotte; Kelsell, David P; Marchès, Olivier

    2013-01-01

    Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.

  11. Baculovirus expression system and method for high throughput expression of genetic material

    DOEpatents

    Clark, Robin; Davies, Anthony

    2001-01-01

    The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

  12. Bacterial detection: from microscope to smartphone.

    PubMed

    Gopinath, Subash C B; Tang, Thean-Hock; Chen, Yeng; Citartan, Marimuthu; Lakshmipriya, Thangavel

    2014-10-15

    The ubiquitous nature of bacteria enables them to survive in a wide variety of environments. Hence, the rise of various pathogenic species that are harmful to human health raises the need for the development of accurate sensing systems. Sensing systems are necessary for diagnosis and epidemiological control of pathogenic organism, especially in the food-borne pathogen and sanitary water treatment facility' bacterial populations. Bacterial sensing for the purpose of diagnosis can function in three ways: bacterial morphological visualization, specific detection of bacterial component and whole cell detection. This paper provides an overview of the currently available bacterial detection systems that ranges from microscopic observation to state-of-the-art smartphone-based detection. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

    PubMed

    Sturm, A; Chrispeels, M J

    1990-11-01

    We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

  14. Bacterial Reduction after Gutta-Percha Removal with Single vs. Multiple Instrument Systems.

    PubMed

    Xavier, Felipe; Nevares, Giselle; Gominho, Luciana; Rodrigues, Renata; Cassimiro, Marcely; Romeiro, Kaline; Albuquerque, Diana

    2018-01-01

    The aim of this study was to evaluate the effectiveness of a reciprocating single-instrument system (Reciproc-REC) compared with combined continuously rotating multiple-instrument systems [D-Race (DR) and BioRace (BR)] in reducing Enterococcus faecalis (E.f.) after gutta-percha removal. Forty-six extracted human maxillary incisors were prepared and contaminated with E.f. strain (ATCC 29212) for 30 days. The samples were obturated and randomly divided into two experimental groups for gutta-percha removal ( n =23): a REC group (R50) and a DR/BR group (DR1, DR2 and BR6). A standardized irrigation with 0.9% saline solution was performed. Root canal samples were taken with paper points before (S1) and after (S2) the removal of gutta-percha to establish bacterial quantification by culture. The time required for gutta-percha removal was also recorded. Positive and negative control groups ( n =6) were used to test bacterial viability and control asepsis, respectively. Data were analysed using t -Student and one-way ANOVA tests (5% margin of error). The mean percentage of bacterial reduction was significantly higher in DR/BR group (84.2%) than in REC group (72.3%) ( P <0.05). The mean time for obturation removal was 74.00 sec in REC group and 107.53 sec in DR/BR group ( P <0.05). The combined continuously rotating multiple-instrument system was more effective in reducing bacteria after the removal of gutta-percha than the single-instrument system. None of the tested systems was able to completely eliminate root canal infection after gutta-percha removal. Thus, additional techniques should be considered.

  15. Interplay of heritage and habitat in the distribution of bacterial signal transduction systems.

    PubMed

    Galperin, Michael Y; Higdon, Roger; Kolker, Eugene

    2010-04-01

    Comparative analysis of the complete genome sequences from a variety of poorly studied organisms aims at predicting ecological and behavioral properties of these organisms and helping in characterizing their habitats. This task requires finding appropriate descriptors that could be correlated with the core traits of each system and would allow meaningful comparisons. Using the relatively simple bacterial models, first attempts have been made to introduce suitable metrics to describe the complexity of organism's signaling machinery, which included introducing the "bacterial IQ" score. Here, we use an updated census of prokaryotic signal transduction systems to improve this parameter and evaluate its consistency within selected bacterial phyla. We also introduce a more elaborate descriptor, a set of profiles of relative abundance of members of each family of signal transduction proteins encoded in each genome. We show that these family profiles are well conserved within each genus and are often consistent within families of bacteria. Thus, they reflect evolutionary relationships between organisms as well as individual adaptations of each organism to its specific ecological niche.

  16. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    PubMed

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  17. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    PubMed

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  18. Selective Infection of Antigen-Specific B Lymphocytes by Salmonella Mediates Bacterial Survival and Systemic Spreading of Infection

    PubMed Central

    de Wit, Jelle; Martinoli, Chiara; Zagato, Elena; Janssen, Hans; Jorritsma, Tineke; Bar-Ephraïm, Yotam E.; Rescigno, Maria; Neefjes, Jacques; van Ham, S. Marieke

    2012-01-01

    Background The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer’s Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR). As it is unclear how Salmonella disseminates systemically, we studied whether Salmonella can use B cells as a transport device for spreading. Methodology/Principal Findings Human primary B cells or Ramos cell line were incubated with GFP-expressing Salmonella. Intracellular survival and escape was studied in vitro by live cell imaging, flow cytometry and flow imaging. HEL-specific B cells were transferred into C57BL/6 mice and HEL-expressing Salmonella spreading in vivo was analyzed investigating mesenteric lymph nodes, spleen and blood. After phagocytosis by B cells, Salmonella survives intracellularly in a non-replicative state which is actively maintained by the B cell. Salmonella is later excreted followed by reproductive infection of other cell types. Salmonella-specific B cells thus act both as a survival niche and a reservoir for reinfection. Adoptive transfer of antigen-specific B cells before oral infection of mice showed that these B cells mediate in vivo systemic spreading of Salmonella to spleen and blood. Conclusions/Significance This is a first example of a pathogenic bacterium that abuses the antigen-specific cells of the adaptive immune system for systemic spreading for dissemination of infection. PMID:23209805

  19. Identifying Bacterial Immune Evasion Proteins Using Phage Display.

    PubMed

    Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

    2017-01-01

    Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

  20. Engineering the bacterial shapes for enhanced inclusion bodies accumulation.

    PubMed

    Jiang, Xiao-Ran; Wang, Huan; Shen, Rui; Chen, Guo-Qiang

    2015-05-01

    Many bacteria can accumulate inclusion bodies such as sulfur, polyphosphate, glycogen, proteins or polyhydroxyalkanoates. To exploit bacteria as factories for effective production of inclusion bodies, a larger intracellular space is needed for more inclusion body accumulation. In this study, polyhydroxybutyrate (PHB) was investigated as an inclusion bodies representative to be accumulated by Escherichia coli JM109SG. Various approaches were taken to increase the bacterial cell sizes including deletion on actin-like protein gene mreB, weak expression of mreB in mreB deletion mutant, and weak expression of mreB in mreB deletion mutant under inducible expression of SulA, the inhibitor of division ring protein FtsZ. All of the methods resulted in different levels of increases in bacterial sizes and PHB granules accumulation. Remarkably, an increase of over 100% PHB accumulation was observed in recombinant E. coli overexpressing mreB in an mreB deletion mutant under inducible expression of FtsZ inhibiting protein SulA. The molecular mechanism of enlarged bacterial size was found to be directly relate to weakened cytoskeleton which was the result of broken skeleton helix. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems.

    PubMed

    Gomaa, Ahmed A; Klumpe, Heidi E; Luo, Michelle L; Selle, Kurt; Barrangou, Rodolphe; Beisel, Chase L

    2014-01-28

    CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of "smart" antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. Controlling the composition of microbial populations is a critical aspect in medicine, biotechnology, and environmental cycles. While different antimicrobial strategies, such as antibiotics, antimicrobial peptides, and lytic bacteriophages, offer partial solutions

  2. Bacterial hybrid histidine kinases in plant-bacteria interactions.

    PubMed

    Borland, Stéphanie; Prigent-Combaret, Claire; Wisniewski-Dyé, Florence

    2016-10-01

    Two-component signal transduction systems are essential for many bacteria to maintain homeostasis and adapt to environmental changes. Two-component signal transduction systems typically involve a membrane-bound histidine kinase that senses stimuli, autophosphorylates in the transmitter region and then transfers the phosphoryl group to the receiver domain of a cytoplasmic response regulator that mediates appropriate changes in bacterial physiology. Although usually found on distinct proteins, the transmitter and receiver modules are sometimes fused into a so-called hybrid histidine kinase (HyHK). Such structure results in multiple phosphate transfers that are believed to provide extra-fine-tuning mechanisms and more regulatory checkpoints than classical phosphotransfers. HyHK-based regulation may be crucial for finely tuning gene expression in a heterogeneous environment such as the rhizosphere, where intricate plant-bacteria interactions occur. In this review, we focus on roles fulfilled by bacterial HyHKs in plant-associated bacteria, providing recent findings on the mechanistic of their signalling properties. Recent insights into understanding additive regulatory properties fulfilled by the tethered receiver domain of HyHKs are also addressed.

  3. Direct analysis by time-of-flight secondary ion mass spectrometry reveals action of bacterial laccase-mediator systems on both hardwood and softwood samples.

    PubMed

    Goacher, Robyn E; Braham, Erick J; Michienzi, Courtney L; Flick, Robert M; Yakunin, Alexander F; Master, Emma R

    2017-12-29

    The modification and degradation of lignin play a vital role in carbon cycling as well as production of biofuels and bioproducts. The possibility of using bacterial laccases for the oxidation of lignin offers a route to utilize existing industrial protein expression techniques. However, bacterial laccases are most frequently studied on small model compounds that do not capture the complexity of lignocellulosic materials. This work studied the action of laccases from Bacillus subtilis and Salmonella typhimurium (EC 1.10.3.2) on ground wood samples from yellow birch (Betula alleghaniensis) and red spruce (Picea rubens). The ability of bacterial laccases to modify wood can be facilitated by small molecule mediators. Herein, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), gallic acid and sinapic acid mediators were tested. Direct analysis of the wood samples was achieved by time-of-flight secondary ion mass spectrometry (ToF-SIMS), a surface sensitive mass spectrometry technique that has characteristic peaks for H, G and S lignin. The action of the bacterial laccases on both wood samples was demonstrated and revealed a strong mediator influence. The ABTS mediator led to delignification, evident in an overall increase of polysaccharide peaks in the residual solid, along with equal loss of G and S-lignin peaks. The gallic acid mediator demonstrated minimal laccase activity. Meanwhile, the sinapic acid mediator altered the S/G peak ratio consistent with mediator attaching to the wood solids. The current investigation demonstrates the action of bacterial laccase-mediator systems directly on woody materials, and the potential of using ToF-SIMS to uncover the fundamental and applied role of bacterial enzymes in lignocellulose conversion. © 2017 Scandinavian Plant Physiology Society.

  4. The bacterial sealing capacity of morse taper implant-abutment systems in vitro.

    PubMed

    Ranieri, Rogerio; Ferreira, Andreia; Souza, Emmanuel; Arcoverde, Joao; Dametto, Fabio; Gade-Neto, Cicero; Seabra, Flavio; Sarmento, Carlos

    2015-05-01

    The use of Morse taper systems in dental implantology has been associated widely with a more precise adaptation between implants and their respective abutments. This may lead to an increase in the stability of the implant system and may also prevent microbial invasion through the implant-abutment interface. The aim of this study was to investigate in vitro the ability of four commercially available Morse taper system units to impede bacterial penetration through their implant-abutment interfaces. Abutments were screwed onto the implants, and the units were subsequently immersed in Streptococcus sanguinis bacterial broth (1 × 10(8) colony forming units/mL) for 48 hours. The units were examined by scanning electron microscopy (SEM) under three conditions: 1) with the implant-abutment components assembled as units to investigate for both the existence of microgaps and the presence of bacteria; 2) with the implants and abutments separated for examination of internal surfaces; and 3) with the implant-abutment components again assembled as units to measure any microgaps detected. The mean size of the microgaps in each unit was determined by measuring, under SEM, their width in four equidistant points. Microgaps were detected in all units with no significant differences in dimension (Kruskal-Wallis test, P >0.05). Within all units, the presence of bacteria was also observed. The seals provided by the interfaces of the commercially available Morse taper implant-abutment units tested were not sufficiently small to shield the implant from bacterial penetration.

  5. Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

    PubMed Central

    Yun, Thomas H.; Cott, Jessica E.; Tapping, Richard I.; Slauch, James M.

    2009-01-01

    The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. PMID:18988866

  6. Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children.

    PubMed

    Wong, Hector R; Cvijanovich, Natalie Z; Hall, Mark; Allen, Geoffrey L; Thomas, Neal J; Freishtat, Robert J; Anas, Nick; Meyer, Keith; Checchia, Paul A; Lin, Richard; Bigham, Michael T; Sen, Anita; Nowak, Jeffrey; Quasney, Michael; Henricksen, Jared W; Chopra, Arun; Banschbach, Sharon; Beckman, Eileen; Harmon, Kelli; Lahni, Patrick; Shanley, Thomas P

    2012-10-29

    Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children. Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis. Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone. Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel

  7. Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children

    PubMed Central

    2012-01-01

    Introduction Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children. Methods Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis. Results Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone. Conclusions Genome-wide expression analysis has provided the foundation

  8. Spatially Correlated Gene Expression in Bacterial Groups: The Role of Lineage History, Spatial Gradients, and Cell-Cell Interactions.

    PubMed

    van Vliet, Simon; Dal Co, Alma; Winkler, Annina R; Spriewald, Stefanie; Stecher, Bärbel; Ackermann, Martin

    2018-04-25

    Gene expression levels in clonal bacterial groups have been found to be spatially correlated. These correlations can partly be explained by the shared lineage history of nearby cells, although they could also arise from local cell-cell interactions. Here, we present a quantitative framework that allows us to disentangle the contributions of lineage history, long-range spatial gradients, and local cell-cell interactions to spatial correlations in gene expression. We study pathways involved in toxin production, SOS stress response, and metabolism in Escherichia coli microcolonies and find for all pathways that shared lineage history is the main cause of spatial correlations in gene expression levels. However, long-range spatial gradients and local cell-cell interactions also contributed to spatial correlations in SOS response, amino acid biosynthesis, and overall metabolic activity. Together, our data show that the phenotype of a cell is influenced by its lineage history and population context, raising the question of whether bacteria can arrange their activities in space to perform functions they cannot achieve alone. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. The CRISPR-Cas system - from bacterial immunity to genome engineering.

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-09-01

    Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease - Cas9 - to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

  10. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

    PubMed

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-12-01

    Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

  11. Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

    PubMed Central

    Rugh, C L; Wilde, H D; Stack, N M; Thompson, D M; Summers, A O; Meagher, R B

    1996-01-01

    With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution. Images Fig. 2 Fig. 3 Fig. 4 PMID:8622910

  12. Quantitative Investigation of the Role of Intra-/Intercellular Dynamics in Bacterial Quorum Sensing.

    PubMed

    Leaman, Eric J; Geuther, Brian Q; Behkam, Bahareh

    2018-04-20

    Bacteria utilize diffusible signals to regulate population density-dependent coordinated gene expression in a process called quorum sensing (QS). While the intracellular regulatory mechanisms of QS are well-understood, the effect of spatiotemporal changes in the population configuration on the sensitivity and robustness of the QS response remains largely unexplored. Using a microfluidic device, we quantitatively characterized the emergent behavior of a population of swimming E. coli bacteria engineered with the lux QS system and a GFP reporter. We show that the QS activation time follows a power law with respect to bacterial population density, but this trend is disrupted significantly by microscale variations in population configuration and genetic circuit noise. We then developed a computational model that integrates population dynamics with genetic circuit dynamics to enable accurate (less than 7% error) quantitation of the bacterial QS activation time. Through modeling and experimental analyses, we show that changes in spatial configuration of swimming bacteria can drastically alter the QS activation time, by up to 22%. The integrative model developed herein also enables examination of the performance robustness of synthetic circuits with respect to growth rate, circuit sensitivity, and the population's initial size and spatial structure. Our framework facilitates quantitative tuning of microbial systems performance through rational engineering of synthetic ribosomal binding sites. We have demonstrated this through modulation of QS activation time over an order of magnitude. Altogether, we conclude that predictive engineering of QS-based bacterial systems requires not only the precise temporal modulation of gene expression (intracellular dynamics) but also accounting for the spatiotemporal changes in population configuration (intercellular dynamics).

  13. cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

    PubMed Central

    Sturm, A; Chrispeels, M J

    1990-01-01

    We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose. PMID:2152110

  14. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  15. Auto-production of biosurfactants reverses the coffee ring effect in a bacterial system

    NASA Astrophysics Data System (ADS)

    Sempels, Wouter; de Dier, Raf; Mizuno, Hideaki; Hofkens, Johan; Vermant, Jan

    2013-04-01

    The deposition of material at the edge of evaporating droplets, known as the ‘coffee ring effect’, is caused by a radially outward capillary flow. This phenomenon is common to a wide array of systems including colloidal and bacterial systems. The role of surfactants in counteracting these coffee ring depositions is related to the occurrence of local vortices known as Marangoni eddies. Here we show that these swirling flows are universal, and not only lead to a uniform deposition of colloids but also occur in living bacterial systems. Experiments on Pseudomonas aeruginosa suggest that the auto-production of biosurfactants has an essential role in creating a homogeneous deposition of the bacteria upon drying. Moreover, at biologically relevant conditions, intricate time-dependent flows are observed in addition to the vortex regime, which are also effective in reversing the coffee ring effect at even lower surfactant concentrations.

  16. Bacterial Community Structure Shifted by Geosmin in Granular Activated Carbon System of Water Treatment Plants.

    PubMed

    Pham, Ngoc Dung; Lee, Eun-Hee; Chae, Seon-Ha; Cho, Yongdeok; Shin, Hyejin; Son, Ahjeong

    2016-01-01

    We investigated the relation between the presence of geosmin in water and the bacterial community structure within the granular activated carbon (GAC) system of water treatment plants in South Korea. GAC samples were collected in May and August of 2014 at three water treatment plants (Sungnam, Koyang, and Yeoncho in Korea). Dissolved organic carbon and geosmin were analyzed before and after GAC treatment. Geosmin was found in raw water from Sungnam and Koyang water treatment plants but not in that from Yeoncho water treatment plant. Interestingly, but not surprisingly, the 16S rRNA clone library indicated that the bacterial communities from the Sungnam and Koyang GAC systems were closely related to geosmin-degrading bacteria. Based on the phylogenetic tree and multidimensional scaling plot, bacterial clones from GAC under the influence of geosmin were clustered with Variovorax paradoxus strain DB 9b and Comamonas sp. DB mg. In other words, the presence of geosmin in water might have inevitably contributed to the growth of geosmin degraders within the respective GAC system.

  17. The enzymes of bacterial census and censorship

    PubMed Central

    Fast, Walter; Tipton, Peter A.

    2011-01-01

    N-Acyl-l-homoserine lactones (AHLs) are a major class of quorum sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide detailed insight into the chemistry and enzymology of bacterial communication. PMID:22099187

  18. Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.; Ott, C. M.; zuBentrup, K. Honer; Ramamurthy R.; Quick, L.; Porwollik, S.; Cheng, P.; McClellan, M.; Tsaprailis, G.; Radabaugh, T.; hide

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth.

  19. Influence of sugarbeet tillage Systems on the rhizoctonia-bacterial root rot complex

    USDA-ARS?s Scientific Manuscript database

    The Rhizoctonia-bacterial root rot complex in sugarbeet caused by Rhizoctonia solani and Leuconostoc mesenteroides can cause significant yield losses. To investigate the impact of different tillage systems on this complex, field studies were conducted from 2009 to 2011. Split blocks with conventio...

  20. A multi-phenotypic imaging screen to identify bacterial effectors by exogenous expression in a HeLa cell line.

    PubMed

    Collins, Adam; Huett, Alan

    2018-05-15

    We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 224 GFP-fused proteins from the Crohn's Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 224 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed.

  1. Phylogenetic and Functional Diversity of Total (DNA) and Expressed (RNA) Bacterial Communities in Urban Green Infrastructure Bioswale Soils.

    PubMed

    Gill, Aman S; Lee, Angela; McGuire, Krista L

    2017-08-15

    New York City (NYC) is pioneering green infrastructure with the use of bioswales and other engineered soil-based habitats to provide stormwater infiltration and other ecosystem functions. In addition to avoiding the environmental and financial costs of expanding traditional built infrastructure, green infrastructure is thought to generate cobenefits in the form of diverse ecological processes performed by its plant and microbial communities. Yet, although plant communities in these habitats are closely managed, we lack basic knowledge about how engineered ecosystems impact the distribution and functioning of soil bacteria. We sequenced amplicons of the 16S ribosomal subunit, as well as seven genes associated with functional pathways, generated from both total (DNA-based) and expressed (RNA) soil communities in the Bronx, NYC, NY, in order to test whether bioswale soils host characteristic bacterial communities with evidence for enriched microbial functioning, compared to nonengineered soils in park lawns and tree pits. Bioswales had distinct, phylogenetically diverse bacterial communities, including taxa associated with nutrient cycling and metabolism of hydrocarbons and other pollutants. Bioswale soils also had a significantly greater diversity of genes involved in several functional pathways, including carbon fixation ( cbbL-R [ cbbL gene, red-like subunit] and apsA ), nitrogen cycling ( noxZ and amoA ), and contaminant degradation ( bphA ); conversely, no functional genes were significantly more abundant in nonengineered soils. These results provide preliminary evidence that urban land management can shape the diversity and activity of soil communities, with positive consequences for genetic resources underlying valuable ecological functions, including biogeochemical cycling and degradation of common urban pollutants. IMPORTANCE Management of urban soil biodiversity by favoring taxa associated with decontamination or other microbial metabolic processes is a

  2. Phylogenetic and Functional Diversity of Total (DNA) and Expressed (RNA) Bacterial Communities in Urban Green Infrastructure Bioswale Soils

    PubMed Central

    Lee, Angela; McGuire, Krista L.

    2017-01-01

    ABSTRACT New York City (NYC) is pioneering green infrastructure with the use of bioswales and other engineered soil-based habitats to provide stormwater infiltration and other ecosystem functions. In addition to avoiding the environmental and financial costs of expanding traditional built infrastructure, green infrastructure is thought to generate cobenefits in the form of diverse ecological processes performed by its plant and microbial communities. Yet, although plant communities in these habitats are closely managed, we lack basic knowledge about how engineered ecosystems impact the distribution and functioning of soil bacteria. We sequenced amplicons of the 16S ribosomal subunit, as well as seven genes associated with functional pathways, generated from both total (DNA-based) and expressed (RNA) soil communities in the Bronx, NYC, NY, in order to test whether bioswale soils host characteristic bacterial communities with evidence for enriched microbial functioning, compared to nonengineered soils in park lawns and tree pits. Bioswales had distinct, phylogenetically diverse bacterial communities, including taxa associated with nutrient cycling and metabolism of hydrocarbons and other pollutants. Bioswale soils also had a significantly greater diversity of genes involved in several functional pathways, including carbon fixation (cbbL-R [cbbL gene, red-like subunit] and apsA), nitrogen cycling (noxZ and amoA), and contaminant degradation (bphA); conversely, no functional genes were significantly more abundant in nonengineered soils. These results provide preliminary evidence that urban land management can shape the diversity and activity of soil communities, with positive consequences for genetic resources underlying valuable ecological functions, including biogeochemical cycling and degradation of common urban pollutants. IMPORTANCE Management of urban soil biodiversity by favoring taxa associated with decontamination or other microbial metabolic processes is a

  3. Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Tanaka, Kan

    2016-10-01

    Regulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employing Vibrio fischeri luciferase to probe the intracellular metabolic condition in Escherichia coli Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells. Intracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here

  4. Role of overexpressed CFA/I fimbriae in bacterial swimming

    NASA Astrophysics Data System (ADS)

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, SangMu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  5. Role of overexpressed CFA/I fimbriae in bacterial swimming.

    PubMed

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, Sangmu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  6. ISYMOD: a knowledge warehouse for the identification, assembly and analysis of bacterial integrated systems.

    PubMed

    Chabalier, Julie; Capponi, Cécile; Quentin, Yves; Fichant, Gwennaele

    2005-04-01

    Complex biological functions emerge from interactions between proteins in stable supra-molecular assemblies and/or through transitory contacts. Most of the time protein partners of the assemblies are composed of one or several domains which exhibit different biochemical functions. Thus the study of cellular process requires the identification of different functional units and their integration in an interaction network; such complexes are referred to as integrated systems. In order to exploit with optimum efficiency the increased release of data, automated bioinformatics strategies are needed to identify, reconstruct and model such systems. For that purpose, we have developed a knowledge warehouse dedicated to the representation and acquisition of bacterial integrated systems involved in the exchange of the bacterial cell with its environment. ISYMOD is a knowledge warehouse that consistently integrates in the same environment the data and the methods used for their acquisition. This is achieved through the construction of (1) a domain knowledge base (DKB) devoted to the storage of the knowledge about the systems, their functional specificities, their partners and how they are related and (2) a methodological knowledge base (MKB) which depicts the task layout used to identify and reconstruct functional integrated systems. Instantiation of the DKB is obtained by solving the tasks of the MKB, whereas some tasks need instances of the DKB to be solved. AROM, an object-based knowledge representation system, has been used to design the DKB, and its task manager, AROMTasks, for developing the MKB. In this study two integrated systems, ABC transporters and two component systems, both involved in adaptation processes of a bacterial cell to its biotope, have been used to evaluate the feasibility of the approach.

  7. Modeling the within-host dynamics of cholera: bacterial-viral interaction.

    PubMed

    Wang, Xueying; Wang, Jin

    2017-08-01

    Novel deterministic and stochastic models are proposed in this paper for the within-host dynamics of cholera, with a focus on the bacterial-viral interaction. The deterministic model is a system of differential equations describing the interaction among the two types of vibrios and the viruses. The stochastic model is a system of Markov jump processes that is derived based on the dynamics of the deterministic model. The multitype branching process approximation is applied to estimate the extinction probability of bacteria and viruses within a human host during the early stage of the bacterial-viral infection. Accordingly, a closed-form expression is derived for the disease extinction probability, and analytic estimates are validated with numerical simulations. The local and global dynamics of the bacterial-viral interaction are analysed using the deterministic model, and the result indicates that there is a sharp disease threshold characterized by the basic reproduction number [Formula: see text]: if [Formula: see text], vibrios ingested from the environment into human body will not cause cholera infection; if [Formula: see text], vibrios will grow with increased toxicity and persist within the host, leading to human cholera. In contrast, the stochastic model indicates, more realistically, that there is always a positive probability of disease extinction within the human host.

  8. Development of a tape transport bacterial detection system

    NASA Technical Reports Server (NTRS)

    Witz, S.; Hartung, W. H.

    1972-01-01

    The feasibility of a tape transport chemiluminescence system for bacterial monitoring of regenerated water was demonstrated using a manually operated laboratory breadboard. The principle of detection is based on measuring the increase in chemiluminescence produced by the catalytic action of bacterial porphyrins on a luminol-hydrogen peroxide mixture. Viable organisms are distinguished from nonviable by comparing the signals of incubated and unincubated water samples. Using optimized protocols, sensitivities were obtained with 400 ml suspensions of E. coli and Cl. sporogenes. The sensitivity of the unincubated cycle E. coli (aerobe) was found to be 30 to 35 cells/m1, and that of the Cl. sporogenes (anaerobe) was 1000 to 10,000 cells/m1. The lower sensitivity toward Cl. sporogenes is attributed to several factors, namely the lower cytochrome content, the tendency to sporulate, long lag periods and the lower growth rate of Clostridia in general. The operational procedures used for processing the incubated and unincubated samples involved the following sequence: (1) concentrating the sample by filtration through a membrane filter, (2) washing with Dextrose-Thioglycollate Broth (3) incubating (0 to 4 hrs as required), (4) washing with 4M Urea, and (5) reacting with reagent in front of a photomultiplier tube. The signal output was recorded on a strip chart recorder.

  9. Identification of a two-component Class IIb bacteriocin in Streptococcus pyogenes by recombinase-based in vivo expression technology.

    PubMed

    Armstrong, Brent D; Herfst, Christine A; Tonial, Nicholas C; Wakabayashi, Adrienne T; Zeppa, Joseph J; McCormick, John K

    2016-11-03

    Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens.

  10. Identification of a two-component Class IIb bacteriocin in Streptococcus pyogenes by recombinase-based in vivo expression technology

    PubMed Central

    Armstrong, Brent D.; Herfst, Christine A.; Tonial, Nicholas C.; Wakabayashi, Adrienne T.; Zeppa, Joseph J.; McCormick, John K.

    2016-01-01

    Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens. PMID:27808235

  11. Seasonal Changes in Bacterial and Archaeal Gene Expression Patterns across Salinity Gradients in the Columbia River Coastal Margin

    PubMed Central

    Smith, Maria W.; Herfort, Lydie; Tyrol, Kaitlin; Suciu, Dominic; Campbell, Victoria; Crump, Byron C.; Peterson, Tawnya D.; Zuber, Peter; Baptista, Antonio M.; Simon, Holly M.

    2010-01-01

    Through their metabolic activities, microbial populations mediate the impact of high gradient regions on ecological function and productivity of the highly dynamic Columbia River coastal margin (CRCM). A 2226-probe oligonucleotide DNA microarray was developed to investigate expression patterns for microbial genes involved in nitrogen and carbon metabolism in the CRCM. Initial experiments with the environmental microarrays were directed toward validation of the platform and yielded high reproducibility in multiple tests. Bioinformatic and experimental validation also indicated that >85% of the microarray probes were specific for their corresponding target genes and for a few homologs within the same microbial family. The validated probe set was used to query gene expression responses by microbial assemblages to environmental variability. Sixty-four samples from the river, estuary, plume, and adjacent ocean were collected in different seasons and analyzed to correlate the measured variability in chemical, physical and biological water parameters to differences in global gene expression profiles. The method produced robust seasonal profiles corresponding to pre-freshet spring (April) and late summer (August). Overall relative gene expression was high in both seasons and was consistent with high microbial abundance measured by total RNA, heterotrophic bacterial production, and chlorophyll a. Both seasonal patterns involved large numbers of genes that were highly expressed relative to background, yet each produced very different gene expression profiles. April patterns revealed high differential gene expression in the coastal margin samples (estuary, plume and adjacent ocean) relative to freshwater, while little differential gene expression was observed along the river-to-ocean transition in August. Microbial gene expression profiles appeared to relate, in part, to seasonal differences in nutrient availability and potential resource competition. Furthermore, our results

  12. Plasma bacterial and mitochondrial DNA distinguish bacterial sepsis from sterile systemic inflammatory response syndrome and quantify inflammatory tissue injury in nonhuman primates.

    PubMed

    Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

    2013-01-01

    Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. Systemic inflammatory response syndrome can cause organ dysfunction and death, but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS, and postinfective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern reflecting cellular injury. Circulating bacterial 16S DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed quantitative polymerase chain reaction assays to quantify these markers, and predicting their plasma levels might help distinguish sterile injury from infection. To study these events in primates, we assayed banked serum from Papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis delta Sterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to "severe" sepsis and death, we studied animals where anthrax was pretreated with drotrecogin alfa (activated protein C), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against nonlethal E. coli bacteremia and against sterile tissue injury from Shiga-like toxin 1. Bacterial DNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Shiga-like toxin 1 increased mtDNA, but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sublethal E. coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal

  13. Biofilm bacterial communities in urban drinking water distribution systems transporting waters with different purification strategies.

    PubMed

    Wu, Huiting; Zhang, Jingxu; Mi, Zilong; Xie, Shuguang; Chen, Chao; Zhang, Xiaojian

    2015-02-01

    Biofilm formation in drinking water distribution systems (DWDS) has many adverse consequences. Knowledge of microbial community structure of DWDS biofilm can aid in the design of an effective control strategy. However, biofilm bacterial community in real DWDS and the impact of drinking water purification strategy remain unclear. The present study investigated the composition and diversity of biofilm bacterial community in real DWDSs transporting waters with different purification strategies (conventional treatment and integrated treatment). High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the diversity and structure of biofilm bacterial community in real DWDS. Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Nitrospirae, and Cyanobacteria were the major components of biofilm bacterial community. Proteobacteria (mainly Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) predominated in each DWDS biofilm, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples. Drinking water purification strategy could shape DWDS biofilm bacterial community. Moreover, Pearson's correlation analysis indicated that Actinobacteria was positively correlated with the levels of total alkalinity and dissolved organic carbon in tap water, while Firmicutes had a significant positive correlation with nitrite nitrogen.

  14. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chen, Hong-Chen; Chao, Yun-Peng; Tzen, Jason T C

    2005-06-15

    Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

  15. Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems

    PubMed Central

    Gomaa, Ahmed A.; Klumpe, Heidi E.; Luo, Michelle L.; Selle, Kurt; Barrangou, Rodolphe; Beisel, Chase L.

    2014-01-01

    ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of “smart” antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. PMID:24473129

  16. Profiling bacterial communities associated with sediment-based aquaculture bioremediation systems under contrasting redox regimes

    NASA Astrophysics Data System (ADS)

    Robinson, Georgina; Caldwell, Gary S.; Wade, Matthew J.; Free, Andrew; Jones, Clifford L. W.; Stead, Selina M.

    2016-12-01

    Deposit-feeding invertebrates are proposed bioremediators in microbial-driven sediment-based aquaculture effluent treatment systems. We elucidate the role of the sediment reduction-oxidation (redox) regime in structuring benthic bacterial communities, having direct implications for bioremediation potential and deposit-feeder nutrition. The sea cucumber Holothuria scabra was cultured on sediments under contrasting redox regimes; fully oxygenated (oxic) and redox stratified (oxic-anoxic). Taxonomically, metabolically and functionally distinct bacterial communities developed between the redox treatments with the oxic treatment supporting the greater diversity; redox regime and dissolved oxygen levels were the main environmental drivers. Oxic sediments were colonised by nitrifying bacteria with the potential to remediate nitrogenous wastes. Percolation of oxygenated water prevented the proliferation of anaerobic sulphate-reducing bacteria, which were prevalent in the oxic-anoxic sediments. At the predictive functional level, bacteria within the oxic treatment were enriched with genes associated with xenobiotics metabolism. Oxic sediments showed the greater bioremediation potential; however, the oxic-anoxic sediments supported a greater sea cucumber biomass. Overall, the results indicate that bacterial communities present in fully oxic sediments may enhance the metabolic capacity and bioremediation potential of deposit-feeder microbial systems. This study highlights the benefits of incorporating deposit-feeding invertebrates into effluent treatment systems, particularly when the sediment is oxygenated.

  17. Profiling bacterial communities associated with sediment-based aquaculture bioremediation systems under contrasting redox regimes

    PubMed Central

    Robinson, Georgina; Caldwell, Gary S.; Wade, Matthew J.; Free, Andrew; Jones, Clifford L. W.; Stead, Selina M.

    2016-01-01

    Deposit-feeding invertebrates are proposed bioremediators in microbial-driven sediment-based aquaculture effluent treatment systems. We elucidate the role of the sediment reduction-oxidation (redox) regime in structuring benthic bacterial communities, having direct implications for bioremediation potential and deposit-feeder nutrition. The sea cucumber Holothuria scabra was cultured on sediments under contrasting redox regimes; fully oxygenated (oxic) and redox stratified (oxic-anoxic). Taxonomically, metabolically and functionally distinct bacterial communities developed between the redox treatments with the oxic treatment supporting the greater diversity; redox regime and dissolved oxygen levels were the main environmental drivers. Oxic sediments were colonised by nitrifying bacteria with the potential to remediate nitrogenous wastes. Percolation of oxygenated water prevented the proliferation of anaerobic sulphate-reducing bacteria, which were prevalent in the oxic-anoxic sediments. At the predictive functional level, bacteria within the oxic treatment were enriched with genes associated with xenobiotics metabolism. Oxic sediments showed the greater bioremediation potential; however, the oxic-anoxic sediments supported a greater sea cucumber biomass. Overall, the results indicate that bacterial communities present in fully oxic sediments may enhance the metabolic capacity and bioremediation potential of deposit-feeder microbial systems. This study highlights the benefits of incorporating deposit-feeding invertebrates into effluent treatment systems, particularly when the sediment is oxygenated. PMID:27941918

  18. Administration of capsule-selective endosialidase E minimizes upregulation of organ gene expression induced by experimental systemic infection with Escherichia coli K1.

    PubMed

    Zelmer, Andrea; Martin, Melissa J; Gundogdu, Ozan; Birchenough, George; Lever, Rebecca; Wren, Brendan W; Luzio, J Paul; Taylor, Peter W

    2010-07-01

    Many neurotropic strains of Escherichia coli cause potentially lethal bacteraemia and meningitis in newborn infants by virtue of their capacity to elaborate the protective polysialic acid (polySia) K1 capsule. Recombinant capsule depolymerase, endosialidase E (endoE), selectively removes polySia from the bacterial surface; when administered intraperitoneally to infected neonatal rats, the enzyme interrupts the transit of E. coli K1 from gut to brain via the blood circulation and prevents death from systemic infection. We now show that experimental E. coli K1 infection is accompanied by extensive modulation of host gene expression in the liver, spleen and brain tissues of neonatal rats. Bacterial invasion of the brain resulted in a threefold or greater upregulation of approximately 400 genes, a large number of which were associated with the induction of inflammation and the immune and stress responses: these included genes encoding C-X-C and C-C chemokines, lipocalins, cytokines, apolipoproteins and enzymes involved in the synthesis of low-molecular-mass inflammatory mediators. Administration of a single dose of endoE, 24 h after initiation of systemic infection, markedly reduced, but did not completely abrogate, these changes in gene expression, suggesting that attenuation of E. coli K1 virulence by removal of the polySia capsule may minimize the attendant inflammatory processes that contribute to poor outcome in these severe systemic infections.

  19. Bacterial 'immunity' against bacteriophages.

    PubMed

    Abedon, Stephen T

    2012-01-01

    Vertebrate animals possess multiple anti-pathogen defenses. Individual mechanisms usually are differentiated into those that are immunologically adaptive vs. more "primitive" anti-pathogen phenomena described as innate responses. Here I frame defenses used by bacteria against bacteriophages as analogous to these animal immune functions. Included are numerous anti-phage defenses in addition to the adaptive immunity associated with CRISPR/cas systems. As these other anti-pathogen mechanisms are non-adaptive they can be described as making up an innate bacterial immunity. This exercise was undertaken in light of the recent excitement over the discovery that CRISPR/cas systems can serve, as noted, as a form of bacterial adaptive immunity. The broader goal, however, is to gain novel insight into bacterial defenses against phages by fitting these mechanisms into considerations of how multicellular organisms also defend themselves against pathogens. This commentary can be viewed in addition as a bid toward integrating these numerous bacterial anti-phage defenses into a more unified immunology.

  20. Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles

    PubMed Central

    Caswell, Clayton C.; Oglesby-Sherrouse, Amanda G.; Murphy, Erin R.

    2014-01-01

    Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators. PMID:25389522

  1. Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

    PubMed

    Caswell, Clayton C; Oglesby-Sherrouse, Amanda G; Murphy, Erin R

    2014-01-01

    Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.

  2. Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein.

    PubMed

    Zhang, J Z; Ng, M H; Xia, N S; Lau, S H; Che, X Y; Chau, T N; Lai, S T; Im, S W

    2001-06-01

    A 23 kDa peptide locating to amino acid residues 394 to 604 of the major Hepatitis E Virus (HEV) structural protein was expressed in E. coli. This peptide was found to interact naturally with one another to form homodimers and it was recognized strongly and commonly in its dimeric form by HEV reactive human sera. The antigenic activity associated with the dimeric form was abrogated when the dimer was dissociated into monomer and the activity was reconstituted after the monomer was re-associated into dimer again. The dimeric form of the peptide elicited a vigorous antibody response in experimental animals and the resulting antisera were found to cross-react against HEV, effecting an efficient immune capture of the virus. These results attributed the antigenic activity associated with the dimeric form of the peptide to conformational antigenic determinants generated as a result of interaction between the peptide molecules. It is suggested that some of these antigenic determinants may be expressed by the HEV capsid and raised the possibility of this bacterially expressed peptide as an HEV vaccine candidate. Copyright 2001 Wiley-Liss, Inc.

  3. Cellulases for biomass degradation: comparing recombinant cellulase expression platforms.

    PubMed

    Garvey, Megan; Klose, Holger; Fischer, Rainer; Lambertz, Camilla; Commandeur, Ulrich

    2013-10-01

    Improvement of cellulase expression has the potential to change the nature of the biofuel industry. Increasing the economic feasibility of cellulase systems would significantly broaden the range of practicable biomass conversion, lowering the environmental impact of our civilisations' fuel needs. Cellulases are derived from certain fungi and bacteria, which are often difficult to culture on an industrial scale. Accordingly, methods to recombinantly express important cellulases and other glycosyl hydrolase (GH) enzymes are under serious investigation. Herein, we examine the latest developments in bacterial, yeast, plant, and fungal expression systems. We discuss current strategies for producing cellulases, and evaluate the benefits and drawbacks in yield, stability, and activity of enzymes from each system, and the overall progress in the field. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1987-05-01

    Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased themore » amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.« less

  5. Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

    PubMed Central

    Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.

    2013-01-01

    Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102

  6. Bacterial DNA indicated as an important inducer of fish cathelicidins.

    PubMed

    Maier, Valerie Helene; Schmitt, Clemens Nikolaus Zeno; Gudmundsdottir, Sigridur; Gudmundsson, Gudmundur Hrafn

    2008-04-01

    Cathelicidins are antimicrobial peptides indicated as important in the control of the natural microflora as well as in the fight against bacterial invasion in mammals. Little is known about cathelicidins in fish and here the Chinook salmon (Oncorhynchus tshawytscha) embryo cell line (CHSE-214) was used as a model system to study the expression of cathelicidins due to fish pathogenic bacteria. The cDNA of cathelicidin from CHSE-214 cells (csCath) was cloned and shown to be closely related to gene 2 of both rainbow trout and Atlantic salmon. The deducted amino acid sequence showed highest sequence identity to rtCath2 with 95% and 72% for the cathelin and the antibacterial part, respectively. Cathelicidin gene expression was studied and various Gram positive and Gram negative bacteria caused the upregulation of the gene (csCath). Bacterial DNA and protein were shown important for the induction of cathelicidin expression in these cells. LPS (Escherichia coli) also causes the upregulation of cathelicidins, but digestion of the LPS with DNase I before incubation of the cells, totally abolished the upregulation of cathelicidin and suggests DNA contamination in the LPS to be the trigger for this effect. These results could explain the limited responsiveness of fish cells towards pure LPS and confirm previous suggestions that fish cells are less sensitive to LPS than mammalian cells.

  7. A facile approach to construct versatile signal amplification system for bacterial detection.

    PubMed

    Qi, Peng; Zhang, Dun; Wan, Yi; Lv, Dandan

    2014-01-01

    In this work, a facile approach to design versatile signal amplification system for bacterial detection has been presented. Bio-recognition elements and signaling molecules can be immobilized on the surface of Fe₃O₄@MnO₂ nanomaterials with the help of bioinspired polydopamine (PDA). Fe₃O₄@MnO₂ nanoplates were chosen as carrier for bio-recognizing and signaling molecules because this kind of nanomaterial was superparamagnetic and the existence of MnO₂ could enhance the polymerization of dopamine due to its strong oxidative ability. This nanocomposite system was versatile because PDA around Fe₃O₄@MnO₂ nanoplates provided a stable and convenient platform for immobilization of biological and chemical materials, and various kinds of bio-recognizing and signaling molecules could be immobilized by reaction with pendant amino groups of dopamine to meet different detection requirements. Since a substantial amount of signaling molecules were immobilized on the surface of the nanocomposites, so the sensitivity of detection would be improved when the prepared nanocomposites were selectively conjugated with target pathogen. In the experimental section, a sandwich-type electrochemical biosensor was developed to verify the amplified bacterial detection sensitivity. Concanavalin A (conA) and ferrocene (Fc) were chosen as bio-recognition elements and signaling molecules for detection of Desulforibrio caledoiensis, respectively. The conA and Fc modified nanocomposites were conjugated on electrode by the selective recognition between conA and target bacteria, and the bacterial population was obtained by quantification of the electrochemical signal of Fc moieties. The experimental results showed that the detection sensitivity for D. caledoiensis was improved by taking advantage of this signal amplification system. © 2013 Elsevier B.V. All rights reserved.

  8. [Autochthonous acute viral and bacterial infections of the central nervous system (meningitis and encephalitis)].

    PubMed

    Pérez-Ruiz, Mercedes; Vicente, Diego; Navarro-Marí, José María

    2008-07-01

    Rapid diagnosis of acute viral and bacterial infections of the central nervous system (meningitis and encephalitis) is highly important for the clinical management of the patient and helps to establish early therapy that may solve life-threatening situations, to avoid unnecessary empirical treatments, to reduce hospital stay, and to facilitate appropriate interventions in the context of public health. Molecular techniques, especially real-time polymerase chain reaction, have become the fastest and most sensitive diagnostic procedures for autochthonous viral meningitis and encephalitis, and their role is becoming increasingly important for the diagnosis and control of most frequent acute bacterial meningitides. Automatic and closed systems may encourage the widespread and systematic use of molecular techniques for the diagnosis of these neurological syndromes in most laboratories.

  9. Bacterial communities in the collection and chlorinated distribution sections of a drinking water system in Budapest, Hungary.

    PubMed

    Homonnay, Zalán G; Török, György; Makk, Judit; Brumbauer, Anikó; Major, Eva; Márialigeti, Károly; Tóth, Erika

    2014-07-01

    Bacterial communities of a bank-filtered drinking water system were investigated by aerobic cultivation and clone library analysis. Moreover, bacterial communities were compared using sequence-aided terminal restriction fragment length polymorphism (T-RFLP) fingerprinting at ten characteristic points located at both the collecting and the distributing part of the water supply system. Chemical characteristics of the samples were similar, except for the presence of chlorine residuals in the distribution system and increased total iron concentration in two of the samples. Assimilable organic carbon (AOC) concentration increased within the collection system, it was reduced by chlorination and it increased again in the distribution system. Neither fecal indicators nor pathogens were detected by standard cultivation techniques. Chlorination reduced bacterial diversity and heterotrophic plate counts. Community structures were found to be significantly different before and after chlorination: the diverse communities in wells and the collection system were dominated by chemolithotrophic (e.g., Gallionella and Nitrospira) and oligocarbophilic-heterotrophic bacteria (e.g., Sphingomonas, Sphingopyxis, and Bradyrhizobium). After chlorination in the distribution system, the most characteristic bacterium was related to the facultative methylotrophic Methylocella spp. Communities changed within the distribution system too, Mycobacterium spp. or Sphingopyxis spp. became predominant in certain samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Inhibition of intracellular bacterial replication in fibroblasts is dependent on the perforin-like protein (Perforin-2) encoded by macrophage expressed gene 1

    PubMed Central

    McCormack, Ryan; de Armas, Lesley R.; Shiratsuchi, Motoaki; Ramos, Jay; Podack, Eckhard R.

    2013-01-01

    Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage expressed gene 1 (mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knock-down of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with an RFP-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria including Gram positive, Gram negative and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEFs damage cell walls of intracellular bacteria by insertion, polymerization and pore-formation of the perforin-like protein, analogous to pore-formers of complement and Perforin-1 of cytolytic lymphocytes. We propose the name Perforin-2. PMID:23257510

  11. The enzymes of bacterial census and censorship.

    PubMed

    Fast, Walter; Tipton, Peter A

    2012-01-01

    N-Acyl-L-homoserine lactones (AHLs) are a major class of quorum-sensing signals used by Gram-negative bacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior. Enzymes capable of generating and catabolizing AHL signals are of significant interest for the study of microbial ecology and quorum-sensing pathways, for understanding the systems that bacteria have evolved to interact with small-molecule signals, and for their possible use in therapeutic and industrial applications. The recent structural and functional studies reviewed here provide a detailed insight into the chemistry and enzymology of bacterial communication. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

    PubMed

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

  13. Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm

    NASA Astrophysics Data System (ADS)

    Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

    2016-08-01

    There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (Dtot) and the displacement distribution functions (P (r ,t ) ) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ , which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ =0.65 , while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ . We also investigate the distribution (P (θ ,t ) ) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

  14. Distribution and diversity of bacterial secretion systems across metagenomic datasets.

    PubMed

    Barret, Matthieu; Egan, Frank; O'Gara, Fergal

    2013-02-01

    Bacteria can manipulate their surrounding environment through the secretion of proteins into other living organisms and into the extracellular milieu. In Gram stain negative bacteria this process is mediated by different types of secretion systems from type I through type VI secretion system (T1SS-T6SS). In this study the prevalence of these secretion systems in 312 publicly available microbiomes derived from a wide range of ecosystems was investigated by a gene-centric approach. Our analysis demonstrates that some secretion systems are over-represented in some specific samples. In addition, some T3SS and T6SS phylogenetic clusters were specifically enriched in particular ecological niches, which could indicate specific bacterial adaptation to these environments. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  15. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi

    2015-01-01

    High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for

  16. Characterization of bacterial coliform occurrences in different zones of a drinking water distribution system.

    PubMed

    Blanch, A R; Galofré, B; Lucena, F; Terradillos, A; Vilanova, X; Ribas, F

    2007-03-01

    To compare the bacterial coliforms detected from occurrences in three zones of a water distribution system supplied by two separate water sources. Conventional and standardized protocols for identifying enterobacterial populations were applied. Additional tests to confirm isolates were included. Analyses of diversity and population similarity were performed using the Phene Plate System, a miniaturized biochemical phenotyping method. Isolates were identified by the API 20E system in tandem with biochemical phenotyping. A total of 16 576 samples were taken from the water distribution system, with 1416 isolates analysed. A low number of coliform occurrences were observed (2%). Escherichia coli was not detected in either water origin or in Zone 2 samples; however, in Zones 1 and 3 a low number of cases of E. coli were recorded. The percentages of E. coli depended on the identification criteria. Eight biochemical profiles for coliform populations were defined according to the results of the confirmative tests. There was a high diversity among these populations in the three zones studied, although no significant variations in their composition (associated with occurrences in the different zones) were observed. Klebsiella oxytoca was the most commonly detected species irrespective of zone, although seven other enterobacterial genera were also found. Analysis of the enzymatic activity of beta-glucuronidase or application of the criteria established in the norm ISO 9308-1, in tandem with thermotolerance was needed to evaluate the occurrence of E. coli in the distribution systems. Detected occurrences of bacterial coliforms could be associated with re-growth patterns for specific sampling points in the distribution system. Seasonal differences, independent of the studied zones, were observed. Biochemical phenotyping of bacterial coliforms was shown to be a useful method on the characterization of occurrences in water distribution systems.

  17. Shrimp alpha-2-macroglobulin prevents the bacterial escape by inhibiting fibrinolysis of blood clots.

    PubMed

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

    2012-01-01

    Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis.

  18. Bacterial Production of Site Specific 13C Labeled Phenylalanine and Methodology for High Level Incorporation into Bacterially Expressed Recombinant Proteins

    PubMed Central

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L.

    2016-01-01

    Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study. PMID:28028744

  19. Influence of tillage systems on Rhizoctonia-bacterial root rot complex in sugar beet

    USDA-ARS?s Scientific Manuscript database

    The Rhizoctonia-bacterial root rot complex on sugarbeet caused by Rhizoctonia solani and Leuconostoc mesenteroides can cause significant yield losses. To investigate the impact of different tillage systems on this complex, field studies were conducted from 2009 to 2011. Split blocks with conventio...

  20. Synthetic analogs of bacterial quorum sensors

    DOEpatents

    Iyer, Rashi [Los Alamos, NM; Ganguly, Kumkum [Los Alamos, NM; Silks, Louis A [Los Alamos, NM

    2011-12-06

    Bacterial quorum-sensing molecule analogs having the following structures: ##STR00001## and methods of reducing bacterial pathogenicity, comprising providing a biological system comprising pathogenic bacteria which produce natural quorum-sensing molecule; providing a synthetic bacterial quorum-sensing molecule having the above structures and introducing the synthetic quorum-sensing molecule into the biological system comprising pathogenic bacteria. Further is provided a method of targeted delivery of an antibiotic, comprising providing a synthetic quorum-sensing molecule; chemically linking the synthetic quorum-sensing molecule to an antibiotic to produce a quorum-sensing molecule-antibiotic conjugate; and introducing the conjugate into a biological system comprising pathogenic bacteria susceptible to the antibiotic.

  1. Synthetic analogs of bacterial quorum sensors

    DOEpatents

    Iyer, Rashi S.; Ganguly, Kumkum; Silks, Louis A.

    2013-01-08

    Bacterial quorum-sensing molecule analogs having the following structures: ##STR00001## and methods of reducing bacterial pathogenicity, comprising providing a biological system comprising pathogenic bacteria which produce natural quorum-sensing molecule; providing a synthetic bacterial quorum-sensing molecule having the above structures and introducing the synthetic quorum-sensing molecule into the biological system comprising pathogenic bacteria. Further is provided a method of targeted delivery of an antibiotic, comprising providing a synthetic quorum-sensing molecule; chemically linking the synthetic quorum-sensing molecule to an antibiotic to produce a quorum-sensing molecule-antibiotic conjugate; and introducing the conjugate into a biological system comprising pathogenic bacteria susceptible to the antibiotic.

  2. Physical stress and bacterial colonization

    PubMed Central

    Otto, Michael

    2014-01-01

    Bacterial surface colonizers are subject to a variety of physical stresses. During the colonization of human epithelia such as on the skin or the intestinal mucosa, bacteria mainly have to withstand the mechanical stress of being removed by fluid flow, scraping, or epithelial turnover. To that end, they express a series of molecules to establish firm attachment to the epithelial surface, such as fibrillar protrusions (pili) and surface-anchored proteins that bind to human matrix proteins. In addition, some bacteria – in particular gut and urinary tract pathogens – use internalization by epithelial cells and other methods such as directed inhibition of epithelial turnover to ascertain continued association with the epithelial layer. Furthermore, many bacteria produce multi-layered agglomerations called biofilms with a sticky extracellular matrix, providing additional protection from removal. This review will give an overview over the mechanisms human bacterial colonizers have to withstand physical stresses with a focus on bacterial adhesion. PMID:25212723

  3. Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection[OPEN

    PubMed Central

    Henry, Elizabeth; Jauneau, Alain; Deslandes, Laurent

    2017-01-01

    To cause disease, diverse pathogens deliver effector proteins into host cells. Pathogen effectors can inhibit defense responses, alter host physiology, and represent important cellular probes to investigate plant biology. However, effector function and localization have primarily been investigated after overexpression in planta. Visualizing effector delivery during infection is challenging due to the plant cell wall, autofluorescence, and low effector abundance. Here, we used a GFP strand system to directly visualize bacterial effectors delivered into plant cells through the type III secretion system. GFP is a beta barrel that can be divided into 11 strands. We generated transgenic Arabidopsis thaliana plants expressing GFP1-10 (strands 1 to 10). Multiple bacterial effectors tagged with the complementary strand 11 epitope retained their biological function in Arabidopsis and tomato (Solanum lycopersicum). Infection of plants expressing GFP1-10 with bacteria delivering GFP11-tagged effectors enabled direct effector detection in planta. We investigated the temporal and spatial delivery of GFP11-tagged effectors during infection with the foliar pathogen Pseudomonas syringae and the vascular pathogen Ralstonia solanacearum. Thus, the GFP strand system can be broadly used to investigate effector biology in planta. PMID:28600390

  4. Flow Chamber System for the Statistical Evaluation of Bacterial Colonization on Materials

    PubMed Central

    Menzel, Friederike; Conradi, Bianca; Rodenacker, Karsten; Gorbushina, Anna A.; Schwibbert, Karin

    2016-01-01

    Biofilm formation on materials leads to high costs in industrial processes, as well as in medical applications. This fact has stimulated interest in the development of new materials with improved surfaces to reduce bacterial colonization. Standardized tests relying on statistical evidence are indispensable to evaluate the quality and safety of these new materials. We describe here a flow chamber system for biofilm cultivation under controlled conditions with a total capacity for testing up to 32 samples in parallel. In order to quantify the surface colonization, bacterial cells were DAPI (4`,6-diamidino-2-phenylindole)-stained and examined with epifluorescence microscopy. More than 100 images of each sample were automatically taken and the surface coverage was estimated using the free open source software g’mic, followed by a precise statistical evaluation. Overview images of all gathered pictures were generated to dissect the colonization characteristics of the selected model organism Escherichia coli W3310 on different materials (glass and implant steel). With our approach, differences in bacterial colonization on different materials can be quantified in a statistically validated manner. This reliable test procedure will support the design of improved materials for medical, industrial, and environmental (subaquatic or subaerial) applications. PMID:28773891

  5. Bench-to-bedside review: Quorum sensing and the role of cell-to-cell communication during invasive bacterial infection

    PubMed Central

    Asad, Shadaba; Opal, Steven M

    2008-01-01

    Bacteria communicate extensively with each other and employ a communal approach to facilitate survival in hostile environments. A hierarchy of cell-to-cell signaling pathways regulates bacterial growth, metabolism, biofilm formation, virulence expression, and a myriad of other essential functions in bacterial populations. The notion that bacteria can signal each other and coordinate their assault patterns against susceptible hosts is now well established. These signaling networks represent a previously unrecognized survival strategy by which bacterial pathogens evade antimicrobial defenses and overwhelm the host. These quorum sensing communication signals can transgress species barriers and even kingdom barriers. Quorum sensing molecules can regulate human transcriptional programs to the advantage of the pathogen. Human stress hormones and cytokines can be detected by bacterial quorum sensing systems. By this mechanism, the pathogen can detect the physiologically stressed host, providing an opportunity to invade when the patient is most vulnerable. These rather sophisticated, microbial communication systems may prove to be a liability to pathogens as they make convenient targets for therapeutic intervention in our continuing struggle to control microbial pathogens. PMID:19040778

  6. Bacterial Modulation of Plant Ethylene Levels

    PubMed Central

    Gamalero, Elisa; Glick, Bernard R.

    2015-01-01

    A focus on the mechanisms by which ACC deaminase-containing bacteria facilitate plant growth.Bacteria that produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, when present either on the surface of plant roots (rhizospheric) or within plant tissues (endophytic), play an active role in modulating ethylene levels in plants. This enzyme activity facilitates plant growth especially in the presence of various environmental stresses. Thus, plant growth-promoting bacteria that express ACC deaminase activity protect plants from growth inhibition by flooding and anoxia, drought, high salt, the presence of fungal and bacterial pathogens, nematodes, and the presence of metals and organic contaminants. Bacteria that express ACC deaminase activity also decrease the rate of flower wilting, promote the rooting of cuttings, and facilitate the nodulation of legumes. Here, the mechanisms behind bacterial ACC deaminase facilitation of plant growth and development are discussed, and numerous examples of the use of bacteria with this activity are summarized. PMID:25897004

  7. Diagnosis of bacterial vaginosis by wet mount identification of bacterial morphotypes in vaginal fluid.

    PubMed

    Schmidt, H; Hansen, J G

    2000-03-01

    In order to develop a more practical way of diagnosing bacterial vaginosis (BV), we evaluated a scoring system, weighting small bacterial morphotypes versus lactobacillary morphotypes in wet mounts, assessed criteria for BV and normalcy from this scoring, and then evaluated their reproducibility and accuracy. We examined 754 women for pH, homogeneous vaginal discharge, amine odour, clue cells and the composite clinical diagnosis. We also examined wet mounts for small bacterial morphotypes and lactobacillary morphotypes, and weighted their quantitative presence as a bacterial morphotype score. The term 'small bacterial morphotypes' denotes a group of small bacillary forms comprising coccobacilli, tiny rods, and mobile curved rods. The different characteristics of BV were all gradually associated with increased bacterial morphotype scoring. We deemed a score of 0-1 as normal, 2-4 as intermediate phase, grade I, 5-6 as intermediate phase, grade II, and 7-8 indicative of BV. Reproducibility of the interpretation was high, both for the new grading system (weighted Kappa 0.90 in women perceiving and 0.81 in women not perceiving abnormal vaginal discharge) and for the new criterion for BV (non-weighted Kappa 0.91 and 0.84 in the 2 groups of women). The new criterion also proved highly concurrent with the composite clinical diagnosis (Kappa 0.91 and 0.81 in the 2 groups). In conclusion, the wet mount bacterial morphotype scoring is valid for grading of the disorder of the vaginal microbial ecosystem, and the new criterion for BV a more practical option than existing diagnostic methods.

  8. Effect of cell-surface hydrophobicity on bacterial conversion of water-immiscible chemicals in two-liquid-phase culture systems.

    PubMed

    Hamada, Takahiro; Maeda, Yusuke; Matsuda, Hiroyuki; Sameshima, Yuka; Honda, Kohsuke; Omasa, Takeshi; Kato, Junichi; Ohtake, Hisao

    2009-08-01

    The effect of bacterial cell-surface hydrophobicity on the bioconversion of water-immiscible chemicals in an aqueous-organic (A/O) two-liquid-phase culture system was investigated. Escherichia coli JM109 and Rhodococcus opacus B-4 were used as hydrophilic and hydrophobic whole-cell catalysts, respectively. Hydroxylation reactions of monoaromatics, including toluene (log P(ow)=2.9), ethylbenzene (3.1), n-propylbenzene (3.4), and sec-butylbenzene (3.7), were employed as model conversions. When the todC1C2BA genes encoding Pseudomonas putida toluene dioxygenase were expressed in E. coli JM109, the yield of hydroxylated monoaromatics decreased with increasing substrate hydrophobicity. By contrast, R. opacus transformants, which expressed the todC1C2BA genes, showed high performance in the hydroxylation of monoaromatics, irrespective of substrate hydrophobicity. When the R. opacus transformants were examined for their ability to hydroxylate monoaromatics in an aqueous single-liquid-phase culture, the reaction velocity was markedly lower than that observed in the A/O two-liquid-phase culture. These results suggested that R. opacus B-4 accessed the hydrophobic substrates in the oil phase, thus making it more effective for the bioconversion reactions.

  9. Changes in diversity, abundance, and structure of soil bacterial communities in Brazilian Savanna under different land use systems.

    PubMed

    Rampelotto, Pabulo Henrique; de Siqueira Ferreira, Adão; Barboza, Anthony Diego Muller; Roesch, Luiz Fernando Wurdig

    2013-10-01

    The Brazilian Savanna, also known as "Cerrado", is the richest and most diverse savanna in the world and has been ranked as one of the main hotspots of biodiversity. The Cerrado is a representative biome in Central Brazil and the second largest biome in species diversity of South America. Nevertheless, large areas of native vegetation have been converted to agricultural land including grain production, livestock, and forestry. In this view, understanding how land use affects microbial communities is fundamental for the sustainable management of agricultural ecosystems. The aim of this work was to analyze and compare the soil bacterial communities from the Brazilian Cerrado associated with different land use systems using high throughput pyrosequencing of 16S rRNA genes. Relevant differences were observed in the abundance and structure of bacterial communities in soils under different land use systems. On the other hand, the diversity of bacterial communities was not relevantly changed among the sites studied. Land use systems had also an important impact on specific bacterial groups in soil, which might change the soil function and the ecological processes. Acidobacteria, Proteobacteria, and Actinobacteria were the most abundant groups in the Brazilian Cerrado. These findings suggest that more important than analyzing the general diversity is to analyze the composition of the communities. Since soil type was the same among the sites, we might assume that land use was the main factor defining the abundance and structure of bacterial communities.

  10. Bacterial Associations with Diatoms Influence Host Health in a Xenic Model System

    NASA Astrophysics Data System (ADS)

    Baker, L.; Kemp, P. F.

    2016-02-01

    Diatoms are photosynthetic unicellular eukaryotes found ubiquitously in aquatic systems. Microorganisms such as bacteria are frequently found attached to diatoms and may influence the fitness of their host. The most commonly used model organisms in studies of diatom-bacterial associations are Alteromonas and Marinobacter. Some strains of Alteromonas are capable of parasitism, producing chitinases or having algicidal interactions; some strains of Marinobacter are capable of mutualism, providing its host with vital nutrients. In this study, multiple strains of Alteromonas and Marinobacter were isolated from the centric diatom Chaetoceros sp KBDT20. Isolates were added back in varying concentration to cultures of their original xenic diatom host, and to cultures of a smaller, xenic naïve host, Chaetoceros sp. KBDT32. The growth rate of the diatom host was monitored using flow cytometry to assess the impact of the added bacterial isolates on host health. Our results suggest that all strains of Alteromonas tested have an antagonistic relationship with both the original as well as the naïve host while all strains of Marinobacter tested have a synergistic relationship with both diatom cultures. The functional basis for these relationships is being explored by supplementing xenic diatom cultures with materials essential for diatom growth that may be contributed by bacteria, such as B-vitamins and bioavailable trace metals. The colonization rates and competitive interactions between bacteria are investigated through surface colonization studies. The goal of this study is to better inform our understanding of how bacterial associates of diatom populations may contribute to their health, success, or failure in aquatic systems.

  11. A distinct bacterial dysbiosis associated skin inflammation in ovine footrot

    NASA Astrophysics Data System (ADS)

    Maboni, Grazieli; Blanchard, Adam; Frosth, Sara; Stewart, Ceri; Emes, Richard; Tötemeyer, Sabine

    2017-03-01

    Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1β and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression.

  12. Bacterial communities in full-scale wastewater treatment systems.

    PubMed

    Cydzik-Kwiatkowska, Agnieszka; Zielińska, Magdalena

    2016-04-01

    Bacterial metabolism determines the effectiveness of biological treatment of wastewater. Therefore, it is important to define the relations between the species structure and the performance of full-scale installations. Although there is much laboratory data on microbial consortia, our understanding of dependencies between the microbial structure and operational parameters of full-scale wastewater treatment plants (WWTP) is limited. This mini-review presents the types of microbial consortia in WWTP. Information is given on extracellular polymeric substances production as factor that is key for formation of spatial structures of microorganisms. Additionally, we discuss data on microbial groups including nitrifiers, denitrifiers, Anammox bacteria, and phosphate- and glycogen-accumulating bacteria in full-scale aerobic systems that was obtained with the use of molecular techniques, including high-throughput sequencing, to shed light on dependencies between the microbial ecology of biomass and the overall efficiency and functional stability of wastewater treatment systems. Sludge bulking in WWTPs is addressed, as well as the microbial composition of consortia involved in antibiotic and micropollutant removal.

  13. Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads.

    PubMed

    Tsfasman, Irina M; Lapteva, Yulia S; Krasovskaya, Ludmila A; Kudryakova, Irina V; Vasilyeva, Natalia V; Granovsky, Igor E; Stepnaya, Olga A

    2015-01-01

    Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms. © 2015 S. Karger AG, Basel.

  14. Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

    PubMed Central

    Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

    2011-01-01

    The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

  15. Transcriptional response of Musca domestica larvae to bacterial infection.

    PubMed

    Tang, Ting; Li, Xiang; Yang, Xue; Yu, Xue; Wang, Jianhui; Liu, Fengsong; Huang, Dawei

    2014-01-01

    The house fly Musca domestica, a cosmopolitan dipteran insect, is a significant vector for human and animal bacterial pathogens, but little is known about its immune response to these pathogens. To address this issue, we inoculated the larvae with a mixture of Escherichia coli and Staphylococcus aureus and profiled the transcriptome 6, 24, and 48 h thereafter. Many genes known to controlling innate immunity in insects were induced following infection, including genes encoding pattern recognition proteins (PGRPs), various components of the Toll and IMD signaling pathways and of the proPO-activating and redox systems, and multiple antimicrobial peptides. Interestingly, we also uncovered a large set of novel immune response genes including two broad-spectrum antimicrobial peptides (muscin and domesticin), which might have evolved to adapt to house-fly's unique ecological environments. Finally, genes mediating oxidative phosphorylation were repressed at 48 h post-infection, suggesting disruption of energy homeostasis and mitochondrial function at the late stages of infection. Collectively, our data reveal dynamic changes in gene expression following bacterial infection in the house fly, paving the way for future in-depth analysis of M. domestica's immune system.

  16. Bacterial growth laws and their applications.

    PubMed

    Scott, Matthew; Hwa, Terence

    2011-08-01

    Quantitative empirical relationships between cell composition and growth rate played an important role in the early days of microbiology. Gradually, the focus of the field began to shift from growth physiology to the ever more elaborate molecular mechanisms of regulation employed by the organisms. Advances in systems biology and biotechnology have renewed interest in the physiology of the cell as a whole. Furthermore, gene expression is known to be intimately coupled to the growth state of the cell. Here, we review recent efforts in characterizing such couplings, particularly the quantitative phenomenological approaches exploiting bacterial 'growth laws.' These approaches point toward underlying design principles that can guide the predictive manipulation of cell behavior in the absence of molecular details. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Bacterial growth laws and their applications

    PubMed Central

    SCOTT, Matthew; HWA, Terence

    2011-01-01

    Quantitative empirical relationships between cell composition and growth rate played an important role in the early days of microbiology. Gradually, the focus of the field began to shift from growth physiology to the ever more elaborate molecular mechanisms of regulation employed by the organisms. Advances in systems biology and biotechnology have renewed interest in the physiology of the cell as a whole. Furthermore, gene expression is known to be intimately coupled to the growth state of the cell. Here, we review recent efforts in characterizing such couplings, particularly the quantitative phenomenological approaches exploiting bacterial `growth laws.' These approaches point toward underlying design principles that can guide the predictive manipulation of cell behavior in the absence of molecular details. PMID:21592775

  18. Suppression of bacterial cell-cell signalling, biofilm formation and type III secretion system by citrus flavonoids.

    PubMed

    Vikram, A; Jayaprakasha, G K; Jesudhasan, P R; Pillai, S D; Patil, B S

    2010-08-01

    This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96-well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi-quantitative PCR. Differential responses for different flavonoids were observed for different cell-cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell-cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. The results of the study indicate a potential modulation of bacterial cell-cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer-mediated cell-cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell-cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.

  19. Postviral Complications: Bacterial Pneumonia.

    PubMed

    Prasso, Jason E; Deng, Jane C

    2017-03-01

    Secondary bacterial pneumonia after viral respiratory infection remains a significant source of morbidity and mortality. Susceptibility is mediated by a variety of viral and bacterial factors, and complex interactions with the host immune system. Prevention and treatment strategies are limited to influenza vaccination and antibiotics/antivirals respectively. Novel approaches to identifying the individuals with influenza who are at increased risk for secondary bacterial pneumonias are urgently needed. Given the threat of further pandemics and the heightened prevalence of these viruses, more research into the immunologic mechanisms of this disease is warranted with the hope of discovering new potential therapies. Published by Elsevier Inc.

  20. Core Principles of Bacterial Autoinducer Systems

    PubMed Central

    2015-01-01

    SUMMARY Autoinduction (AI), the response to self-produced chemical signals, is widespread in the bacterial world. This process controls vastly different target functions, such as luminescence, nutrient acquisition, and biofilm formation, in different ways and integrates additional environmental and physiological cues. This diversity raises questions about unifying principles that underlie all AI systems. Here, we suggest that such core principles exist. We argue that the general purpose of AI systems is the homeostatic control of costly cooperative behaviors, including, but not limited to, secreted public goods. First, costly behaviors require preassessment of their efficiency by cheaper AI signals, which we encapsulate in a hybrid “push-pull” model. The “push” factors cell density, diffusion, and spatial clustering determine when a behavior becomes effective. The relative importance of each factor depends on each species' individual ecological context and life history. In turn, “pull” factors, often stress cues that reduce the activation threshold, determine the cellular demand for the target behavior. Second, control is homeostatic because AI systems, either themselves or through accessory mechanisms, not only initiate but also maintain the efficiency of target behaviors. Third, AI-controlled behaviors, even seemingly noncooperative ones, are generally cooperative in nature, when interpreted in the appropriate ecological context. The escape of individual cells from biofilms, for example, may be viewed as an altruistic behavior that increases the fitness of the resident population by reducing starvation stress. The framework proposed here helps appropriately categorize AI-controlled behaviors and allows for a deeper understanding of their ecological and evolutionary functions. PMID:25694124

  1. Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria.

    PubMed

    Gebhardt, Michael J; Jacobson, Rachael K; Shuman, Howard A

    2017-01-01

    The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires' Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community.

  2. Transcriptome landscape of a bacterial pathogen under plant immunity.

    PubMed

    Nobori, Tatsuya; Velásquez, André C; Wu, Jingni; Kvitko, Brian H; Kremer, James M; Wang, Yiming; He, Sheng Yang; Tsuda, Kenichi

    2018-03-27

    Plant pathogens can cause serious diseases that impact global agriculture. The plant innate immunity, when fully activated, can halt pathogen growth in plants. Despite extensive studies into the molecular and genetic bases of plant immunity against pathogens, the influence of plant immunity in global pathogen metabolism to restrict pathogen growth is poorly understood. Here, we developed RNA sequencing pipelines for analyzing bacterial transcriptomes in planta and determined high-resolution transcriptome patterns of the foliar bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana with a total of 27 combinations of plant immunity mutants and bacterial strains. Bacterial transcriptomes were analyzed at 6 h post infection to capture early effects of plant immunity on bacterial processes and to avoid secondary effects caused by different bacterial population densities in planta We identified specific "immune-responsive" bacterial genes and processes, including those that are activated in susceptible plants and suppressed by plant immune activation. Expression patterns of immune-responsive bacterial genes at the early time point were tightly linked to later bacterial growth levels in different host genotypes. Moreover, we found that a bacterial iron acquisition pathway is commonly suppressed by multiple plant immune-signaling pathways. Overexpression of a P. syringae sigma factor gene involved in iron regulation and other processes partially countered bacterial growth restriction during the plant immune response triggered by AvrRpt2. Collectively, this study defines the effects of plant immunity on the transcriptome of a bacterial pathogen and sheds light on the enigmatic mechanisms of bacterial growth inhibition during the plant immune response.

  3. Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

    PubMed Central

    Rondon, Michelle R.; Raffel, Sandra J.; Goodman, Robert M.; Handelsman, Jo

    1999-01-01

    As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes. PMID:10339608

  4. Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system.

    PubMed

    Klammt, Christian; Schwarz, Daniel; Fendler, Klaus; Haase, Winfried; Dötsch, Volker; Bernhard, Frank

    2005-12-01

    Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.

  5. N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

    PubMed Central

    Nothaft, Harald; Zheng, Jing

    2013-01-01

    Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

  6. Superantigens Modulate Bacterial Density during Staphylococcus aureus Nasal Colonization

    PubMed Central

    Xu, Stacey X.; Kasper, Katherine J.; Zeppa, Joseph J.; McCormick, John K.

    2015-01-01

    Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization. PMID:26008236

  7. Cellular effects of bacterial N-3-Oxo-dodecanoyl-L-Homoserine lactone on the sponge Suberites domuncula (Olivi, 1792): insights into an intimate inter-kingdom dialogue.

    PubMed

    Gardères, Johan; Henry, Joël; Bernay, Benoit; Ritter, Andrès; Zatylny-Gaudin, Céline; Wiens, Matthias; Müller, Werner E G; Le Pennec, Gaël

    2014-01-01

    Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and β subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on

  8. [Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

    PubMed

    Minamino, Tohru

    2015-01-01

    The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

  9. Arabidopsis EF-Tu receptor enhances bacterial disease resistance in transgenic wheat.

    PubMed

    Schoonbeek, Henk-Jan; Wang, Hsi-Hua; Stefanato, Francesca L; Craze, Melanie; Bowden, Sarah; Wallington, Emma; Zipfel, Cyril; Ridout, Christopher J

    2015-04-01

    Perception of pathogen (or microbe)-associated molecular patterns (PAMPs/MAMPs) by pattern recognition receptors (PRRs) is a key component of plant innate immunity. The Arabidopsis PRR EF-Tu receptor (EFR) recognizes the bacterial PAMP elongation factor Tu (EF-Tu) and its derived peptide elf18. Previous work revealed that transgenic expression of AtEFR in Solanaceae confers elf18 responsiveness and broad-spectrum bacterial disease resistance. In this study, we developed a set of bioassays to study the activation of PAMP-triggered immunity (PTI) in wheat. We generated transgenic wheat (Triticum aestivum) plants expressing AtEFR driven by the constitutive rice actin promoter and tested their response to elf18. We show that transgenic expression of AtEFR in wheat confers recognition of elf18, as measured by the induction of immune marker genes and callose deposition. When challenged with the cereal bacterial pathogen Pseudomonas syringae pv. oryzae, transgenic EFR wheat lines had reduced lesion size and bacterial multiplication. These results demonstrate that AtEFR can be transferred successfully from dicot to monocot species, further revealing that immune signalling pathways are conserved across these distant phyla. As novel PRRs are identified, their transfer between plant families represents a useful strategy for enhancing resistance to pathogens in crops. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  10. The Effect of a Low Fluoride Delivery System on Bacterial Metabolism.

    DTIC Science & Technology

    1981-08-25

    of motile to non-motile bacteria in subgingival plaque samples is approximately 1:50; whereas in periodontally diseased sites, the ratio is approxi...parameters are indicated to assess the feasibility of this system as an adjunct in the control of caries and periodontal disease . 32 Antiplaque Determinants...of SnF2: pH and Ions INTRODUCTION The pathology of dental caries and periodontal disease is associated with the accumulation of bacterial plaque on

  11. A Metaproteomics Approach to Elucidate Host and Pathogen Protein Expression during Catheter-Associated Urinary Tract Infections (CAUTIs)

    PubMed Central

    Lassek, Christian; Burghartz, Melanie; Chaves-Moreno, Diego; Otto, Andreas; Hentschker, Christian; Fuchs, Stephan; Bernhardt, Jörg; Jauregui, Ruy; Neubauer, Rüdiger; Becher, Dörte; Pieper, Dietmar H.; Jahn, Martina; Jahn, Dieter; Riedel, Katharina

    2015-01-01

    Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections

  12. A metaproteomics approach to elucidate host and pathogen protein expression during catheter-associated urinary tract infections (CAUTIs).

    PubMed

    Lassek, Christian; Burghartz, Melanie; Chaves-Moreno, Diego; Otto, Andreas; Hentschker, Christian; Fuchs, Stephan; Bernhardt, Jörg; Jauregui, Ruy; Neubauer, Rüdiger; Becher, Dörte; Pieper, Dietmar H; Jahn, Martina; Jahn, Dieter; Riedel, Katharina

    2015-04-01

    Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections

  13. Shrimp Alpha-2-Macroglobulin Prevents the Bacterial Escape by Inhibiting Fibrinolysis of Blood Clots

    PubMed Central

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

    2012-01-01

    Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis. PMID:23082160

  14. Airway fungal colonization compromises the immune system allowing bacterial pneumonia to prevail.

    PubMed

    Roux, Damien; Gaudry, Stéphane; Khoy-Ear, Linda; Aloulou, Meryem; Phillips-Houlbracq, Mathilde; Bex, Julie; Skurnik, David; Denamur, Erick; Monteiro, Renato C; Dreyfuss, Didier; Ricard, Jean-Damien

    2013-09-01

    To study the correlation between fungal colonization and bacterial pneumonia and to test the effect of antifungal treatments on the development of bacterial pneumonia in colonized rats. Experimental animal investigation. University research laboratory. Pathogen-free male Wistar rats weighing 250-275 g. Rats were colonized by intratracheal instillation of Candida albicans. Fungal clearance from the lungs and immune response were measured. Both colonized and noncolonized animals were secondarily instilled with different bacterial species (Pseudomonas aeruginosa, Escherichia coli, or Staphylococcus aureus). Bacterial phagocytosis by alveolar macrophages was evaluated in the presence of interferon-gamma, the main cytokine produced during fungal colonization. The effect of antifungal treatments on fungal colonization and its immune response were assessed. The prevalence of P. aeruginosa pneumonia was compared in antifungal treated and control colonized rats. C. albicans was slowly cleared and induced a Th1-Th17 immune response with very high interferon-gamma concentrations. Airway fungal colonization favored the development of bacterial pneumonia. Interferon-gamma was able to inhibit the phagocytosis of unopsonized bacteria by alveolar macrophages. Antifungal treatment decreased airway fungal colonization, lung interferon-gamma levels and, consequently, the prevalence of subsequent bacterial pneumonia. C. albicans airway colonization elicited a Th1-Th17 immune response that favored the development of bacterial pneumonia via the inhibition of bacterial phagocytosis by alveolar macrophages. Antifungal treatment decreased the risk of bacterial pneumonia in colonized rats.

  15. Culturable bacterial diversity from a feed water of a reverse osmosis system, evaluation of biofilm formation and biocontrol using phages.

    PubMed

    Belgini, D R B; Dias, R S; Siqueira, V M; Valadares, L A B; Albanese, J M; Souza, R S; Torres, A P R; Sousa, M P; Silva, C C; De Paula, S O; Oliveira, V M

    2014-10-01

    Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.

  16. Bacterial Prostatitis: Bacterial Virulence, Clinical Outcomes, and New Directions.

    PubMed

    Krieger, John N; Thumbikat, Praveen

    2016-02-01

    Four prostatitis syndromes are recognized clinically: acute bacterial prostatitis, chronic bacterial prostatitis, chronic prostatitis/chronic pelvic pain syndrome, and asymptomatic prostatitis. Because Escherichia coli represents the most common cause of bacterial prostatitis, we investigated the importance of bacterial virulence factors and antimicrobial resistance in E. coli strains causing prostatitis and the potential association of these characteristics with clinical outcomes. A structured literature review revealed that we have limited understanding of the virulence-associated characteristics of E. coli causing acute prostatitis. Therefore, we completed a comprehensive microbiological and molecular investigation of a unique strain collection isolated from healthy young men. We also considered new data from an animal model system suggesting certain E. coli might prove important in the etiology of chronic prostatitis/chronic pelvic pain syndrome. Our human data suggest that E. coli needs multiple pathogenicity-associated traits to overcome anatomic and immune responses in healthy young men without urological risk factors. The phylogenetic background and accumulation of an exceptional repertoire of extraintestinal pathogenic virulence-associated genes indicate that these E. coli strains belong to a highly virulent subset of uropathogenic variants. In contrast, antibiotic resistance confers little added advantage to E. coli strains in these healthy outpatients. Our animal model data also suggest that certain pathogenic E. coli may be important in the etiology of chronic prostatitis/chronic pelvic pain syndrome through mechanisms that are dependent on the host genetic background and the virulence of the bacterial strain.

  17. Bacterial Pili exploit integrin machinery to promote immune activation and efficient blood-brain barrier penetration

    PubMed Central

    Banerjee, Anirban; Kim, Brandon J.; Carmona, Ellese M.; Cutting, Andrew S.; Gurney, Michael A.; Carlos, Chris; Feuer, Ralph; Prasadarao, Nemani V.; Doran, Kelly S.

    2011-01-01

    Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages, known as pili, have been recently described in streptococcal pathogens, including GBS. The pilus tip adhesin, PilA, contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the host receptor and the contribution of PilA in central nervous system (CNS) disease pathogenesis are unknown. Here we show that PilA binds collagen, which promotes GBS interaction with the α2β1 integrin resulting in activation of host chemokine expression and neutrophil recruitment during infection. Mice infected with the PilA-deficient mutant exhibit delayed mortality, a decrease in neutrophil infiltration and bacterial CNS dissemination. We find that PilA-mediated virulence is dependent on neutrophil influx as neutrophil depletion results in a decrease in BBB permeability and GBS–BBB penetration. Our results suggest that the bacterial pilus, specifically the PilA adhesin, has a dual role in immune activation and bacterial entry into the CNS. PMID:21897373

  18. Serum procalcitonin has negative predictive value for bacterial infection in active systemic lupus erythematosus.

    PubMed

    Bador, K M; Intan, S; Hussin, S; Gafor, A H A

    2012-10-01

    Previous studies in systemic lupus erythematosus (SLE) patients have produced conflicting results regarding the diagnostic utility of procalcitonin (PCT). The aim of this study was to determine predictive values of PCT and C-reactive protein (CRP) for bacterial infection in SLE patients. This was a cross-sectional study of clinic and hospitalized SLE patients with and without bacterial infection recruited over 18 months. Bacterial infection was defined as positive culture results. SLE disease activity was measured using SLEDAI. PCT and CRP were measured by automated immunoassays. Sixty-eight patients (57 females) were studied. Ten patients (15%) had infection. The areas under the receiver operating characteristic curves for PCT and CRP were not significantly different [0.797 (CI 0.614-0.979) versus 0.755 (CI 0.600-0.910)]. In lupus flare patients, PCT but not CRP was higher with infection (p = 0.019 versus 0.195). A PCT of <0.17 ng/ml ruled out infection with 94% negative predictive value (NPV). In remission patients, CRP but not PCT was elevated with infection (p = 0.036 versus 0.103). CRP < 0.57 mg/dl had 96% NPV. PCT may be a better marker to rule out bacterial infection in lupus flare but not in remission or general screening.

  19. Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development.

    PubMed

    Wu, Shan; Zhang, Xiaofeng; He, Yongqiang; Shuai, Jiangbing; Chen, Xiaomei; Ling, Erjun

    2010-11-01

    Although Bombyx mori systematic immunity is extensively studied, little is known about the silkworm's intestine-specific responses to bacterial infection. Antimicrobial peptides (AMPs) gene expression analysis of B. mori intestinal tissue to oral infection with the Gram-positive (Staphylococcus aureus) and -negative (Escherichia coli) bacteria revealed that there is specificity in the interaction between host immune responses and parasite types. Neither Att1 nor Leb could be stimulated by S. aureus and E. coli. However, CecA1, Glo1, Glo2, Glo3, Glo4 and Lys, could only be trigged by S. aureus. On the contrary, E. coli stimulation caused the decrease in the expression of CecA1, Glo3 and Glo4 in some time points. Interestingly, there is regional specificity in the silkworm local gut immunity. During the immune response, the increase in Def, Hem and LLP3 was only detected in the foregut and midgut. For CecB1, CecD, LLP2 and Mor, after orally administered with E. coli, the up-regulation was only limited in the midgut and hindgut. CecE was the only AMP that positively responses to the both bacteria in all the testing situations. With development, the expression levels of the AMPs were also changed dramatically. That is, at spinning and prepupa stages, a large increase in the expression of CecA1, CecB1, CecD, CecE, Glo1, Glo2, Glo3, Glo4, Leb, Def, Hem, Mor and Lys was detected in the gut. Unexpectedly, in addition to the IMD pathway genes, the Toll and JAK/STAT pathway genes in the silkworm gut can also be activated by microbial oral infection. But in the developmental course, corresponding to the increase in expression of AMPs at spinning and prepupa stages, only the Toll pathway genes in the gut exhibit the similar increasing trend. Our results imply that the immune responses in the silkworm gut are synergistically regulated by the Toll, JAK/STAT and IMD pathways. However, as the time for approaching pupation, the Toll pathway may play a role in the AMPs expression

  20. Burkholderia Type VI Secretion Systems Have Distinct Roles in Eukaryotic and Bacterial Cell Interactions

    PubMed Central

    Schwarz, Sandra; West, T. Eoin; Boyer, Frédéric; Chiang, Wen-Chi; Carl, Mike A.; Hood, Rachel D.; Rohmer, Laurence; Tolker-Nielsen, Tim; Skerrett, Shawn J.; Mougous, Joseph D.

    2010-01-01

    Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections. PMID:20865170

  1. Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism

    PubMed Central

    Shimizu, Kazuyuki

    2013-01-01

    It is quite important to understand the overall metabolic regulation mechanism of bacterial cells such as Escherichia coli from both science (such as biochemistry) and engineering (such as metabolic engineering) points of view. Here, an attempt was made to clarify the overall metabolic regulation mechanism by focusing on the roles of global regulators which detect the culture or growth condition and manipulate a set of metabolic pathways by modulating the related gene expressions. For this, it was considered how the cell responds to a variety of culture environments such as carbon (catabolite regulation), nitrogen, and phosphate limitations, as well as the effects of oxygen level, pH (acid shock), temperature (heat shock), and nutrient starvation. PMID:25937963

  2. Expression systems for therapeutic glycoprotein production.

    PubMed

    Durocher, Yves; Butler, Michael

    2009-12-01

    There are slightly over 165 recombinant pharmaceuticals currently approved for human use. Another 500 protein candidates are in preclinical and clinical development, about 70% of these being glycosylated proteins. The need for expression systems allowing the efficient manufacturing of high quality glycoproteins is thus becoming imperative. Recent developments with CHO cells, the predominant mammalian expression system, have focused on either increasing cell specific productivity or prolonging the life span of cells in culture that translates to high integrated viable cell densities. These two factors have allowed volumetric productivities in excess of 5 g/L under conditions of controlled nutrient feeding. In addition to glycoengineering strategies, which are offering considerable advantage in producing proteins with enhanced therapeutic properties, several alternative expression systems are being developed for their manufacture, each with their advantages and limitations.

  3. Induction of plant defense gene expression by plant activators and Pseudomonas syringae pv. tomato in greenhouse-grown tomatoes.

    PubMed

    Herman, M A B; Davidson, J K; Smart, C D

    2008-11-01

    Plant activators provide an appealing management option for bacterial diseases of greenhouse-grown tomatoes. Two types of plant activators, one that induces systemic acquired resistance (SAR) and a second that activates induced systemic resistance (ISR), were evaluated for control of Pseudomonas syringae pv. tomato and effect on plant defense gene activation. Benzothiadiazole (BTH, SAR-inducing compound) effectively reduced bacterial speck incidence and severity, both alone and in combination with the ISR-inducing product. Application of BTH also led to elevated activation of salicylic acid and ethylene-mediated responses, based on real-time polymerase chain reaction analysis of marker gene expression levels. In contrast, the ISR-inducing product (made up of plant growth-promoting rhizobacteria) inconsistently modified defense gene expression and did not provide disease control to the same level as did BTH. No antagonism was observed by combining the two activators as control of bacterial speck was similar to or better than BTH alone.

  4. Impact of Bioreactor Environment and Recovery Method on the Profile of Bacterial Populations from Water Distribution Systems.

    PubMed

    Luo, Xia; Jellison, Kristen L; Huynh, Kevin; Widmer, Giovanni

    2015-01-01

    Multiple rotating annular reactors were seeded with biofilms flushed from water distribution systems to assess (1) whether biofilms grown in bioreactors are representative of biofilms flushed from the water distribution system in terms of bacterial composition and diversity, and (2) whether the biofilm sampling method affects the population profile of the attached bacterial community. Biofilms were grown in bioreactors until thickness stabilized (9 to 11 weeks) and harvested from reactor coupons by sonication, stomaching, bead-beating, and manual scraping. High-throughput sequencing of 16S rRNA amplicons was used to profile bacterial populations from flushed biofilms seeded into bioreactors as well as biofilms recovered from bioreactor coupons by different methods. β diversity between flushed and reactor biofilms was compared to β diversity between (i) biofilms harvested from different reactors and (ii) biofilms harvested by different methods from the same reactor. These analyses showed that average diversity between flushed and bioreactor biofilms was double the diversity between biofilms from different reactors operated in parallel. The diversity between bioreactors was larger than the diversity associated with different biofilm recovery methods. Compared to other experimental variables, the method used to recover biofilms had a negligible impact on the outcome of water biofilm analyses based on 16S amplicon sequencing. Results from this study show that biofilms grown in reactors over 9 to 11 weeks are not representative models of the microbial populations flushed from a distribution system. Furthermore, the bacterial population profile of biofilms grown in replicate reactors from the same flushed water are likely to diverge. However, four common sampling protocols, which differ with respect to disruption of bacterial cells, provide similar information with respect to the 16S rRNA population profile of the biofilm community.

  5. Phytoplankton-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge.

    PubMed

    Bertrand, Erin M; McCrow, John P; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B; Delmont, Tom O; Post, Anton F; Sipler, Rachel E; Spackeen, Jenna L; Xu, Kai; Bronk, Deborah A; Hutchins, David A; Allen, Andrew E

    2015-08-11

    Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton-bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton-bacterial interactions in both positive and negative feedback loops.

  6. Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge

    PubMed Central

    Bertrand, Erin M.; McCrow, John P.; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B.; Delmont, Tom O.; Post, Anton F.; Sipler, Rachel E.; Spackeen, Jenna L.; Xu, Kai; Bronk, Deborah A.; Hutchins, David A.; Allen, Andrew E.

    2015-01-01

    Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton–bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton–bacterial interactions in both positive and negative feedback loops. PMID:26221022

  7. Metabolic sensor governing bacterial virulence in Staphylococcus aureus.

    PubMed

    Ding, Yue; Liu, Xing; Chen, Feifei; Di, Hongxia; Xu, Bin; Zhou, Lu; Deng, Xin; Wu, Min; Yang, Cai-Guang; Lan, Lefu

    2014-11-18

    An effective metabolism is essential to all living organisms, including the important human pathogen Staphylococcus aureus. To establish successful infection, S. aureus must scavenge nutrients and coordinate its metabolism for proliferation. Meanwhile, it also must produce an array of virulence factors to interfere with host defenses. However, the ways in which S. aureus ties its metabolic state to its virulence regulation remain largely unknown. Here we show that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, binds to and activates the catabolite control protein E (CcpE) of S. aureus. Using structural and site-directed mutagenesis studies, we demonstrate that two arginine residues (Arg145 and Arg256) within the putative inducer-binding cavity of CcpE are important for its allosteric activation by citrate. Microarray analysis reveals that CcpE tunes the expression of 126 genes that comprise about 4.7% of the S. aureus genome. Intriguingly, although CcpE is a major positive regulator of the TCA-cycle activity, its regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Moreover, inactivation of CcpE results in increased staphyloxanthin production, improved ability to acquire iron, increased resistance to whole-blood-mediated killing, and enhanced bacterial virulence in a mouse model of systemic infection. This study reveals CcpE as an important metabolic sensor that allows S. aureus to sense and adjust its metabolic state and subsequently to coordinate the expression of virulence factors and bacterial virulence.

  8. Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora.

    PubMed

    Mäe, A; Montesano, M; Koiv, V; Palva, E T

    2001-09-01

    Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

  9. Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

    PubMed Central

    Shaw, Jennifer L. A.; Weyrich, Laura S.; Sawade, Emma; Drikas, Mary; Cooper, Alan J.

    2015-01-01

    Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. PMID:26162884

  10. CHANGES IN BACTERIAL COMPOSITION OF BIOFILM IN A ...

    EPA Pesticide Factsheets

    This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e., groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. The biofilm community was characterized using 16S rRNA gene clone libraries and functional potential analysis, generated from total DNA extracted from coupons in biofilm annular reactors fed with onsite drinking water for up to eighteen months. Significant differences in the bacterial community structure were observed between GW and SW. Representatives that explained the dissimilarity between service areas were associated with Betaproteobacteria, Alphaproteobacteria, Actinobacteria, Gammaproteobacteria, and Firmicutes. After nine months the biofilm bacterial community from both areas were dominated by Mycobacterium species. The distribution of the dominant OTU (Mycobacterium) positively correlated with the drinking water distribution system (DWDS) temperature, but no clear relationship was seen with free chlorine residual, pH, turbidity or total organic carbon (TOC). The results suggest that biofilm microbial communities harbor distinct and diverse bacterial communities, and that source water, treatment processes and environmental conditions may play an important role in shaping the bacterial community in the distribution system. On the other hand, several bacterial groups were present i

  11. Analysis of the Impact of Rosuvastatin on Bacterial Mevalonate Production Using a UPLC-Mass Spectrometry Approach.

    PubMed

    Nolan, J A; Kinsella, M; Hill, C; Joyce, S A; Gahan, C G M

    2016-07-01

    Statins are widely prescribed cholesterol-lowering medications and act through inhibition of the human enzyme 3-methylglutaryl coenzyme A reductase (HMG-R) which produces mevalonate (MVAL), a key substrate for cholesterol biosynthesis. Some important microbial species also express an isoform of HMG-R; however, the nature of the interaction between statins and bacteria is currently unclear and studies would benefit from protocols to quantify MVAL in complex microbial environments. The objective of this study was to develop a protocol for the analytical quantification of MVAL in bacterial systems and to utilise this approach to analyse the effects of Rosuvastatin (RSV) on bacterial MVAL formation. To determine the effective concentration range of RSV, we examined the dose-dependent inhibition of growth in the HMG-R(+) bacterial pathogens Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecium at various concentrations of pure RSV. Growth inhibition generally correlated with a reduction in bacterial MVAL levels, particularly in culture supernatants at high RSV concentrations, as determined using our ultra-performance liquid chromatography mass spectrometry protocol. This work therefore outlines a refined protocol for the analysis of MVAL in microbial cultures and provides evidence for statin-mediated inhibition of bacterial HMG-R. Furthermore, we show that MVAL is readily transported and secreted from bacterial cells into the growth media.

  12. Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system.

    PubMed

    Lu, Yun-Yueh; Franz, Bettina; Truttmann, Matthias C; Riess, Tanja; Gay-Fraret, Jérémie; Faustmann, Marco; Kempf, Volkhard A J; Dehio, Christoph

    2013-05-01

    The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae - each displaying multiple functions in host cell interaction - have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). BadA mediates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translocates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowledge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both virulence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep translocation and invasome formation by likely preventing close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of

  13. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System

    PubMed Central

    Prest, E. I.; Weissbrodt, D. G.; Hammes, F.; van Loosdrecht, M. C. M.; Vrouwenvelder, J. S.

    2016-01-01

    Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1–3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson’s correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously

  14. Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System.

    PubMed

    Prest, E I; Weissbrodt, D G; Hammes, F; van Loosdrecht, M C M; Vrouwenvelder, J S

    2016-01-01

    Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1-3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson's correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously

  15. Bacterial pneumonia as an influenza complication.

    PubMed

    Martin-Loeches, Ignacio; van Someren Gréve, Frank; Schultz, Marcus J

    2017-04-01

    The pathogenesis and impact of coinfection, in particular bacterial coinfection, in influenza are incompletely understood. This review summarizes results from studies on bacterial coinfection in the recent pandemic influenza outbreak. Systemic immune mechanisms play a key role in the development of coinfection based on the complexity of the interaction of the host and the viral and bacterial pathogens. Several studies were performed to determine the point prevalence of bacterial coinfection in influenza. Coinfection in influenza is frequent in critically ill patients with Streptococcus pneumoniae being the most frequent bacterial pathogen and higher rates of potentially resistant pathogens over the years. Bacterial pneumonia is certainly an influenza complication. The recent epidemiology findings have helped to partially resolve the contribution of different pathogens. Immunosuppression is a risk factor for bacterial coinfection in influenza, and the epidemiology of coinfection has changed over the years during the last influenza pandemic, and these recent findings should be taken into account during present outbreaks.

  16. Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components.

    PubMed

    Gao, Quanxin; Yue, Yanfeng; Min, Minghua; Peng, Shiming; Shi, Zhaohong; Sheng, Wenquan; Zhang, Tao

    2018-06-08

    Toll like receptor (TLR) 5 and 9 are important members of the TLR family that play key roles in innate immunity in all vertebrates. In this study, paTLR5 and paTLR9 were identified in silver pomfret (Pampus argenteus), a marine teleost of great economic value. Open reading frames (ORFs) of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively. Sequence analysis revealed several conserved characteristic features, including signal peptides, leucine-rich repeat (LRR) motifs, and a Toll/interleukin-I receptor (TIR) domain. Sequence, phylogenetic and synteny analysis revealed high sequence identity with counterparts in other teleosts, confirming their correct nomenclature and conservation during evolution. Quantitative real-time PCR revealed that the that both TLRs were ubiquitously expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin. In vitro immunostimulation experiments revealed that Aeromonas hydrophila lipopolysaccharide (LPS) and Vibrio anguillarum flagellin induced higher levels of paTLR9 and paTLR5 mRNA expression in isolated fish intestinal epithelial cells (FIECs) than Lactobacillus plantarum lipoteichoic acid (LTA), but all increased the secretion of IL-6 and TNF-α and induced cell apoptosis and necrosis. Together, these results indicate that paTLR5 and paTLR9 may function in the response to bacterial pathogens. Our findings enhance our understanding of the function of TLRs in the innate immune system of silver pomfret and other teleosts. Copyright © 2018. Published by Elsevier Ltd.

  17. Exchange of rotor components in functioning bacterial flagellar motor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fukuoka, Hajime; Inoue, Yuichi; Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai 980-8577

    2010-03-26

    The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP,more » and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s{sup -1}, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.« less

  18. Combing bacterial turbulence.

    NASA Astrophysics Data System (ADS)

    Sokolov, Andrey; Nishiguchi, Daiki; Aronson, Igor

    Living systems represented by ensembles of motile organisms demonstrate a transition from a chaotic motion to a highly ordered state. Examples of such living systems include suspensions of bacteria, schools of fish, flocks of birds and even crowds of people. In spite of significant differences in interacting mechanisms and motion scales, ordered living systems have many similarities: short-range alignment of organism, turbulent-like motion, emergence of large-scale flows and dynamic vortices. In this work, we rectify a turbulent dynamics in suspensions of swimming bacteria Bacillus subtilis by imposing periodical constraints on bacterial motion. Bacteria, swimming between periodically placed microscopic vertical pillars, may self-organize in a stable lattice of vortices. We demonstrate the emergence of a strong anti-ferromagnetic order of bacterial vortices in a rectangular lattice of pillars. Hydrodynamic interaction between vortices increases the stability of an emerged pattern. The highest stability of vortices in the anti-ferromagnetic lattice and the fastest vortices speed were observed in structures with the periods comparable with a correlation length of bacterial unconstrained motion. A.S and I.A were supported by the US DOE, Office of Basic Energy Sciences, Division of Materials Science And Engineering, under contract No. DE AC02-06CH11357 and D.N was supported by ALPS and JSPS Grant No. 26-9915.

  19. Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens.

    PubMed

    Zink, Steven D; Van Slyke, Greta A; Palumbo, Michael J; Kramer, Laura D; Ciota, Alexander T

    2015-10-27

    Complex interactions between microbial residents of mosquitoes and arboviruses are likely to influence many aspects of vectorial capacity and could potentially have profound effects on patterns of arbovirus transmission. Such interactions have not been well studied for West Nile virus (WNV; Flaviviridae, Flavivirus) and Culex spp. mosquitoes. We utilized next-generation sequencing of 16S ribosomal RNA bacterial genes derived from Culex pipiens Linnaeus following WNV exposure and/or infection and compared bacterial populations and broad immune responses to unexposed mosquitoes. Our results demonstrate that WNV infection increases the diversity of bacterial populations and is associated with up-regulation of classical invertebrate immune pathways including RNA interference (RNAi), Toll, and Jak-STAT (Janus kinase-Signal Transducer and Activator of Transcription). In addition, WNV exposure alone, without the establishment of infection, results in similar alterations to microbial and immune signatures, although to a lesser extent. Multiple bacterial genera were found in greater abundance inWNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia.

  20. Variation in the ovine cortisol response to systemic bacterial endotoxin challenge is predominantly determined by signalling within the hypothalamic-pituitary-adrenal axis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    You Qiumei; Karrow, Niel A.; Cao Honghe

    Bi-directional communication between the neuroendocrine and immune systems is designed, in part, to maintain or restore homeostasis during physiological stress. Exposure to endotoxin during Gram-negative bacterial infection for example, elicits the release of pro-inflammatory cytokines that activate the hypothalamic-pituitary-adrenal axis (HPAA). The secretion of adrenal glucocorticoids subsequently down regulates the host inflammatory response, minimizing potential tissue damage. Sequence and epigenetic variants in genes involved in regulating the neuroendocrine and immune systems are likely to contribute to individual differences in the HPAA response, and this may influence the host anti-inflammatory response to toxin exposure and susceptibility to inflammatory disease. In thismore » study, high (HCR) and low (LCR) cortisol responders were selected from a normal population of 110 female sheep challenged iv with Escherichia coli endotoxin (400 ng/kg) to identify potential determinants that contribute to variation in the cortisol response phenotype. This phenotype was stable over several years in the HCR and LCR animals, and did not appear to be attributed to differences in expression of hepatic immune-related genes or systemic pro-inflammatory cytokine concentrations. Mechanistic studies using corticotrophin-releasing factor (0.5 {mu}g/kg body weight), arginine vasopressin (0.5 {mu}g/kg), and adrenocorticotropic hormone (0.5 {mu}g/kg) administered iv demonstrated that variation in this phenotype is largely determined by signalling within the HPAA. Future studies will use this ovine HCR/LCR model to investigate potential genetic and epigenetic variants that may contribute to variation in cortisol responsiveness to bacterial endotoxin.« less

  1. Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems.

    PubMed

    Shaw, Jennifer L A; Monis, Paul; Weyrich, Laura S; Sawade, Emma; Drikas, Mary; Cooper, Alan J

    2015-09-01

    Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. PHYLOGENETIC AFFILIATION OF WATER DISTRIBUTION SYSTEM BACTERIAL ISOLATES USING 16S RDNA SEQUENCE ANALYSIS

    EPA Science Inventory

    In a previously described study, only 15% of the bacterial strains isolated from a water distribution system (WDS) grown on R2A agar were identifiable using fatty acid methyl esthers (FAME) profiling. The lack of success was attributed to the use of fatty acid databases of bacter...

  3. Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq

    PubMed Central

    Wilson, J. W.; Ott, C. M.; zu Bentrup, K. Höner; Ramamurthy, R.; Quick, L.; Porwollik, S.; Cheng, P.; McClelland, M.; Tsaprailis, G.; Radabaugh, T.; Hunt, A.; Fernandez, D.; Richter, E.; Shah, M.; Kilcoyne, M.; Joshi, L.; Nelman-Gonzalez, M.; Hing, S.; Parra, M.; Dumars, P.; Norwood, K.; Bober, R.; Devich, J.; Ruggles, A.; Goulart, C.; Rupert, M.; Stodieck, L.; Stafford, P.; Catella, L.; Schurr, M. J.; Buchanan, K.; Morici, L.; McCracken, J.; Allen, P.; Baker-Coleman, C.; Hammond, T.; Vogel, J.; Nelson, R.; Pierson, D. L.; Stefanyshyn-Piper, H. M.; Nickerson, C. A.

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth. PMID:17901201

  4. Systems Biophysics of Gene Expression

    PubMed Central

    Vilar, Jose M.G.; Saiz, Leonor

    2013-01-01

    Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365

  5. Diagnostic test accuracy of a 2-transcript host RNA signature for discriminating bacterial vs viral infection in febrile children

    PubMed Central

    Shailes, Hannah; Eleftherohorinou, Hariklia; Hoggart, Clive J; Cebey-Lopez, Miriam; Carter, Michael J; Janes, Victoria A; Gormley, Stuart; Shimizu, Chisato; Tremoulet, Adriana H; Barendregt, Anouk M; Salas, Antonio; Kanegaye, John; Pollard, Andrew J; Faust, Saul N; Patel, Sanjay; Kuijpers, Taco; Martinon-Torres, Federico; Burns, Jane C; Coin, Lachlan JM; Levin, Michael

    2018-01-01

    Importance As clinical features do not reliably distinguish bacterial from viral infection, many children worldwide receive unnecessary antibiotic treatment whilst bacterial infection is missed in others. Objective To identify a blood RNA expression signature that distinguishes bacterial from viral infection in febrile children. Design Febrile children presenting to participating hospitals in UK, Spain, Netherlands and USA between 2009-2013 were prospectively recruited, comprising a discovery group and validation group. Each group was classified after microbiological investigation into definite bacterial, definite viral infection or indeterminate infection. RNA expression signatures distinguishing definite bacterial from viral infection were identified in the discovery group and diagnostic performance assessed in the validation group. Additional validation was undertaken in separate studies of children with meningococcal disease (n=24) inflammatory diseases (n=48), and on published gene expression datasets. Exposures A 2-transcript RNA expression signature distinguishing bacterial infection from viral infection was evaluated against clinical and microbiological diagnosis. Main Outcomes Definite Bacterial and viral infection was confirmed by culture or molecular detection of the pathogens. Performance of the RNA signature was evaluated in the definite bacterial and viral group, and the indeterminate group. Results The discovery cohort of 240 children (median age 19 months, 62% males) included 52 with definite bacterial infection of whom 36 (69%) required intensive care; and 92 with definite viral infection of whom 32 (35%) required intensive care. 96 children had indeterminate infection. Bioinformatic analysis of RNA expression data identified a 38-transcript signature distinguishing bacterial from viral infection. A smaller (2-transcript) signature (FAM89A and IFI44L) was identified by removing highly correlated transcripts. When this 2-transcript signature was

  6. Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

    PubMed Central

    Atilano, Magda Luciana; Pereira, Pedro Matos; Vaz, Filipa; Catalão, Maria João; Reed, Patricia; Grilo, Inês Ramos; Sobral, Rita Gonçalves; Ligoxygakis, Petros; Pinho, Mariana Gomes; Filipe, Sérgio Raposo

    2014-01-01

    Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001 PMID:24692449

  7. Repressor-mediated tissue-specific gene expression in plants

    DOEpatents

    Meagher, Richard B [Athens, GA; Balish, Rebecca S [Oxford, OH; Tehryung, Kim [Athens, GA; McKinney, Elizabeth C [Athens, GA

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  8. Genome-wide identification, characterization and expression analyses of two TNFRs in Yesso scallop (Patinopecten yessoensis) provide insight into the disparity of responses to bacterial infections and heat stress in bivalves.

    PubMed

    Xing, Qiang; Yu, Qian; Dou, Huaiqian; Wang, Jing; Li, Ruojiao; Ning, Xianhui; Wang, Ruijia; Wang, Shi; Zhang, Lingling; Hu, Xiaoli; Bao, Zhenmin

    2016-05-01

    Tumor necrosis factors receptors (TNFRs) comprise a superfamily of proteins characterized by a unique cysteine-rich domain (CRD) and play important roles in diverse physiological and pathological processes in the innate immune system, including inflammation, apoptosis, autoimmunity and organogenesis. Although significant effects of TNFRs on immunity have been reported in most vertebrates as well as some invertebrates, the complete TNFR superfamily has not been systematically characterized in scallops. In this study, two different types of TNFR-like genes, including PyTNFR1 and PyTNFR2 genes were identified from Yesso scallop (Patinopecten yessoensis, Jay, 1857) through whole-genome scanning. Phylogenetic and protein structural analyses were carried out to determine the identities and evolutionary relationships of the two genes. The expression profiling of PyTNFRs was performed at different development stages, in healthy adult tissues and in hemocytes after bacterial infection and heat stress. Expression analysis revealed that both PyTNFRs were significantly induced during the acute phase (3 h) after infection with Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacteria, though much more dramatic chronic-phase (24 h) changes were observed after V. anguillarum challenge. For heat stress, only PyTNFR2 displayed significant elevation at 12 h and 24 h, which suggests a functional difference in the two PyTNFRs. Collectively, this study provides novel insight into the PyTNFRs and the specific role and response of TNFR-involved pathways in host immune responses against different bacterial pathogens and heat stress in bivalves. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. A bacterial genetic selection system for ubiquitylation cascade discovery.

    PubMed

    Levin-Kravets, Olga; Tanner, Neta; Shohat, Noa; Attali, Ilan; Keren-Kaplan, Tal; Shusterman, Anna; Artzi, Shay; Varvak, Alexander; Reshef, Yael; Shi, Xiaojing; Zucker, Ori; Baram, Tamir; Katina, Corine; Pilzer, Inbar; Ben-Aroya, Shay; Prag, Gali

    2016-11-01

    About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

  10. L-arginine mediated renaturation enhances yield of human, α6 type IV collagen non-collagenous domain from bacterial inclusion bodies

    PubMed Central

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Akul Sudhakar, Yakkanti

    2012-01-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated. PMID:22512648

  11. L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

    PubMed

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

    2012-10-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

  12. Warfare between Host Immunity and Bacterial Weapons.

    PubMed

    Yu, Manda; Lai, Erh-Min

    2017-01-11

    Bacterial pathogens deploy protein secretion systems to facilitate infection and colonization of their hosts. In this issue of Cell Host & Microbe, Chen et al. (2017) report a new role for a type VI secretion effector in promoting bacterial colonization by preventing inflammasome activation induced by a type III secretion system. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Characterization of microfouling and corrosive bacterial community of a firewater distribution system.

    PubMed

    Palaniappan, Balamurugan; Toleti, Subba Rao

    2016-04-01

    This investigation provides generic information on the culturable corrosive and the microfouling bacterial community in a firewater distribution system that uses freshwater. Conventional microbiological methods were used for the selective isolation of the major microfouling bacteria. The isolates were characterized by 16S rRNA gene sequencing and the biofilm as well as the corrosion characteristics of the isolates were evaluated. Pseudomonas aeruginosa and Bacillus cereus were predominantly observed in all the samples analysed. Denaturing gradient gel electrophoresis (DGGE) was carried out for the various samples of firewater system (FWS) and the high intensity bands were sequenced to identify the predominant bacteria. Bacterial groups such as Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes were identified. Biofilm thickness was recorded using confocal scanning laser microscopy (CSLM). This was the first study to report Lysinibacillus fusiformis in a firewater system and its role in iron corrosion. Sulphidogenic bacteria Tissierella sp. and Clostridium bifermentans generated sulphides in the range of 400-900 ppm. Significant corrosion rates of carbon steel (CS) coupons were observed up to 4.3 mpy. C. bifermentans induced more localized corrosion in CS with a pit diameter of 50 μm. Overall, the data on the characterization of the fouling bacteria, their biofilm forming potential and subsequent metal deterioration studies supported in designing an effective water treatment program. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Gene expression systems in corynebacteria.

    PubMed

    Srivastava, Preeti; Deb, J K

    2005-04-01

    Corynebacterium belongs to a group of gram-positive bacteria having moderate to high G+C content, the other members being Mycobacterium, Nocardia, and Rhodococcus. Considerable information is now available on the plasmids, gene regulatory elements, and gene expression in corynebacteria, especially in soil corynebacteria such as Corynebacterium glutamicum. These bacteria are non-pathogenic and, unlike Bacillus and Streptomyces, are low in proteolytic activity and thus have the potential of becoming attractive systems for expression of heterologous proteins. This review discusses recent advances in our understanding of the organization of various regulatory elements, such as promoters, transcription terminators, and development of vectors for cloning and gene expression.

  15. Gene and transcript abundances of bacterial type III secretion systems from the rumen microbiome are correlated with methane yield in sheep.

    PubMed

    Kamke, Janine; Soni, Priya; Li, Yang; Ganesh, Siva; Kelly, William J; Leahy, Sinead C; Shi, Weibing; Froula, Jeff; Rubin, Edward M; Attwood, Graeme T

    2017-08-08

    Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH 4 /kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. This is the first report of bacterial type III secretion system genes being associated with high

  16. Genetic analysis of a bacterial genetic exchange element: The gene transfer agent of Rhodobacter capsulatus

    PubMed Central

    Lang, Andrew S.; Beatty, J. T.

    2000-01-01

    An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell. PMID:10639170

  17. Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis

    PubMed Central

    Cui, Zhouqi; Jin, Guoqiang; Li, Bin; Kakar, Kaleem Ullah; Ojaghian, Mohammad Reza; Wang, Yangli; Xie, Guanlin; Sun, Guochang

    2015-01-01

    Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H2O2 and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice. PMID:26378528

  18. Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

    PubMed

    Bahar, Ofir; Mordukhovich, Gideon; Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Jehle, Anna Kristina; Felix, Georg; Ronald, Pamela C

    2016-05-01

    Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

  19. Genome-wide identification and characterization of five MyD88 duplication genes in Yesso scallop (Patinopecten yessoensis) and expression changes in response to bacterial challenge.

    PubMed

    Ning, Xianhui; Wang, Ruijia; Li, Xue; Wang, Shuyue; Zhang, Mengran; Xing, Qiang; Sun, Yan; Wang, Shi; Zhang, Lingling; Hu, Xiaoli; Bao, Zhenmin

    2015-10-01

    Myeloid differentiation factor 88 (MyD88) is a pivotal adaptor in the TLR/IL-1R signaling pathway, which plays an important role in activating the innate immune system. Although MyD88 genes have been identified in a variety of species, they have not been systematically characterized in scallops. In this study, five MyD88 genes were identified in Yesso scallop (Patinopecten yessoensis), PyMyD88-1, PyMyD88-2a, PyMyD88-2b, PyMyD88-3 and PyMyD88-4, which consisted of two pairs of tandem duplications located on the same chromosome. To our knowledge, this is the largest number of MyD88 genes found in an invertebrate. Phylogenetic and protein structural analyses were carried out to determine the identities and evolutionary relationships of these genes. PyMyD88s have highly conserved structures compared to MyD88 genes from other invertebrate species, except for PyMyD88-4, which contains only a DD domain, suggesting the evolutionarily conserved form of this particular gene member. We investigated the expression profiles of PyMyD88 genes at different developmental stages and in healthy adult tissues and hemocytes after Micrococcus luteus and Vibrio anguillarum infection using quantitative real-time PCR (qRT-PCR). The expression of most PyMyD88s was significantly induced in the acute phase (3-6 h) after infection with both gram-positive (M. luteus) and gram-negative (V. anguillarum) bacteria, with much more dramatic changes in PyMyD88 expression being observed after V. anguillarum challenge. Collectively, the abundance of MyD88s and their specific expression patterns provide insight into their versatile roles in the response of the bivalve innate immune system to gram-negative bacterial pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

    PubMed

    Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

    2015-12-30

    Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

  1. Application of a microcomputer-based system to control and monitor bacterial growth.

    PubMed

    Titus, J A; Luli, G W; Dekleva, M L; Strohl, W R

    1984-02-01

    A modular microcomputer-based system was developed to control and monitor various modes of bacterial growth. The control system was composed of an Apple II Plus microcomputer with 64-kilobyte random-access memory; a Cyborg ISAAC model 91A multichannel analog-to-digital and digital-to-analog converter; paired MRR-1 pH, pO(2), and foam control units; and in-house-designed relay, servo control, and turbidimetry systems. To demonstrate the flexibility of the system, we grew bacteria under various computer-controlled and monitored modes of growth, including batch, turbidostat, and chemostat systems. The Apple-ISAAC system was programmed in Labsoft BASIC (extended Applesoft) with an average control program using ca. 6 to 8 kilobytes of memory and up to 30 kilobytes for datum arrays. This modular microcomputer-based control system was easily coupled to laboratory scale fermentors for a variety of fermentations.

  2. Application of a Microcomputer-Based System to Control and Monitor Bacterial Growth

    PubMed Central

    Titus, Jeffrey A.; Luli, Gregory W.; Dekleva, Michael L.; Strohl, William R.

    1984-01-01

    A modular microcomputer-based system was developed to control and monitor various modes of bacterial growth. The control system was composed of an Apple II Plus microcomputer with 64-kilobyte random-access memory; a Cyborg ISAAC model 91A multichannel analog-to-digital and digital-to-analog converter; paired MRR-1 pH, pO2, and foam control units; and in-house-designed relay, servo control, and turbidimetry systems. To demonstrate the flexibility of the system, we grew bacteria under various computer-controlled and monitored modes of growth, including batch, turbidostat, and chemostat systems. The Apple-ISAAC system was programmed in Labsoft BASIC (extended Applesoft) with an average control program using ca. 6 to 8 kilobytes of memory and up to 30 kilobytes for datum arrays. This modular microcomputer-based control system was easily coupled to laboratory scale fermentors for a variety of fermentations. PMID:16346462

  3. T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System.

    PubMed

    Tay, Daniel Ming Ming; Govindarajan, Kunde Ramamoorthy; Khan, Asif M; Ong, Terenze Yao Rui; Samad, Hanif M; Soh, Wei Wei; Tong, Minyan; Zhang, Fan; Tan, Tin Wee

    2010-10-15

    Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of

  4. Behind the lines–actions of bacterial type III effector proteins in plant cells

    PubMed Central

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715

  5. [Risk factors associated with bacterial growth in derivative systems from cerebrospinal liquid in pediatric patients].

    PubMed

    de Jesús Vargas-Lares, José; Andrade-Aguilera, Angélica Rocío; Díaz-Peña, Rafael; Barrera de León, Juan Carlos

    2015-01-01

    To determine risk factors associated with bacterial growth in systems derived from cerebrospinal fluid in pediatric patients. Case and controls study from January to December 2012, in patients aged <16 years who were carriers of hydrocephalus and who required placement or replacement of derivative system. Cases were considered as children with cultures with bacterial growth and controls with negative bacterial growth. Inferential statistics with Chi-squared and Mann-Whitney U tests. Association of risk with odds ratio. We reviewed 746 registries, cases n=99 (13%) and controls n=647 (87%). Masculine gender 58 (57%) vs. feminine gender 297 (46%) (p=0.530). Age of cases: median, five months and controls, one year (p=0.02). Median weight, 7 vs. 10 kg (p=0.634). Surgical interventions: median n=2 (range, 1-8) vs. n=1 (range, 1-7). Infection rate, 13.2%. Main etiology ductal stenosis, n=29 (29%) vs. n=50 (23%) (p=0.530). Non-communicating, n=50 (51%) vs. 396 (61%) (p=0.456). Predominant microorganisms: enterobacteria, pseudomonas, and enterococcus. Non-use of iodized dressing OR=2.6 (range, 1.8-4.3), use of connector OR=6.8 (range, 1.9-24.0), System replacement OR=2.0 (range, 1.3-3.1), assistant without surgical facemask OR=9.7 (range, 2.3-42.0). Being a breastfeeding infant, of low weight, non-application of iodized dressing, use of connector, previous derivation, and lack of adherence to aseptic technique were all factors associated with ependymitis.

  6. Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance.

    PubMed

    Cao, Qi; Feng, Fenfen; Wang, Huan; Xu, Xiaojuan; Chen, Huanchun; Cai, Xuwang; Wang, Xiangru

    2018-01-01

    Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. [Bacterial translocation: gap in the shield].

    PubMed

    Rosero, Olivér; Kovács, Tibor; Onody, Péter; Harsányi, László; Szijártó, Attila

    2014-02-23

    The gastrointestinal tract is not only regarded as a system where nutrient absorption takes place, but also as a vital barrier against intraluminal pathogens entering the circulation and the maintenance of immune homeostasis. Bacterial translocation is defined as the penetration of viable bacteria or bacterial compounds from the gastrointestinal tract to extraintestinal sites. This disorder has been described in several clinical conditions. The main promoting factors for bacterial translocation have been proposed to be changes in the intestinal microflora, mucosal barrier failure and defects in host immunity. The presence of bacterial translocation has been associated with higher complications and mortality rates; therefore it should be taken into account in the therapeutic strategies of patients with predisposing factors.

  8. Preventive effects of Schistosoma japonicum ova on trinitrobenzenesulfonic acid-induced colitis and bacterial translocation in mice.

    PubMed

    Zhao, Yuan; Zhang, Shuncai; Jiang, Li; Jiang, Jie; Liu, Hongchun

    2009-11-01

    To evaluate the preventive effects of Schistosoma japonicum ova on trinitrobenzenesulfonic acid (TNBS)-induced colitis and bacterial translocation in mice. BALB/c mice were randomly divided into three groups: control group; TNBS(+)Ova(-) group; and TNBS(+)Ova(+) group. Mice of the TNBS(+)Ova(+) group were exposed to 10 000 freeze-killed S. japonicum ova by i.p. injection on day 1 and day 11. On day 15, mice were challenged with TNBS to induce colitis. The following variables were assessed: colon pathological changes; serum expression of tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and interleukin-10 (IL-10); expression of Toll-like receptor 4 (TLR4) in colon; IFN-gamma, IL-10 and TLR4 mRNA expression in colon; and the bacterial translocation rate. Compared to TNBS(+)Ova(-) group, the colonic inflammation in the TNBS(+)Ova(+) group were relieved. A highly significant elevation of IFN-gamma and TNF-alpha were observed in the TNBS-induced colitis group. After exposure to the eggs, IFN-gamma was significantly decreased, while TNF-alpha was similar to that of the TNBS(+)ova(-) group. No obvious variation was seen in IL-10 expression in TNBS-induced colitis, compared to the controls. Exposure to the eggs led to a significant upregulation of IL-10 expression. TLR4 expression was elevated after injected with TNBS and was downregulated in the eggs group. Less intestinal bacterial translocation frequency was observed when exposed to eggs. S. japonicum ova can prevent the TNBS-induced colitis and reduce the bacterial translocation frequency in mice. The mechanisms were supposed to be due to the regulation of T-helper cell 1/2 balance and TLR4 expression.

  9. Effect of TiO2 nanoparticles on aerobic granulation of algal-bacterial symbiosis system and nutrients removal from synthetic wastewater.

    PubMed

    Li, Bing; Huang, Wenli; Zhang, Chao; Feng, Sisi; Zhang, Zhenya; Lei, Zhongfang; Sugiura, Norio

    2015-01-01

    The influence of TiO2 nanoparticles (TiO2-NPs) (10-50mg/L) on aerobic granulation of algal-bacterial symbiosis system was investigated by using two identical sequencing batch reactors (SBRs). Although little adverse effect was observed on their nitritation efficiency (98-100% in both reactors), algal-bacterial granules in the control SBR (Rc) gradually lost stability mainly brought about by algae growth. TiO2-NPs addition to RT was found to enhance the granulation process achieving stable and compact algal-bacterial granules with remarkably improved nitratation thus little nitrite accumulation in RT when influent TiO2-NPs⩾30mg/L. Despite almost similar organics and phosphorus removals obtained in both reactors, the stably high nitratation efficiency in addition to much stable granular structure in RT suggests that TiO2-NPs addition might be a promising remedy for the long-term operation of algal-bacterial granular system, most probably attributable to the stimulated excretion of extracellular polymeric substances and less filamentous TM7. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages.

    PubMed

    Qin, Wanhai; Wang, Lei; Zhai, Ruidong; Ma, Qiuyue; Liu, Jianfang; Bao, Chuntong; Zhang, Hu; Sun, Changjiang; Feng, Xin; Gu, Jingmin; Du, Chongtao; Han, Wenyu; Langford, P R; Lei, Liancheng

    2016-01-01

    Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.

  11. Bacterial communities associated with an occurrence of colored water in an urban drinking water distribution system.

    PubMed

    Wu, Hui Ting; Mi, Zi Long; Zhang, Jing Xu; Chen, Chao; Xie, Shu Guang

    2014-08-01

    This study aimed to investigate bacterial community in an urban drinking water distribution system (DWDS) during an occurrence of colored water. Variation in the bacterial community diversity and structure was observed among the different waters, with the predominance of Proteobacteria. While Verrucomicrobia was also a major phylum group in colored water. Limnobacter was the major genus group in colored water, but Undibacterium predominated in normal tap water. The coexistence of Limnobacter as well as Sediminibacterium and Aquabacterium might contribute to the formation of colored water. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  12. Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions.

    PubMed

    diCenzo, George C; Finan, Turlough M

    2018-01-01

    The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArray™ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.

  13. Patterns of Bacterial and Archaeal Gene Expression through the Lower Amazon River

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Satinsky, Brandon M.; Smith, Christa B.; Sharma, Shalabh

    Analysis of metatranscriptomic and metagenomic datasets from the lower reaches of the Amazon River between Obidos and the river mouth revealed microbial transcript and gene pools dominated by Actinobacteria, Thaumarchaeota, Bacteroidetes, Acidobacteria, Betaproteobacteria, and Planctomycetes. Three mainstem stations spanning a 625 km reach had similar gene expression patterns (transcripts gene copy-1) across a diverse suite of element cycling genes, but two tributary-influenced stations at the mouth of the Tapajos River and near the Tocantins River at Belem had distinct transcriptome composition and expression ratios, particularly for genes encoding light-related energy capture (higher) and iron acquisition and ammonia oxidation (lower). Environmentalmore » parameters that were useful predictors of gene expression ratios included concentrations of lignin phenols, suspended sediments, nitrate, phosphate, and particulate organic carbon and nitrogen. Similar to the gene expression data, these chemical properties reflected highly homogeneous mainstem stations punctuated by distinct tributary- influenced stations at Tapajos and Belem. Although heterotrophic processes were expected to dominate in the lower Amazon, transcripts from photosynthetic bacteria were abundant in tributary-influenced regions, and transcripts from Thaumarcheota taxa genetically capable of chemosynthetic ammonia oxidation accounted for up to 21% of the transcriptome at others. Based on regressions of transcript numbers against gene numbers, expression ratios of Thaumarchaeota populations were largely unchanged within the mainstem, suggesting a relatively minor role for gene regulation. These quantitative gene and transcript inventories detail a diverse array of energy acquisition strategies and metabolic capabilities for bacteria and archaea populations of the world’s largest river system.« less

  14. Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration.

    PubMed

    Douterelo, I; Sharpe, R; Boxall, J

    2014-07-01

    To characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). Biofilm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Biofilm cell cover was monitored by fluorescent microscopy and a fingerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed significant changes in the bacterial community structure over time (P < 0·05). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on fingerprinting results, the hydraulic regimes did not affect the bacteriological composition of biofilms, but they did influence their mechanical stability. This study gives a better insight into the early stages of biofilm formation in DWDS and will contribute to the improvement of management strategies to control the formation of biofilms and the risk of discolouration. © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.

  15. Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration

    PubMed Central

    Douterelo, I; Sharpe, R; Boxall, J

    2014-01-01

    Aims To characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). Methods and Results Biofilm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Biofilm cell cover was monitored by fluorescent microscopy and a fingerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed significant changes in the bacterial community structure over time (P < 0·05). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. Conclusions Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on fingerprinting results, the hydraulic regimes did not affect the bacteriological composition of biofilms, but they did influence their mechanical stability. Significance and Importance of the Study This study gives a better insight into the early stages of biofilm formation in DWDS and will contribute to the improvement of management strategies to control the formation of biofilms and the risk of discolouration. PMID:24712449

  16. Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations: How bacterial secretion systems bias Brownian motion for efficient translocation of macromolecules.

    PubMed

    Hepp, Christof; Maier, Berenike

    2017-10-01

    Secretion systems enable bacteria to import and secrete large macromolecules including DNA and proteins. While most components of these systems have been identified, the molecular mechanisms of macromolecular transport remain poorly understood. Recent findings suggest that various bacterial secretion systems make use of the translocation ratchet mechanism for transporting polymers across the cell envelope. Translocation ratchets are powered by chemical potential differences generated by concentration gradients of ions or molecules that are specific to the respective secretion systems. Bacteria employ these potential differences for biasing Brownian motion of the macromolecules within the conduits of the secretion systems. Candidates for this mechanism include DNA import by the type II secretion/type IV pilus system, DNA export by the type IV secretion system, and protein export by the type I secretion system. Here, we propose that these three secretion systems employ different molecular implementations of the translocation ratchet mechanism. © 2017 The Authors. BioEssays Published by WILEY Periodicals, Inc.

  17. Pathogen espionage: multiple bacterial adrenergic sensors eavesdrop on host communication systems.

    PubMed

    Karavolos, Michail H; Winzer, Klaus; Williams, Paul; Khan, C M Anjam

    2013-02-01

    The interactions between bacterial pathogens and their eukaryotic hosts are vital in determining the outcome of infections. Bacterial pathogens employ molecular sensors to detect and facilitate adaptation to changes in their niche. The sensing of these extracellular signals enables the pathogen to navigate within mammalian hosts. Intercellular bacterial communication is facilitated by the production and sensing of autoinducer (AI) molecules via quorum sensing. More recently, AI-3 and the host neuroendocrine (NE) hormones adrenaline and noradrenaline were reported to display cross-talk for the activation of the same signalling pathways. Remarkably, there is increasing evidence to suggest that enteric bacteria sense and respond to the host NE stress hormones adrenaline and noradrenaline to modulate virulence. These responses can be inhibited by α and β-adrenergic receptor antagonists implying a bacterial receptor-based sensing and signalling cascade. In Escherichia coli O157:H7 and Salmonella, QseC has been proposed as the adrenergic receptor. Strikingly, there is an increasing body of evidence that not all the bacterial adrenergic responses require signalling through QseC. Here we provide additional hypotheses to reconcile these observations implicating the existence of alternative adrenergic receptors including BasS, QseE and CpxA and their associated signalling cascades with major roles in interkingdom communication. © 2012 Blackwell Publishing Ltd.

  18. Succession of bacterial community composition over two consecutive years in two aquatic systems: a natural lake and a lake-reservoir.

    PubMed

    Boucher, Delphine; Jardillier, Ludwig; Debroas, Didier

    2006-01-01

    The succession in bacterial community composition was studied over two years in the epilimnion and hypolimnion of two freshwater systems: a natural lake (Pavin Lake) and a lake-reservoir (Sep Reservoir). The bacterial community composition was determined by cloning-sequencing of 16S rRNA and by terminal restriction fragment length polymorphism. Despite large hydrogeological differences, in the Sep Reservoir and Pavin Lake the dominant bacteria were from the same taxonomic divisions, particularly Actinobacteria and Betaproteobacteria. In both ecosystems, these major bacterial divisions showed temporal fluctuations that were much less marked than those occurring at a finer phylogenetic scale. Nutrient availability and mortality factors, the nature of which differed from one lake to another, covaried with the temporal variations in the bacterial community composition at all sampling depths, whereas factors related to seasonal forces (temperature and outflow for Sep Reservoir) seemed to account only for the variation of the hypolimnion bacterial community composition. No seasonal reproducibility in temporal evolution of bacterial community from one year to the next was observed.

  19. [Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane].

    PubMed

    Ziakun, A M; Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

    2007-01-01

    Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.

  20. Survivial Strategies in Bacterial Range Expansions

    NASA Astrophysics Data System (ADS)

    Frey, Erwin

    2014-03-01

    Bacterial communities represent complex and dynamic ecological systems. Different environmental conditions as well as bacterial interactions determine the establishment and sustainability of bacterial diversity. In this talk we discuss the competition of three Escherichia coli strains during range expansions on agar plates. In this bacterial model system, a colicin E2 producing strain C competes with a colicin resistant strain R and with a colicin sensitive strain S for new territory. Genetic engineering allows us to tune the growth rates of the strains and to study distinct ecological scenarios. These scenarios may lead to either single-strain dominance, pairwise coexistence, or to the coexistence of all three strains. In order to elucidate the survival mechanisms of the individual strains, we also developed a stochastic agent-based model to capture the ecological scenarios in silico. In a combined theoretical and experimental approach we are able to show that the level of biodiversity depends crucially on the composition of the inoculum, on the relative growth rates of the three strains, and on the effective reach of colicin toxicity.

  1. Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

    USDA-ARS?s Scientific Manuscript database

    Transcription activator-like (TAL) effectors found in Xanthomonas spp. promote bacterial growth and plant susceptibility by binding specific DNA sequences or, effector-binding elements (EBEs), and inducing host gene expression. In this study, we have found substantially different transcriptional pro...

  2. Dynamics of Wolbachia pipientis Gene Expression Across the Drosophila melanogaster Life Cycle

    PubMed Central

    Gutzwiller, Florence; Carmo, Catarina R.; Miller, Danny E.; Rice, Danny W.; Newton, Irene L. G.; Hawley, R. Scott; Teixeira, Luis; Bergman, Casey M.

    2015-01-01

    Symbiotic interactions between microbes and their multicellular hosts have manifold biological consequences. To better understand how bacteria maintain symbiotic associations with animal hosts, we analyzed genome-wide gene expression for the endosymbiotic α-proteobacteria Wolbachia pipientis across the entire life cycle of Drosophila melanogaster. We found that the majority of Wolbachia genes are expressed stably across the D. melanogaster life cycle, but that 7.8% of Wolbachia genes exhibit robust stage- or sex-specific expression differences when studied in the whole-organism context. Differentially-expressed Wolbachia genes are typically up-regulated after Drosophila embryogenesis and include many bacterial membrane, secretion system, and ankyrin repeat-containing proteins. Sex-biased genes are often organized as small operons of uncharacterized genes and are mainly up-regulated in adult Drosophila males in an age-dependent manner. We also systematically investigated expression levels of previously-reported candidate genes thought to be involved in host-microbe interaction, including those in the WO-A and WO-B prophages and in the Octomom region, which has been implicated in regulating bacterial titer and pathogenicity. Our work provides comprehensive insight into the developmental dynamics of gene expression for a widespread endosymbiont in its natural host context, and shows that public gene expression data harbor rich resources to probe the functional basis of the Wolbachia-Drosophila symbiosis and annotate the transcriptional outputs of the Wolbachia genome. PMID:26497146

  3. The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival

    PubMed Central

    Moulin, Pauline; Patron, Kévin; Cano, Camille; Zorgani, Mohamed Amine; Camiade, Emilie; Borezée-Durant, Elise; Rosenau, Agnès; Mereghetti, Laurent

    2016-01-01

    ABSTRACT The Lmb protein of Streptococcus agalactiae is described as an adhesin that binds laminin, a component of the human extracellular matrix. In this study, we revealed a new role for this protein in zinc uptake. We also identified two Lmb homologs, AdcA and AdcAII, redundant binding proteins that combine with the AdcCB translocon to form a zinc-ABC transporter. Expression of this transporter is controlled by the zinc concentration in the medium through the zinc-dependent regulator AdcR. Triple deletion of lmb, adcA, and adcAII, or that of the adcCB genes, impaired growth and cell separation in a zinc-restricted environment. Moreover, we found that this Adc zinc-ABC transporter promotes S. agalactiae growth and survival in some human biological fluids, suggesting that it contributes to the infection process. These results indicated that zinc has biologically vital functions in S. agalactiae and that, under the conditions tested, the Adc/Lmb transporter constitutes the main zinc acquisition system of the bacterium. IMPORTANCE A zinc transporter, composed of three redundant binding proteins (Lmb, AdcA, and AdcAII), was characterized in Streptococcus agalactiae. This system was shown to be essential for bacterial growth and morphology in zinc-restricted environments, including human biological fluids. PMID:27672194

  4. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    DOE PAGES

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; ...

    2015-01-19

    Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

  5. Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila.

    PubMed

    Fontana, Mary F; Banga, Simran; Barry, Kevin C; Shen, Xihui; Tan, Yunhao; Luo, Zhao-Qing; Vance, Russell E

    2011-02-01

    The intracellular bacterial pathogen Legionella pneumophila causes an inflammatory pneumonia called Legionnaires' Disease. For virulence, L. pneumophila requires a Dot/Icm type IV secretion system that translocates bacterial effectors to the host cytosol. L. pneumophila lacking the Dot/Icm system is recognized by Toll-like receptors (TLRs), leading to a canonical NF-κB-dependent transcriptional response. In addition, L. pneumophila expressing a functional Dot/Icm system potently induces unique transcriptional targets, including proinflammatory genes such as Il23a and Csf2. Here we demonstrate that this Dot/Icm-dependent response, which we term the effector-triggered response (ETR), requires five translocated bacterial effectors that inhibit host protein synthesis. Upon infection of macrophages with virulent L. pneumophila, these five effectors caused a global decrease in host translation, thereby preventing synthesis of IκB, an inhibitor of the NF-κB transcription factor. Thus, macrophages infected with wildtype L. pneumophila exhibited prolonged activation of NF-κB, which was associated with transcription of ETR target genes such as Il23a and Csf2. L. pneumophila mutants lacking the five effectors still activated TLRs and NF-κB, but because the mutants permitted normal IκB synthesis, NF-κB activation was more transient and was not sufficient to fully induce the ETR. L. pneumophila mutants expressing enzymatically inactive effectors were also unable to fully induce the ETR, whereas multiple compounds or bacterial toxins that inhibit host protein synthesis via distinct mechanisms recapitulated the ETR when administered with TLR ligands. Previous studies have demonstrated that the host response to bacterial infection is induced primarily by specific microbial molecules that activate TLRs or cytosolic pattern recognition receptors. Our results add to this model by providing a striking illustration of how the host immune response to a virulent pathogen can also

  6. Bacterial disease management: challenges, experience, innovation and future prospects: Challenges in Bacterial Molecular Plant Pathology.

    PubMed

    Sundin, George W; Castiblanco, Luisa F; Yuan, Xiaochen; Zeng, Quan; Yang, Ching-Hong

    2016-12-01

    Plant diseases caused by bacterial pathogens place major constraints on crop production and cause significant annual losses on a global scale. The attainment of consistent effective management of these diseases can be extremely difficult, and management potential is often affected by grower reliance on highly disease-susceptible cultivars because of consumer preferences, and by environmental conditions favouring pathogen development. New and emerging bacterial disease problems (e.g. zebra chip of potato) and established problems in new geographical regions (e.g. bacterial canker of kiwifruit in New Zealand) grab the headlines, but the list of bacterial disease problems with few effective management options is long. The ever-increasing global human population requires the continued stable production of a safe food supply with greater yields because of the shrinking areas of arable land. One major facet in the maintenance of the sustainability of crop production systems with predictable yields involves the identification and deployment of sustainable disease management solutions for bacterial diseases. In addition, the identification of novel management tactics has also come to the fore because of the increasing evolution of resistance to existing bactericides. A number of central research foci, involving basic research to identify critical pathogen targets for control, novel methodologies and methods of delivery, are emerging that will provide a strong basis for bacterial disease management into the future. Near-term solutions are desperately needed. Are there replacement materials for existing bactericides that can provide effective disease management under field conditions? Experience should inform the future. With prior knowledge of bactericide resistance issues evolving in pathogens, how will this affect the deployment of newer compounds and biological controls? Knowledge is critical. A comprehensive understanding of bacterial pathosystems is required to not

  7. Expression and in vitro functional analyses of recombinant Gam1 protein

    PubMed Central

    Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

    2014-01-01

    Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

  8. Responses of Bacterial Communities in Arable Soils in a Rice-Wheat Cropping System to Different Fertilizer Regimes and Sampling Times

    PubMed Central

    Zhao, Jun; Ni, Tian; Li, Yong; Xiong, Wu; Ran, Wei; Shen, Biao; Shen, Qirong; Zhang, Ruifu

    2014-01-01

    Soil physicochemical properties, soil microbial biomass and bacterial community structures in a rice-wheat cropping system subjected to different fertilizer regimes were investigated in two seasons (June and October). All fertilizer regimes increased the soil microbial biomass carbon and nitrogen. Both fertilizer regime and time had a significant effect on soil physicochemical properties and bacterial community structure. The combined application of inorganic fertilizer and manure organic-inorganic fertilizer significantly enhanced the bacterial diversity in both seasons. The bacterial communities across all samples were dominated by Proteobacteria, Acidobacteria and Chloroflexi at the phylum level. Permutational multivariate analysis confirmed that both fertilizer treatment and season were significant factors in the variation of the composition of the bacterial community. Hierarchical cluster analysis based on Bray-Curtis distances further revealed that bacterial communities were separated primarily by season. The effect of fertilizer treatment is significant (P = 0.005) and accounts for 7.43% of the total variation in bacterial community. Soil nutrients (e.g., available K, total N, total P and organic matter) rather than pH showed significant correlation with the majority of abundant taxa. In conclusion, both fertilizer treatment and seasonal changes affect soil properties, microbial biomass and bacterial community structure. The application of NPK plus manure organic-inorganic fertilizer may be a sound fertilizer practice for sustainable food production. PMID:24465530

  9. Method for construction of bacterial strains with increased succinic acid production

    DOEpatents

    Donnelly, Mark I.; Sanville-Millard, Cynthia; Chatterjee, Ranjini

    2000-01-01

    A fermentation process for producing succinic acid is provided comprising selecting a bacterial strain that does not produce succinic acid in high yield, disrupting the normal regulation of sugar metabolism of said bacterial strain, and combining the mutant bacterial strain and selected sugar in anaerobic conditions to facilitate production of succinic acid. Also provided is a method for changing low yield succinic acid producing bacteria to high yield succinic acid producing bacteria comprising selecting a bacterial strain having a phosphotransferase system and altering the phosphotransferase system so as to allow the bacterial strain to simultaneously metabolize different sugars.

  10. LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    PubMed Central

    Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A.; Briggs, Winslow R.; Goldbaum, Fernando A.; Paris, Gastón

    2015-01-01

    Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms. PMID:25993430

  11. Human Tear Fluid Reduces Culturability of Contact Lens Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence Associated Type III Secretion System

    PubMed Central

    Wu, Yvonne T.; Tam, Connie; Zhu, Lucia S.; Evans, David J.; Fleiszig, Suzanne M. J.

    2017-01-01

    Purpose The type III secretion system (T3SS) is a significant virulence determinant for Pseudomonas aeruginosa. Using a rodent model, we found that contact lens (CL)-related corneal infections were associated with lens surface biofilms. Here, we studied the impact of human tear fluid on CL-associated biofilm growth and T3SS expression. Methods P. aeruginosa biofilms were formed on contact lenses for up to 7 days with or without human tear fluid, then exposed to tear fluid for 5 or 24 h. Biofilms were imaged using confocal microscopy. Bacterial culturability was quantified by viable counts, and T3SS gene expression measured by RT-qPCR. Controls included trypticase soy broth, PBS and planktonic bacteria. Results With or without tear fluid, biofilms grew to ~108 cfu viable bacteria by 24 h. Exposing biofilms to tear fluid after they had formed without it on lenses reduced bacterial culturability ~180-fold (p<.001). CL growth increased T3SS gene expression versus planktonic bacteria [5.46 ± 0.24-fold for T3SS transcriptional activitor exsA (p=.02), and 3.76 ± 0.36-fold for T3SS effector toxin exoS (p=.01)]. Tear fluid further enhanced exsA and exoS expression in CL-grown biofilms, but not planktonic bacteria, by 2.09 ± 0.38-fold (p = 0.04) and 1.89 ± 0.26-fold (p<.001), respectively. Conclusions Considering the pivitol role of the T3SS in P. aeruginosa infections, its induction in CL-grown P. aeruginosa biofilms by tear fluid might contribute to the pathogenesis of CL-related P. aeruginosa keratitis. PMID:27670247

  12. Exploring bacterial infections: theoretical and experimental studies of the bacterial population dynamics and antibiotic treatment

    NASA Astrophysics Data System (ADS)

    Shao, Xinxian

    Bacterial infections are very common in human society. Thus extensive research has been conducted to reveal the molecular mechanisms of the pathogenesis and to evaluate the antibiotics' efficacy against bacteria. Little is known, however, about the population dynamics of bacterial populations and their interactions with the host's immune system. In this dissertation, a stochatic model is developed featuring stochastic phenotypic switching of bacterial individuals to explain the single-variant bottleneck discovered in multi strain bacterial infections. I explored early events in a bacterial infection establishment using classical experiments of Moxon and Murphy on neonatal rats. I showed that the minimal model and its simple variants do not work. I proposed modifications to the model that could explain the data quantitatively. The bacterial infections are also commonly established in physical structures, as biofilms or 3-d colonies. In contrast, most research on antibiotic treatment of bacterial infections has been conducted in well-mixed liquid cultures. I explored the efficacy of antibiotics to treat such bacterial colonies, a broadly applicable method is designed and evaluated where discrete bacterial colonies on 2-d surfaces were exposed to antibiotics. I discuss possible explanations and hypotheses for the experimental results. To verify these hypotheses, we investigated the dynamics of bacterial population as 3-d colonies. We showed that a minimal mathematical model of bacterial colony growth in 3-d was able to account for the experimentally observed presence of a diffusion-limited regime. The model further revealed highly loose packing of the cells in 3-d colonies and smaller cell sizes in colonies than plancktonic cells in corresponding liquid culture. Further experimental tests of the model predictions have revealed that the ratio of the cell size in liquid culture to that in colony cultures was consistent with the model prediction, that the dead cells

  13. Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores.

    PubMed

    Stachowiak, Jeanne C; Shugard, Erin E; Mosier, Bruce P; Renzi, Ronald F; Caton, Pamela F; Ferko, Scott M; Van de Vreugde, James L; Yee, Daniel D; Haroldsen, Brent L; VanderNoot, Victoria A

    2007-08-01

    For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.

  14. Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.

    PubMed

    Hill, Heather E; Pioszak, Augen A

    2013-03-01

    Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

    PubMed

    Hayashi, Tsuyoshi; Nakamichi, Masahiro; Naitou, Hirotaka; Ohashi, Norio; Imai, Yasuyuki; Miyake, Masaki

    2010-07-22

    Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

  16. Serum soluble ST2 as diagnostic marker of systemic inflammatory reactive syndrome of bacterial etiology in children.

    PubMed

    Calò Carducci, Francesca Ippolita; Aufiero, Lelia Rotondi; Folgori, Laura; Vittucci, Anna Chiara; Amodio, Donato; De Luca, Maia; Li Pira, Giuseppina; Bergamini, Alberto; Pontrelli, Giuseppe; Finocchi, Andrea; D'Argenio, Patrizia

    2014-02-01

    Accurate and timely diagnosis of community-acquired bacterial versus viral infections in children with systemic inflammatory response syndrome (SIRS) remains challenging both for clinician and laboratory. In the quest of new biochemical markers to distinguish bacterial from viral infection, we have explored the possible role of the soluble secreted form of ST2 (sST2). This explorative prospective cohort study included children with SIRS who were suspected of having community-acquired infections. Plasma samples for sST2 measurement were obtained from 64 hospitalized children, 41 of whom had SIRS of bacterial etiology and 23 SIRS of viral etiology, and from 20 healthy, age- and sex-matched control children. sST2 measurement was carried out by enzyme-linked immunosorbent assay in parallel with standard measurements of procalcitonin (PCT) and C reactive protein (CRP). Our findings demonstrate that children with SIRS associated with bacterial infection present significantly increased levels of sST2, when compared with patients with SIRS of viral etiology and healthy children. More important, receiver operating characteristic curve analysis indicated that sST2 has a significant diagnostic performance with respect to early identification of SIRS of bacterial etiology, which was similar to that of PCT and greater than that of CRP. Finally, the combination of sST2 plus PCT and/or CRP, and PCT plus CRP increased their sensitivity and negative predictive value compared with sST2, PCT and CRP alone. In conclusion, sST2 level may prove useful in predicting bacterial etiology in children with SIRS.

  17. Bacterial water quality and network hydraulic characteristics: a field study of a small, looped water distribution system using culture-independent molecular methods.

    PubMed

    Sekar, R; Deines, P; Machell, J; Osborn, A M; Biggs, C A; Boxall, J B

    2012-06-01

    To determine the spatial and temporal variability in the abundance, structure and composition of planktonic bacterial assemblages sampled from a small, looped water distribution system and to interpret results with respect to hydraulic conditions. Water samples were collected from five sampling points, twice a day at 06:00 h and 09:00 h on a Monday (following low weekend demand) and a Wednesday (higher midweek demand). All samples were fully compliant with current regulated parameter standards. This study did not show obvious changes in bacterial abundance (DAPI count) or community structure Denaturing gradient gel electrophoresis analysis with respect to sample site and hence to water age; however, the study did show temporal variability with respect to both sampling day and sample times. Data suggests that variations in the bacterial assemblages may be associated with the local system hydraulics: the bacterial composition and numbers, over short durations, are governed by the interaction of the bulk water and the biofilm influenced by the hydraulic conditions. This study demonstrates general stability in bacterial abundance, community structure and composition within the system studied. Trends and patterns supporting the transfer of idealized understanding to the real world were evident. Ultimately, such work will help to safeguard potable water quality, fundamental to public health. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  18. Bacterial nucleotide-based second messengers.

    PubMed

    Pesavento, Christina; Hengge, Regine

    2009-04-01

    In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

  19. Stable Host Gene Expression in the Gut of Adult Drosophila melanogaster with Different Bacterial Mono-Associations

    PubMed Central

    Zhang, Vivian; Ludington, William B.; Eisen, Michael B.

    2016-01-01

    There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but we still do not understand how they accomplish this. Here, we evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. We observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, we found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, we found whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. Our data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body. PMID:27898741

  20. Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth.

    PubMed

    Merali, Zara; Mayer, Melinda J; Parker, Mary L; Michael, Anthony J; Smith, Andrew C; Waldron, Keith W

    2012-06-01

    Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications. Copyright © Physiologia Plantarum 2012.

  1. Bacterial community characterization and biogeochemistry of sediments from a tropical upwelling system (Cabo Frio, Southeastern Brazil)

    NASA Astrophysics Data System (ADS)

    Castelo-Branco, R.; Barreiro, A.; Silva, F. S.; Carvalhal-Gomes, S. B. V.; Fontana, L. F.; Mendonça-Filho, J. G.; Vasconcelos, V.

    2016-11-01

    The Cabo Frio Upwelling System is one of the largest and most productive areas in southeastern Brazil. Although it is well-known that bacterial communities play a crucial role in the biogeochemical cycles and food chain of marine ecosystems, little is known regarding the microbial communities in the sediments of this upwelling region. In this research, we address the effect of different hydrological conditions on the biogeochemistry of sediments and the diversity of bacterial communities. Biogeochemistry profiles of sediments from four sampling stations along an inner-outer transect on the continental shelf were evaluated and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments was used to study the bacterial community composition in these sediments. Our sequencing analysis of excised bands identified Alpha- and Gammaproteobacteria, Bacteroidetes and bacteria belonging to the Firmicutes phyla as the phylogenetic groups, indicating the existence of great diversity in these marine sediments. In this multidisciplinary study, the use of multivariate analysis was crucial for understanding how biogeochemical profiles influence bacterial community distribution. A Principal Component Analysis (PCA) indicated that the biogeochemical variables exhibited a clear spatial pattern that is mainly related to hydrological conditions. A Correspondence Analysis (CA) revealed an important association between certain taxonomic groups and specific sampling locations. Canonical Correspondence Analysis (CCA) demonstrated that the biogeochemistry influences the structure of the bacterial community in sediments. Among the bacterial groups identified, the most taxonomically diverse classes (Alphaproteobacteria and Gammaproteobacteria) were found to be distributed regardless of any studied biogeochemical variables influences, whereas other groups responded to biogeochemical conditions which, in turn, were influenced by hydrological conditions. This finding

  2. A stable live bacterial vaccine.

    PubMed

    Kunda, Nitesh K; Wafula, Denis; Tram, Meilinn; Wu, Terry H; Muttil, Pavan

    2016-06-01

    Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Proposal of Self-Learning and Recognition System of Facial Expression

    NASA Astrophysics Data System (ADS)

    Ogawa, Yukihiro; Kato, Kunihito; Yamamoto, Kazuhiko

    We describe realization of more complicated function by using the information acquired from some equipped unripe functions. The self-learning and recognition system of the human facial expression, which achieved under the natural relation between human and robot, are proposed. The robot with this system can understand human facial expressions and behave according to their facial expressions after the completion of learning process. The system modelled after the process that a baby learns his/her parents’ facial expressions. Equipping the robot with a camera the system can get face images and equipping the CdS sensors on the robot’s head the robot can get the information of human action. Using the information of these sensors, the robot can get feature of each facial expression. After self-learning is completed, when a person changed his facial expression in front of the robot, the robot operates actions under the relevant facial expression.

  4. Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

    PubMed

    Philip, Naomi H; DeLaney, Alexandra; Peterson, Lance W; Santos-Marrero, Melanie; Grier, Jennifer T; Sun, Yan; Wynosky-Dolfi, Meghan A; Zwack, Erin E; Hu, Baofeng; Olsen, Tayla M; Rongvaux, Anthony; Pope, Scott D; López, Carolina B; Oberst, Andrew; Beiting, Daniel P; Henao-Mejia, Jorge; Brodsky, Igor E

    2016-10-01

    Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense.

  5. Isolation of prawn ( Exopalaemon carinicauda) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

    NASA Astrophysics Data System (ADS)

    Ge, Qianqian; Li, Jian; Duan, Yafei; Li, Jitao; Sun, Ming; Zhao, Fazhen

    2016-04-01

    The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn ( Exopalaemon carinicauda) ( EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

  6. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

    PubMed

    Vercoe, Reuben B; Chang, James T; Dy, Ron L; Taylor, Corinda; Gristwood, Tamzin; Clulow, James S; Richter, Corinna; Przybilski, Rita; Pitman, Andrew R; Fineran, Peter C

    2013-04-01

    In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

  7. The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato.

    PubMed

    Jacobs, Jonathan M; Babujee, Lavanya; Meng, Fanhong; Milling, Annett; Allen, Caitilyn

    2012-01-01

    Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 10(8) to 10(9) CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 10(8) CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical

  8. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

    PubMed

    Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

  9. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

    PubMed Central

    Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

  10. Detection and inhibition of bacterial cell-cell communication.

    PubMed

    Rice, Scott A; McDougald, Diane; Givskov, Michael; Kjelleberg, Staffan

    2008-01-01

    Bacteria communicate with other members of their community through the secretion and perception of small chemical cues or signals. The recognition of a signal normally leads to the expression of a large suite of genes, which in some bacteria are involved in the regulation of virulence factors, and as a result, these signaling compounds are key regulatory factors in many disease processes. Thus, it is of interest when studying pathogens to understand the mechanisms used to control the expression of virulence genes so that strategies might be devised for the control of those pathogens. Clearly, the ability to interfere with this process of signaling represents a novel approach for the treatment of bacterial infections. There is a broad range of compounds that bacteria can use for signaling purposes, including fatty acids, peptides, N-acylated homoserine lactones, and the signals collectively called autoinducer 2 (AI-2). This chapter will focus on the latter two signaling systems as they are present in a range of medically relevant bacteria, and here we describe assays for determining whether an organism produces a particular signal and assays that can be used to identify inhibitors of the signaling cascade. Lastly, the signal detection and inhibition assays will be directly linked to the expression of virulence factors of specific pathogens.

  11. Potential and use of bacterial small RNAs to combat drug resistance: a systematic review

    PubMed Central

    Liu, Xiaodong; Zhang, Lin; Wong, Sunny Hei; Chan, Matthew TV; Wu, William KK

    2017-01-01

    Background Over the decades, new antibacterial agents have been developed in an attempt to combat drug resistance, but they remain unsuccessful. Recently, a novel class of bacterial gene expression regulators, bacterial small RNAs (sRNAs), has received increasing attention toward their involvement in antibiotic resistance. This systematic review aimed to discuss the potential of these small molecules as antibacterial drug targets. Methods Two investigators performed a comprehensive search of MEDLINE, EmBase, and ISI Web of Knowledge from inception to October 2016, without restriction on language. We included all in vitro and in vivo studies investigating the role of bacterial sRNA in antibiotic resistance. Risk of bias of the included studies was assessed by a modified guideline of Systematic Review Center for Laboratory Animal Experimentation (SYRCLE). Results Initial search yielded 432 articles. After exclusion of non-original articles, 20 were included in this review. Of these, all studies examined bacterial-type strains only. There were neither relevant in vivo nor clinical studies. The SYRCLE scores ranged from to 5 to 7, with an average of 5.9. This implies a moderate risk of bias. sRNAs influenced the antibiotics susceptibility through modulation of gene expression relevant to efflux pumps, cell wall synthesis, and membrane proteins. Conclusion Preclinical studies on bacterial-type strains suggest that modulation of sRNAs could enhance bacterial susceptibility to antibiotics. Further studies on clinical isolates and in vivo models are needed to elucidate the therapeutic value of sRNA modulation on treatment of multidrug-resistant bacterial infection. PMID:29290689

  12. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  13. Immunomodulation with bacterial extracts in respiratory diseases.

    PubMed

    Palma-Carlos, A G; Palma-Carlos, M L

    1990-01-01

    A lyophilized bacterial extract (Broncho-Vaxom) has been studied in a large number of models and found to induce specific and nonspecific responses by oral administration. It stimulates the systemic and local immune response. It activates the macrophages that play a key part in the immune system, modulates the immunoglobulin level, and potentiates the lymphocyte response to phytohemagglutinin (PHA) and other mitogens. The effect of this bacterial extract on T-lymphocyte subpopulations is currently under study.

  14. Klebsiella pneumoniae Siderophores Induce Inflammation, Bacterial Dissemination, and HIF-1α Stabilization during Pneumonia.

    PubMed

    Holden, Victoria I; Breen, Paul; Houle, Sébastien; Dozois, Charles M; Bachman, Michael A

    2016-09-13

    Klebsiella pneumoniae is a Gram-negative pathogen responsible for a wide range of infections, including pneumonia and bacteremia, and is rapidly acquiring antibiotic resistance. K. pneumoniae requires secretion of siderophores, low-molecular-weight, high-affinity iron chelators, for bacterial replication and full virulence. The specific combination of siderophores secreted by K. pneumoniae during infection can impact tissue localization, systemic dissemination, and host survival. However, the effect of these potent iron chelators on the host during infection is unknown. In vitro, siderophores deplete epithelial cell iron, induce cytokine secretion, and activate the master transcription factor hypoxia inducible factor-1α (HIF-1α) protein that controls vascular permeability and inflammatory gene expression. Therefore, we hypothesized that siderophore secretion by K. pneumoniae directly contributes to inflammation and bacterial dissemination during pneumonia. To examine the effects of siderophore secretion independently of bacterial growth, we performed infections with tonB mutants that persist in vivo but are deficient in siderophore import. Using a murine model of pneumonia, we found that siderophore secretion by K. pneumoniae induces the secretion of interleukin-6 (IL-6), CXCL1, and CXCL2, as well as bacterial dissemination to the spleen, compared to siderophore-negative mutants at an equivalent bacterial number. Furthermore, we determined that siderophore-secreting K. pneumoniae stabilized HIF-1α in vivo and that bacterial dissemination to the spleen required alveolar epithelial HIF-1α. Our results indicate that siderophores act directly on the host to induce inflammatory cytokines and bacterial dissemination and that HIF-1α is a susceptibility factor for bacterial invasion during pneumonia. Klebsiella pneumoniae causes a wide range of bacterial diseases, including pneumonia, urinary tract infections, and sepsis. To cause infection, K. pneumoniae steals

  15. Bacterial community structure in the drinking water microbiome is governed by filtration processes.

    PubMed

    Pinto, Ameet J; Xi, Chuanwu; Raskin, Lutgarde

    2012-08-21

    The bacterial community structure of a drinking water microbiome was characterized over three seasons using 16S rRNA gene based pyrosequencing of samples obtained from source water (a mix of a groundwater and a surface water), different points in a drinking water plant operated to treat this source water, and in the associated drinking water distribution system. Even though the source water was shown to seed the drinking water microbiome, treatment process operations limit the source water's influence on the distribution system bacterial community. Rather, in this plant, filtration by dual media rapid sand filters played a primary role in shaping the distribution system bacterial community over seasonal time scales as the filters harbored a stable bacterial community that seeded the water treatment processes past filtration. Bacterial taxa that colonized the filter and sloughed off in the filter effluent were able to persist in the distribution system despite disinfection of finished water by chloramination and filter backwashing with chloraminated backwash water. Thus, filter colonization presents a possible ecological survival strategy for bacterial communities in drinking water systems, which presents an opportunity to control the drinking water microbiome by manipulating the filter microbial community. Grouping bacterial taxa based on their association with the filter helped to elucidate relationships between the abundance of bacterial groups and water quality parameters and showed that pH was the strongest regulator of the bacterial community in the sampled drinking water system.

  16. Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

    PubMed

    de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

    2004-08-15

    A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

  17. Endocarditis in adults with bacterial meningitis.

    PubMed

    Lucas, Marjolein J; Brouwer, Matthijs C; van der Ende, Arie; van de Beek, Diederik

    2013-05-21

    Endocarditis may precede or complicate bacterial meningitis, but the incidence and impact of endocarditis in bacterial meningitis are unknown. We assessed the incidence and clinical characteristics of patients with meningitis and endocarditis from a nationwide cohort study of adults with community-acquired bacterial meningitis in the Netherlands from 2006 to 2012. Endocarditis was identified in 24 of 1025 episodes (2%) of bacterial meningitis. Cultures yielded Streptococcus pneumoniae in 13 patients, Staphylococcus aureus in 8 patients, and Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus salivarius in 1 patient each. Clues leading to the diagnosis of endocarditis were cardiac murmurs, persistent or recurrent fever, a history of heart valve disease, and S aureus as the causative pathogen of bacterial meningitis. Treatment consisted of prolonged antibiotic therapy in all patients and surgical valve replacement in 10 patients (42%). Two patients were treated with oral anticoagulants, and both developed life-threatening intracerebral hemorrhage. Systemic (70%) and neurological (54%) complications occurred frequently, leading to a high proportion of patients with unfavorable outcome (63%). Seven of 24 patients (29%) with meningitis and endocarditis died. Endocarditis is an uncommon coexisting condition in bacterial meningitis but is associated with a high rate of unfavorable outcome.

  18. Bacterial contamination of platelet components not detected by BacT/ALERT®.

    PubMed

    Abela, M A; Fenning, S; Maguire, K A; Morris, K G

    2018-02-01

    To investigate the possible causes for false negative results in BacT/ALERT ® 3D Signature System despite bacterial contamination of platelet units. The Northern Ireland Blood Transfusion Service (NIBTS) routinely extends platelet component shelf life to 7 days. Components are sampled and screened for bacterial contamination using an automated microbial detection system, the BacT/ALERT ® 3D Signature System. We report on three platelet components with confirmed bacterial contamination, which represent false negative BacT/ALERT ® results and near-miss serious adverse events. NIBTS protocols for risk reduction of bacterial contamination of platelet components are described. The methodology for bacterial detection using BacT/ALERT ® is outlined. Laboratory tests, relevant patient details and relevant follow-up information are analysed. In all three cases, Staphylococcus aureus was isolated from the platelet residue and confirmed on terminal sub-culture using BacT/ALERT ® . In two cases, S. aureus with similar genetic makeup was isolated from the donors. Risk reduction measures for bacterial contamination of platelet components are not always effective. Automated bacterial culture detection does not eliminate the risk of bacterial contamination. Visual inspection of platelet components prior to release, issue and administration remains an important last line of defence. © 2017 British Blood Transfusion Society.

  19. Role of bacterial adhesion in the microbial ecology of biofilms in cooling tower systems.

    PubMed

    Liu, Yang; Zhang, Wei; Sileika, Tadas; Warta, Richard; Cianciotto, Nicholas P; Packman, Aaron

    2009-01-01

    The fate of the three heterotrophic biofilm forming bacteria, Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. in pilot scale cooling towers was evaluated both by observing the persistence of each species in the recirculating water and the formation of biofilms on steel coupons placed in each cooling tower water reservoir. Two different cooling tower experiments were performed: a short-term study (6 days) to observe the initial bacterial colonization of the cooling tower, and a long-term study (3 months) to observe the ecological dynamics with repeated introduction of the test strains. An additional set of batch experiments (6 days) was carried out to evaluate the adhesion of each strain to steel surfaces under similar conditions to those found in the cooling tower experiments. Substantial differences were observed in the microbial communities that developed in the batch systems and cooling towers. P. aeruginosa showed a low degree of adherence to steel surfaces both in batch and in the cooling towers, but grew much faster than K. pneumoniae and Flavobacterium in mixed-species biofilms and ultimately became the dominant organism in the closed batch systems. However, the low degree of adherence caused P. aeruginosa to be rapidly washed out of the open cooling tower systems, and Flavobacterium became the dominant microorganism in the cooling towers in both the short-term and long-term experiments. These results indicate that adhesion, retention and growth on solid surfaces play important roles in the bacterial community that develops in cooling tower systems.

  20. Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pinzon, NM; Aukema, KG; Gralnick, JA

    A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone productionmore » as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high

  1. Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

    PubMed

    Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

    2013-11-01

    Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

  2. Bacterial Responses to Reactive Chlorine Species

    PubMed Central

    Gray, Michael J.; Wholey, Wei-Yun; Jakob, Ursula

    2013-01-01

    Hypochlorous acid (HOCl), the active ingredient of household bleach, is the most common disinfectant in medical, industrial, and domestic use and plays an important role in microbial killing in the innate immune system. Given the critical importance of the antimicrobial properties of chlorine to public health, it is surprising how little is known about the ways in which bacteria sense and respond to reactive chlorine species (RCS). Although the literature on bacterial responses to reactive oxygen species (ROS) is enormous, work addressing bacterial responses to RCS has begun only recently. Transcriptomic and proteomic studies now provide new insights into how bacteria mount defenses against this important class of antimicrobial compounds. In this review, we summarize the current knowledge, emphasizing the overlaps between RCS stress responses and other more well-characterized bacterial defense systems, and identify outstanding questions that represent productive avenues for future research. PMID:23768204

  3. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  4. Succession of bacterial and fungal communities within biofilms of a chlorinated drinking water distribution system.

    PubMed

    Douterelo, I; Fish, K E; Boxall, J B

    2018-09-15

    Understanding the temporal dynamics of multi-species biofilms in Drinking Water Distribution Systems (DWDS) is essential to ensure safe, high quality water reaches consumers after it passes through these high surface area reactors. This research studied the succession characteristics of fungal and bacterial communities under controlled environmental conditions fully representative of operational DWDS. Microbial communities were observed to increase in complexity after one month of biofilm development but they did not reach stability after three months. Changes in cell numbers were faster at the start of biofilm formation and tended to decrease over time, despite the continuing changes in bacterial community composition. Fungal diversity was markedly less than bacterial diversity and had a lag in responding to temporal dynamics. A core-mixed community of bacteria including Pseudomonas, Massillia and Sphingomonas and the fungi Acremonium and Neocosmopora were present constantly and consistently in the biofilms over time and conditions studied. Monitoring and managing biofilms and such ubiquitous core microbial communities are key control strategies to ensuring the delivery of safe drinking water via the current ageing DWDS infrastructure. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Claudin-3 expression in radiation-exposed rat models: A potential marker for radiation-induced intestinal barrier failure

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shim, Sehwan; Lee, Jong-geol; Bae, Chang-hwan

    2015-01-02

    Highlights: • Irradiation increased intestinal bacterial translocation, accompanied by claudin protein expression in rats. • Neurotensin decreased the bacterial translocation and restored claudin-3 expression. • Claudin-3 can be used as a marker in evaluating radiation induced intestinal injury. - Abstract: The molecular events leading to radiation-induced intestinal barrier failure are not well known. The influence of the expression of claudin proteins in the presence and absence of neurotensin was investigated in radiation-exposed rat intestinal epithelium. Wistar rats were randomly divided into control, irradiation, and irradiation + neurotensin groups, and bacterial translocation to the mesenteric lymph node and expression of claudinsmore » were determined. Irradiation led to intestinal barrier failure as demonstrated by significant bacterial translocation. In irradiated terminal ilea, expression of claudin-3 and claudin-4 was significantly decreased, and claudin-2 expression was increased. Administration of neurotensin significantly reduced bacterial translocation and restored the structure of the villi as seen by histologic examination. Among the three subtype of claudins, only claudin-3 expression was restored. These results suggest that the therapeutic effect of neurotensin on the disruption of the intestinal barrier is associated with claudin-3 alteration and that claudin-3 could be used as a marker in evaluating radiation-induced intestinal injury.« less

  6. Bacterial prostatitis.

    PubMed

    Gill, Bradley C; Shoskes, Daniel A

    2016-02-01

    The review provides the infectious disease community with a urologic perspective on bacterial prostatitis. Specifically, the article briefly reviews the categorization of prostatitis by type and provides a distillation of new findings published on bacterial prostatitis over the past year. It also highlights key points from the established literature. Cross-sectional prostate imaging is becoming more common and may lead to more incidental diagnoses of acute bacterial prostatitis. As drug resistance remains problematic in this condition, the reemergence of older antibiotics such as fosfomycin, has proven beneficial. With regard to chronic bacterial prostatitis, no clear clinical risk factors emerged in a large epidemiological study. However, bacterial biofilm formation has been associated with more severe cases. Surgery has a limited role in bacterial prostatitis and should be reserved for draining of a prostatic abscess or the removal of infected prostatic stones. Prostatitis remains a common and bothersome clinical condition. Antibiotic therapy remains the basis of treatment for both acute and chronic bacterial prostatitis. Further research into improving prostatitis treatment is indicated.

  7. [Analysis of false-positive reaction for bacterial detection of blood samples with the automated microbial detection system BacT/ALERT 3D].

    PubMed

    Zhu, Li-Wei; Yang, Xue-Mei; Xu, Xiao-Qin; Xu, Jian; Lu, Huang-Jun; Yan, Li-Xing

    2008-10-01

    This study was aimed to analyze the results of false positive reaction in bacterial detection of blood samples with BacT/ALERT 3D system, to evaluate the specificity of this system, and to decrease the false positive reaction. Each reaction flasks in past five years were processed for bacteria isolation and identification. When the initial cultures were positive, the remaining samples and the corresponding units were recultured if still available. 11395 blood samples were detected. It is worthy of note that the incubator temperature should be stabilized, avoiding fluctuation; when the cultures were alarmed, the reaction flasks showed be kept some hours for further incubation so as to trace a sharply increasing signal to support the judgement of true bacterial growth. The results indicated that 122 samples (1.07%) wee positive at initial culture, out of them 107 samples (88.7%) were found bacterial, and 15 samples (12.3%) were found nothing. The detection curves of positive samples resulted from bacterial growth showed ascent. In conclusion, maintenance of temperature stability and avoidance of temperature fluctuation in incubator could decrease the occurrence of false-positive reaction in detection process. The reaction flasks with positive results at initial culture should be recultured, and whether existence of a sharply ascending logarilhimic growth phase in bacterial growth curve should be further detected, which are helpful to distinguish false-positive reactions from true positive, and thus increase the specificity of the BacT/ALERT system.

  8. Cone-morse implant connection system significantly reduces bacterial leakage between implant and abutment: an in vitro study.

    PubMed

    Baj, A; Bolzoni, A; Russillo, A; Lauritano, D; Palmieri, A; Cura, F; Silvestre, F J; Giannì, A B

    2017-01-01

    Osseointegrated implants are very popular dental treatments today in the world. In osseointegrated implants, the occlusal forces are transmitted from prosthesis through an abutment to a dental implant. The abutment is connected to the implant by mean of a screw. A screw is the most used mean for connecting an implant to an abutment. Frequently the screws break and are lost. There is an alternative to screw retained abutment systems: the cone-morse connection (CMC). The CMC, thanks to the absence of the abutment screw, guarantees no micro-gaps, no micro-movements, and a reduction of bacterial leakage between implant and abutment. As P. gingivalis and T. forsythia penetration might have clinical relevance, it was the purpose of this investigation to evaluate molecular leakage of these two bacteria in a new CMC implants systems (Leone Spa®, Florence, Italy). To identify the capability of the implant to protect the internal space from the external environment, the passage of genetically modified Escherichia coli across implant-abutment interface was evaluated. Four cone-morse Leone implants (Leone® Spa, Florence, Italy) were immerged in a bacterial culture for 24 h and bacteria amount was then measured inside implant-abutment interface with Real-time PCR. Bacteria were detected inside all studied implants, with a median percentage of 3% for P. gingivalis and 4% for T. forsythia. Cone-morse connection implant system has very low bacterial leakage percentage and is similar to one-piece implants.

  9. The New Kid on the Block: A Specialized Secretion System during Bacterial Sporulation.

    PubMed

    Morlot, Cécile; Rodrigues, Christopher D A

    2018-02-02

    The transport of proteins across the bacterial cell envelope is mediated by protein complexes called specialized secretion systems. These nanomachines exist in both Gram-positive and Gram-negative bacteria and have been categorized into different types based on their structural components and function. Interestingly, multiple studies suggest the existence of a protein complex in endospore-forming bacteria that appears to be a new type of specialized secretion system. This protein complex is called the SpoIIIA-SpoIIQ complex and is an exception to the categorical norm since it appears to be a hybrid composed of different parts from well-defined specialized secretion systems. Here we summarize and discuss the current understanding of this complex and its potential role as a specialized secretion system. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. S1PR3 Signaling Drives Bacterial Killing and Is Required for Survival in Bacterial Sepsis.

    PubMed

    Hou, JinChao; Chen, QiXing; Wu, XiaoLiang; Zhao, DongYan; Reuveni, Hadas; Licht, Tamar; Xu, MengLong; Hu, Hu; Hoeft, Andreas; Ben-Sasson, Shmuel A; Shu, Qiang; Fang, XiangMing

    2017-12-15

    Efficient elimination of pathogenic bacteria is a critical determinant in the outcome of sepsis. Sphingosine-1-phosphate receptor 3 (S1PR3) mediates multiple aspects of the inflammatory response during sepsis, but whether S1PR3 signaling is necessary for eliminating the invading pathogens remains unknown. To investigate the role of S1PR3 in antibacterial immunity during sepsis. Loss- and gain-of-function experiments were performed using cell and murine models. S1PR3 levels were determined in patients with sepsis and healthy volunteers. S1PR3 protein levels were up-regulated in macrophages upon bacterial stimulation. S1pr3 -/- mice showed increased mortality and increased bacterial burden in multiple models of sepsis. The transfer of wild-type bone marrow-derived macrophages rescued S1pr3 -/- mice from lethal sepsis. S1PR3-overexpressing macrophages further ameliorated the mortality rate of sepsis. Loss of S1PR3 led to markedly decreased bacterial killing in macrophages. Enhancing endogenous S1PR3 activity using a peptide agonist potentiated the macrophage bactericidal function and improved survival rates in multiple models of sepsis. Mechanically, the reactive oxygen species levels were decreased and phagosome maturation was delayed in S1pr3 -/- macrophages due to impaired recruitment of vacuolar protein-sorting 34 to the phagosomes. In addition, S1RP3 expression levels were elevated in monocytes from patients with sepsis. Higher levels of monocytic S1PR3 were associated with efficient intracellular bactericidal activity, better immune status, and preferable outcomes. S1PR3 signaling drives bacterial killing and is essential for survival in bacterial sepsis. Interventions targeting S1PR3 signaling could have translational implications for manipulating the innate immune response to combat pathogens.

  11. Bacterial Sialidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Data shows that elevated sialidase in bacterial vaginosis patients correlates to premature births in women. Bacterial sialidase also plays a significant role in the unusual colonization of Pseudomonas aeruginosa in cystic fibrosis patients. Crystals of Salmonella sialidase have been reproduced and are used for studying the inhibitor-enzyme complexes. These inhibitors may also be used to inhibit a trans-sialidase of Trypanosome cruzi, a very similar enzyme to bacterial sialidase, therefore preventing T. cruzi infection, the causitive agent of Chagas' disease. The Center for Macromolecular Crystallography suggests that inhibitors of bacterial sialidases can be used as prophylactic drugs to prevent bacterial infections in these critical cases.

  12. Characterization of successional changes in bacterial community composition during bioremediation of used motor oil-contaminated soil in a boreal climate.

    PubMed

    Yan, Lijuan; Sinkko, Hanna; Penttinen, Petri; Lindström, Kristina

    2016-01-15

    The widespread use of motor oil makes it a notable risk factor to cause scattered contamination in soil. The monitoring of microbial community dynamics can serve as a comprehensive tool to assess the ecological impact of contaminants and their disappearance in the ecosystem. Hence, a field study was conducted to monitor the ecological impact of used motor oil under different perennial cropping systems (fodder galega, brome grass, galega-brome grass mixture and bare fallow) in a boreal climate zone. Length heterogeneity PCR characterized a successional pattern in bacterial community following oil contamination over a four-year bioremediation period. Soil pH and electrical conductivity were associated with the shifts in bacterial community composition. Crops had no detectable effect on bacterial community composition or complexity. However, the legume fodder galega increased soil microbial biomass, expressed as soil total DNA. Oil contamination induced an abrupt change in bacterial community composition at the early stage, yet the effect did not last as long as the oil in soil. The successional variation in bacterial community composition can serve as a sensitive ecological indicator of oil contamination and remediation in situ. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Dynamics of Bacterial Gene Regulatory Networks.

    PubMed

    Shis, David L; Bennett, Matthew R; Igoshin, Oleg A

    2018-05-20

    The ability of bacterial cells to adjust their gene expression program in response to environmental perturbation is often critical for their survival. Recent experimental advances allowing us to quantitatively record gene expression dynamics in single cells and in populations coupled with mathematical modeling enable mechanistic understanding on how these responses are shaped by the underlying regulatory networks. Here, we review how the combination of local and global factors affect dynamical responses of gene regulatory networks. Our goal is to discuss the general principles that allow extrapolation from a few model bacteria to less understood microbes. We emphasize that, in addition to well-studied effects of network architecture, network dynamics are shaped by global pleiotropic effects and cell physiology.

  14. Gut microbial translocation corrupts myeloid cell function to control bacterial infection during liver cirrhosis.

    PubMed

    Hackstein, Carl-Philipp; Assmus, Lisa Mareike; Welz, Meike; Klein, Sabine; Schwandt, Timo; Schultze, Joachim; Förster, Irmgard; Gondorf, Fabian; Beyer, Marc; Kroy, Daniela; Kurts, Christian; Trebicka, Jonel; Kastenmüller, Wolfgang; Knolle, Percy A; Abdullah, Zeinab

    2017-03-01

    Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity. Experimental liver fibrosis in mice induced by bile duct ligation or CCl 4 application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections. In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with Listeria that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis. In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  15. SIV Infection-mediated Changes in Gastrointestinal Bacterial Microbiome and Virome are Associated With Immunodeficiency and Prevented by Vaccination

    PubMed Central

    Handley, Scott A.; Desai, Chandni; Zhao, Guoyan; Droit, Lindsay; Monaco, Cynthia L.; Schroeder, Andrew C.; Nkolola, Joseph P.; Norman, Megan E.; Miller, Andrew D.; Wang, David; Barouch, Dan H.; Virgin, Herbert W.

    2016-01-01

    SUMMARY AIDS caused by simian immunodeficiency virus (SIV) infection is associated with gastrointestinal disease, systemic immune activation and, in cross sectional studies, changes in the enteric virome. Here we performed a longitudinal study of a vaccine cohort to define the natural history of changes in the fecal metagenome in SIV-infected monkeys. Matched rhesus macaques were either uninfected or intrarectally challenged with SIV, with a subset receiving the Ad26 vaccine, an adenovirus vector expressing the viral Env/Gag/Pol antigens. Progression of SIV infection to AIDS was associated with increased detection of potentially pathogenic viruses and bacterial enteropathogens. Specifically, adenoviruses were associated with an increased incidence of gastrointestinal disease and AIDS-related mortality. Viral and bacterial enteropathogens were largely absent from animals protected by the vaccine. These data suggest that the SIV-associated gastrointestinal disease is associated with the presence of both viral and bacterial enteropathogens and protection against SIV infection by vaccination prevents enteropathogen emergence. PMID:26962943

  16. A role for bacterial urease in gut dysbiosis and Crohn’s disease

    PubMed Central

    Ni, Josephine; Shen, Ting-Chin David; Chen, Eric Z.; Bittinger, Kyle; Bailey, Aubrey; Roggiani, Manuela; Sirota-Madi, Alexandra; Friedman, Elliot S.; Chau, Lillian; Lin, Andrew; Nissim, Ilana; Scott, Justin; Lauder, Abigail; Hoffmann, Christian; Rivas, Gloriany; Albenberg, Lindsey; Baldassano, Robert N.; Braun, Jonathan; Xavier, Ramnik J.; Clish, Clary B.; Yudkoff, Marc; Li, Hongzhe; Goulian, Mark; Bushman, Frederic D.; Lewis, James D.; Wu, Gary D.

    2018-01-01

    Gut dysbiosis during inflammatory bowel disease involves alterations in the gut microbiota associated with inflammation of the host gut. We used a combination of shotgun metagenomic sequencing and metabolomics to analyze fecal samples from pediatric patients with Crohn’s disease and found an association between disease severity, gut dysbiosis, and bacterial production of free amino acids. Nitrogen flux studies using 15N in mice showed that activity of bacterial urease, an enzyme that releases ammonia by hydrolysis of host urea, led to the transfer of murine host-derived nitrogen to the gutmicrobiota where it was used for amino acid synthesis. Inoculation of a conventional murine host (pretreated with antibiotics and polyethylene glycol) with commensal Escherichia coli engineered to express urease led to dysbiosis of the gut microbiota, resulting in a predominance of Proteobacteria species. This was associated with a worsening of immune-mediated colitis in these animals. A potential role for altered urease expression and nitrogen flux in the development of gut dysbiosis suggests that bacterial urease may be a potential therapeutic target for inflammatory bowel diseases. PMID:29141885

  17. Membrane Lipids as Indicators for Viable Bacterial Communities Inhabiting Petroleum Systems.

    PubMed

    Gruner, Andrea; Mangelsdorf, Kai; Vieth-Hillebrand, Andrea; Horsfield, Brian; van der Kraan, Geert M; Köhler, Thomas; Janka, Christoph; Morris, Brandon E L; Wilkes, Heinz

    2017-08-01

    Microbial activity in petroleum reservoirs has been implicated in a suite of detrimental effects including deterioration of petroleum quality, increases in oil sulfur content, biofouling of steel pipelines and other infrastructures, and well plugging. Here, we present a biogeochemical approach, using phospholipid fatty acids (PLFAs), for detecting viable bacteria in petroleum systems. Variations within the bacterial community along water flow paths (producing well, topside facilities, and injection well) can be elucidated in the field using the same technique, as shown here within oil production plants in the Molasse Basin of Upper Austria. The abundance of PLFAs is compared to total cellular numbers, as detected by qPCR of the 16S rDNA gene, to give an overall comparison between the resolutions of both methods in a true field setting. Additionally, the influence of biocide applications on lipid- and DNA-based quantification was investigated. The first oil field, Trattnach, showed significant PLFA abundances and cell numbers within the reservoir and topside facilities. In contrast, the second field (Engenfeld) showed very low PLFA levels overall, likely due to continuous treatment of the topside facilities with a glutaraldehyde-based antimicrobial. In comparison, Trattnach is dosed once per week in a batch fashion. Changes within PLFA compositions across the flow path, throughout the petroleum production plants, point to cellular adaptation within the system and may be linked to shifts in the dominance of certain bacterial types in oil reservoirs versus topside facilities. Overall, PLFA-based monitoring provides a useful tool to assess the abundance and high-level taxonomic diversity of viable microbial populations in oil production wells, topside infrastructure, pipelines, and other related facilities.

  18. Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans.

    PubMed

    Fujita, Rie; Koizumi, Nobuo; Sugiyama, Hiromu; Tomizawa, Rina; Sato, Ryoichi; Ohnishi, Makoto

    2015-01-01

    Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis

  19. Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans

    PubMed Central

    Fujita, Rie; Koizumi, Nobuo; Sugiyama, Hiromu; Tomizawa, Rina; Sato, Ryoichi; Ohnishi, Makoto

    2015-01-01

    Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis

  20. Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System

    PubMed Central

    Bhatnagar, Srijak; Eisen, Jonathan A.; Kopp, Artyom

    2011-01-01

    Drosophila melanogaster is emerging as an important model of non-pathogenic host–microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal–microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host–microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host–microbe interactions. Bacterial taxa used in experimental studies are rare or absent in

  1. Acute Bacterial Prostatitis: Diagnosis and Management.

    PubMed

    Coker, Timothy J; Dierfeldt, Daniel M

    2016-01-15

    Acute bacterial prostatitis is an acute infection of the prostate gland that causes pelvic pain and urinary tract symptoms, such as dysuria, urinary frequency, and urinary retention, and may lead to systemic symptoms, such as fevers, chills, nausea, emesis, and malaise. Although the true incidence is unknown, acute bacterial prostatitis is estimated to comprise approximately 10% of all cases of prostatitis. Most acute bacterial prostatitis infections are community acquired, but some occur after transurethral manipulation procedures, such as urethral catheterization and cystoscopy, or after transrectal prostate biopsy. The physical examination should include abdominal, genital, and digital rectal examination to assess for a tender, enlarged, or boggy prostate. Diagnosis is predominantly made based on history and physical examination, but may be aided by urinalysis. Urine cultures should be obtained in all patients who are suspected of having acute bacterial prostatitis to determine the responsible bacteria and its antibiotic sensitivity pattern. Additional laboratory studies can be obtained based on risk factors and severity of illness. Radiography is typically unnecessary. Most patients can be treated as outpatients with oral antibiotics and supportive measures. Hospitalization and broad-spectrum intravenous antibiotics should be considered in patients who are systemically ill, unable to voluntarily urinate, unable to tolerate oral intake, or have risk factors for antibiotic resistance. Typical antibiotic regimens include ceftriaxone and doxycycline, ciprofloxacin, and piperacillin/tazobactam. The risk of nosocomial bacterial prostatitis can be reduced by using antibiotics, such as ciprofloxacin, before transrectal prostate biopsy.

  2. Bacterial carbon utilization in vertical subsurface flow constructed wetlands.

    PubMed

    Tietz, Alexandra; Langergraber, Günter; Watzinger, Andrea; Haberl, Raimund; Kirschner, Alexander K T

    2008-03-01

    Subsurface vertical flow constructed wetlands with intermittent loading are considered as state of the art and can comply with stringent effluent requirements. It is usually assumed that microbial activity in the filter body of constructed wetlands, responsible for the removal of carbon and nitrogen, relies mainly on bacterially mediated transformations. However, little quantitative information is available on the distribution of bacterial biomass and production in the "black-box" constructed wetland. The spatial distribution of bacterial carbon utilization, based on bacterial (14)C-leucine incorporation measurements, was investigated for the filter body of planted and unplanted indoor pilot-scale constructed wetlands, as well as for a planted outdoor constructed wetland. A simple mass-balance approach was applied to explain the bacterially catalysed organic matter degradation in this system by comparing estimated bacterial carbon utilization rates with simultaneously measured carbon reduction values. The pilot-scale constructed wetlands proved to be a suitable model system for investigating microbial carbon utilization in constructed wetlands. Under an ideal operating mode, the bulk of bacterial productivity occurred within the first 10cm of the filter body. Plants seemed to have no significant influence on productivity and biomass of bacteria, as well as on wastewater total organic carbon removal.

  3. Genetic reprogramming of host cells by bacterial pathogens.

    PubMed

    Tran Van Nhieu, Guy; Arbibe, Laurence

    2009-10-29

    During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

  4. Bacterial antisense RNAs are mainly the product of transcriptional noise.

    PubMed

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

    2016-03-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

  5. Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

    PubMed Central

    Vercoe, Reuben B.; Chang, James T.; Dy, Ron L.; Taylor, Corinda; Gristwood, Tamzin; Clulow, James S.; Richter, Corinna; Przybilski, Rita; Pitman, Andrew R.; Fineran, Peter C.

    2013-01-01

    In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity. PMID:23637624

  6. Hemovigilance monitoring of platelet septic reactions with effective bacterial protection systems.

    PubMed

    Benjamin, Richard J; Braschler, Thomas; Weingand, Tina; Corash, Laurence M

    2017-12-01

    Delayed, large-volume bacterial culture and amotosalen/ultraviolet-A light pathogen reduction are effective at reducing the risk of bacterial proliferation in platelet concentrates (PCs). Hemovigilance programs continue to receive reports of suspected septic transfusion reactions, most with low imputability. Here, we compile national hemovigilance data to determine the relative efficacy of these interventions. Annual reports from the United Kingdom, France, Switzerland, and Belgium were reviewed between 2005 and 2016 to assess the risk of bacterial contamination and septic reactions. Approximately 1.65 million delayed, large-volume bacterial culture-screened PCs in the United Kingdom and 2.3 million amotosalen/ultraviolet-A-treated PCs worldwide were issued with no reported septic fatalities. One definite, one possible, and 12 undetermined/indeterminate septic reactions and eight contaminated "near misses" were reported with delayed, large-volume bacterial cultures between 2011 and 2016, for a lower false-negative culture rate than that in the previous 5 years (5.4 vs. 16.3 per million: odds ratio, 3.0; 95% confidence interval, 1.4-6.5). Together, the Belgian, Swiss, and French hemovigilance programs documented zero probable or definite/certain septic reactions with 609,290 amotosalen/ultraviolet-A-treated PCs (<1.6 per million). The rates were significantly lower than those reported with concurrently transfused, nonpathogen-reduced PCs in Belgium (<4.4 vs. 35.6 per million: odds ratio, 8.1; 95% confidence interval,1.1-353.3) and with historic septic reaction rates in Switzerland (<6.0 vs. 82.9 per million: odds ratio, 13.9; 95% confidence interval, 2.1-589.2), and the rates tended to be lower than those from concurrently transfused, nonpathogen-reduced PCs in France (<4.7 vs. 19.0 per million: odds ratio, 4.1; 95% confidence interval, 0.7-164.3). Pathogen reduction and bacterial culture both reduced the incidence of septic reactions, although under-reporting and

  7. Expression and in vitro functional analyses of recombinant Gam1 protein.

    PubMed

    Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

    2015-01-01

    Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival.

    PubMed

    Moulin, Pauline; Patron, Kévin; Cano, Camille; Zorgani, Mohamed Amine; Camiade, Emilie; Borezée-Durant, Elise; Rosenau, Agnès; Mereghetti, Laurent; Hiron, Aurélia

    2016-12-15

    The Lmb protein of Streptococcus agalactiae is described as an adhesin that binds laminin, a component of the human extracellular matrix. In this study, we revealed a new role for this protein in zinc uptake. We also identified two Lmb homologs, AdcA and AdcAII, redundant binding proteins that combine with the AdcCB translocon to form a zinc-ABC transporter. Expression of this transporter is controlled by the zinc concentration in the medium through the zinc-dependent regulator AdcR. Triple deletion of lmb, adcA, and adcAII, or that of the adcCB genes, impaired growth and cell separation in a zinc-restricted environment. Moreover, we found that this Adc zinc-ABC transporter promotes S. agalactiae growth and survival in some human biological fluids, suggesting that it contributes to the infection process. These results indicated that zinc has biologically vital functions in S. agalactiae and that, under the conditions tested, the Adc/Lmb transporter constitutes the main zinc acquisition system of the bacterium. A zinc transporter, composed of three redundant binding proteins (Lmb, AdcA, and AdcAII), was characterized in Streptococcus agalactiae This system was shown to be essential for bacterial growth and morphology in zinc-restricted environments, including human biological fluids. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates

    PubMed Central

    Faron, Matthew L.; Buchan, Blake W.; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L.; Granato, Paul A.; Wilson, Deborah A.; Procop, Gary W.; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A.

    2015-01-01

    The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria. PMID:26529504

  10. Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

    PubMed

    Faron, Matthew L; Buchan, Blake W; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L; Granato, Paul A; Wilson, Deborah A; Procop, Gary W; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A

    2015-01-01

    The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

  11. Characterisation of the bacterial community in expressed prostatic secretions from patients with chronic prostatitis/chronic pelvic pain syndrome and infertile men: a preliminary investigation

    PubMed Central

    Hou, Dong-Sheng; Long, Wen-Min; Shen, Jian; Zhao, Li-Ping; Pang, Xiao-Yan; Xu, Chen

    2012-01-01

    The expressed prostatic secretions (EPSs) of men with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), infertile men and normal men were subjected to microbiological study. EPSs were collected from the subjects, which included 26 normal men, 11 infertile patients and 51 CP/CPPS patients. DNA was extracted from each specimen, and the V3 regions of the 16S rRNA genes were amplified using universal bacterial primers. The results showed that the EPS 16S rRNA gene-positive rate in the CP/CPPS and infertile patients was much higher than in the normal men, but without any difference among the three patient groups. The denaturing gradient gel electrophoresis (DGGE) method was used to characterize the EPS bacterial community structure of the prostate fluid from patients with CP/CPPS or infertility issues. Principal component analysis (PCA) and partial least squares (PLS) analyses of PCR-DGGE profiles revealed that the EPS bacterial community structure differed among the three groups. Three bands were identified as the key factors responsible for the discrepancy between CP/CPPS patients and infertile patients (P<0.05). Two bands were identified as priority factors in the discrepancy of category IIIA and category IIIB prostatitis patients (P<0.05). According to this research, the ecological balance of the prostate and low urethra tract, when considered as a microenvironment, might play an important role in the maintenance of a healthy male reproductive tract. PMID:22635162

  12. Predictive value of the APACHE II, SAPS II, SOFA and GCS scoring systems in patients with severe purulent bacterial meningitis.

    PubMed

    Pietraszek-Grzywaczewska, Iwona; Bernas, Szymon; Łojko, Piotr; Piechota, Anna; Piechota, Mariusz

    2016-01-01

    Scoring systems in critical care patients are essential for predicting of the patient outcome and evaluating the therapy. In this study, we determined the value of the Acute Physiology and Chronic Health Evaluation II (APACHE II), Simplified Acute Physiology Score II (SAPS II), Sequential Organ Failure Assessment (SOFA) and Glasgow Coma Scale (GCS) scoring systems in the prediction of mortality in adult patients admitted to the intensive care unit (ICU) with severe purulent bacterial meningitis. We retrospectively analysed data from 98 adult patients with severe purulent bacterial meningitis who were admitted to the single ICU between March 2006 and September 2015. Univariate logistic regression identified the following risk factors of death in patients with severe purulent bacterial meningitis: APACHE II, SAPS II, SOFA, and GCS scores, and the lengths of ICU stay and hospital stay. The independent risk factors of patient death in multivariate analysis were the SAPS II score, the length of ICU stay and the length of hospital stay. In the prediction of mortality according to the area under the curve, the SAPS II score had the highest accuracy followed by the APACHE II, GCS and SOFA scores. For the prediction of mortality in a patient with severe purulent bacterial meningitis, SAPS II had the highest accuracy.

  13. Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

    PubMed

    Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Kralicek, Andrew V; Shaw, Martin L; Lewis, John G; Conner, Anthony J; Barrell, Philippa J

    2014-11-04

    The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

  14. Expression of virulence factors by Staphylococcus aureus grown in serum.

    PubMed

    Oogai, Yuichi; Matsuo, Miki; Hashimoto, Masahito; Kato, Fuminori; Sugai, Motoyuki; Komatsuzawa, Hitoshi

    2011-11-01

    Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.

  15. Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems.

    PubMed

    Douillard, François P; Mahony, Jennifer; Campanacci, Valérie; Cambillau, Christian; van Sinderen, Douwe

    2011-09-01

    Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

    PubMed

    Hoffmann, E; Streichert, K; Nischan, N; Seitz, C; Brunner, T; Schwagerus, S; Hackenberger, C P R; Rubini, M

    2016-05-24

    The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

  17. MicroRNA-155 regulates host immune response to postviral bacterial pneumonia via IL-23/IL-17 pathway.

    PubMed

    Podsiad, Amy; Standiford, Theodore J; Ballinger, Megan N; Eakin, Richard; Park, Pauline; Kunkel, Steven L; Moore, Bethany B; Bhan, Urvashi

    2016-03-01

    Postinfluenza bacterial pneumonia is associated with significant mortality and morbidity. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. miR-155 has recently emerged as a crucial regulator of innate immunity and inflammatory responses and is induced in macrophages during infection. We hypothesized upregulation of miR-155 inhibits IL-17 and increases susceptibility to secondary bacterial pneumonia. Mice were challenged with 100 plaque-forming units H1N1 intranasally and were infected with 10(7) colony-forming units of MRSA intratracheally at day 5 postviral challenge. Lungs were harvested 24 h later, and expression of miR-155, IL-17, and IL-23 was measured by real-time RT-PCR. Induction of miR-155 was 3.6-fold higher in dual-infected lungs compared with single infection. miR-155(-/-) mice were protected with significantly lower (4-fold) bacterial burden and no differences in viral load, associated with robust induction of IL-23 and IL-17 (2.2- and 4.8-fold, respectively) postsequential challenge with virus and bacteria, compared with WT mice. Treatment with miR-155 antagomir improved lung bacterial clearance by 4.2-fold compared with control antagomir postsequential infection with virus and bacteria. Moreover, lung macrophages collected from patients with postviral bacterial pneumonia also had upregulation of miR-155 expression compared with healthy controls, consistent with observations in our murine model. This is the first demonstration that cellular miRNAs regulate postinfluenza immune response to subsequent bacterial challenge by suppressing the IL-17 pathway in the lung. Our findings suggest that antagonizing certain microRNA might serve as a potential therapeutic strategy against secondary bacterial infection. Copyright © 2016 the American Physiological Society.

  18. Selected Lactic Acid-Producing Bacterial Isolates with the Capacity to Reduce Salmonella Translocation and Virulence Gene Expression in Chickens

    PubMed Central

    Yang, Xiaojian; Brisbin, Jennifer; Yu, Hai; Wang, Qi; Yin, Fugui; Zhang, Yonggang; Sabour, Parviz; Sharif, Shayan; Gong, Joshua

    2014-01-01

    Background Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. Methodology/Principal Findings In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3–1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (106–7 CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (104 CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. Conclusions/Significance The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one

  19. Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

    PubMed

    Yang, Xiaojian; Brisbin, Jennifer; Yu, Hai; Wang, Qi; Yin, Fugui; Zhang, Yonggang; Sabour, Parviz; Sharif, Shayan; Gong, Joshua

    2014-01-01

    Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3-1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7) CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (10(4) CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one of the strategies for controlling Salmonella infection in chickens.

  20. Double-tagged fluorescent bacterial bioreporter for the study of polycyclic aromatic hydrocarbon diffusion and bioavailability.

    PubMed

    Tecon, Robin; Binggeli, Olivier; van der Meer, Jan R

    2009-09-01

    Bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), ubiquitous contaminants from oil and coal, is typically limited by poor accessibility of the contaminant to the bacteria. In order to measure PAH availability in complex systems, we designed a number of diffusion-based assays with a double-tagged bacterial reporter strain Burkholderia sartisoli RP037-mChe. The reporter strain is capable of mineralizing phenanthrene (PHE) and induces the expression of enhanced green fluorescent protein (eGFP) as a function of the PAH flux to the cell. At the same time, it produces a second autofluorescent protein (mCherry) in constitutive manner. Quantitative epifluorescence imaging was deployed in order to record reporter signals as a function of PAH availability. The reporter strain expressed eGFP proportionally to dosages of naphthalene or PHE in batch liquid cultures. To detect PAH diffusion from solid materials the reporter cells were embedded in 2 cm-sized agarose gel patches, and fluorescence was recorded over time for both markers as a function of distance to the PAH source. eGFP fluorescence gradients measured on known amounts of naphthalene or PHE served as calibration for quantifying PAH availability from contaminated soils. To detect reporter gene expression at even smaller diffusion distances, we mixed and immobilized cells with contaminated soils in an agarose gel. eGFP fluorescence measurements confirmed gel patch diffusion results that exposure to 2-3 mg lampblack soil gave four times higher expression than to material contaminated with 10 or 1 (mg PHE) g(-1).

  1. High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

    PubMed

    Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

    2016-11-14

    Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

  2. Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

    PubMed

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

    2014-06-27

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    NASA Astrophysics Data System (ADS)

    Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

    2017-01-01

    Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

  4. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    PubMed Central

    Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

    2017-01-01

    Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids. PMID:28028245

  5. Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

    PubMed Central

    Wang, Ying; Hougaard, Anni B.; Paulander, Wilhelm; Skibsted, Leif H.

    2015-01-01

    Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals. PMID:26150471

  6. Facial expression system on video using widrow hoff

    NASA Astrophysics Data System (ADS)

    Jannah, M.; Zarlis, M.; Mawengkang, H.

    2018-03-01

    Facial expressions recognition is one of interesting research. This research contains human feeling to computer application Such as the interaction between human and computer, data compression, facial animation and facial detection from the video. The purpose of this research is to create facial expression system that captures image from the video camera. The system in this research uses Widrow-Hoff learning method in training and testing image with Adaptive Linear Neuron (ADALINE) approach. The system performance is evaluated by two parameters, detection rate and false positive rate. The system accuracy depends on good technique and face position that trained and tested.

  7. The Effect of a Low Fluoride Delivery System on Bacterial Metabolism.

    DTIC Science & Technology

    1980-09-01

    Fluorides, an4 -Ique mechanisms, slow release delivery, temporary restora- tions, bacterial attachment, Streptococcus mutans , bacterial metabo’ilsm...concentrations of NaF, SnF 4 , Na2SnF6 , TiF 4 , and SnCI2 on altering plaque formation by Streptococcus mutans NCTC 10449. Specific tests were...preparation. Microorganisms, Growth Media, and Growth A streptomycin resistant mutant of Streptococcus mutans NCTC 10449 (Bratthall serotype c) has been

  8. A Straightforward Approach for 3D Bacterial Printing

    PubMed Central

    2017-01-01

    Sustainable and personally tailored materials production is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different molecules in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally friendly materials production techniques. In this paper, we describe the first steps toward the 3-dimensional printing of bacterial cultures for materials production and patterning. This methodology combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a commercial 3D printer was modified for bacterial systems, and new alginate-based bioink chemistry was developed. Printing temperature, printhead speed, and bioink extrusion rate were all adapted and customized to maximize bacterial health and spatial resolution of printed structures. Our combination of 3D printing technology with biological systems enables a sustainable approach for the production of numerous new materials. PMID:28225616

  9. A Straightforward Approach for 3D Bacterial Printing.

    PubMed

    Lehner, Benjamin A E; Schmieden, Dominik T; Meyer, Anne S

    2017-07-21

    Sustainable and personally tailored materials production is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different molecules in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally friendly materials production techniques. In this paper, we describe the first steps toward the 3-dimensional printing of bacterial cultures for materials production and patterning. This methodology combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a commercial 3D printer was modified for bacterial systems, and new alginate-based bioink chemistry was developed. Printing temperature, printhead speed, and bioink extrusion rate were all adapted and customized to maximize bacterial health and spatial resolution of printed structures. Our combination of 3D printing technology with biological systems enables a sustainable approach for the production of numerous new materials.

  10. 21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cardiac allograft gene expression profiling test... Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system. (a) Identification. A cardiac allograft gene expression profiling test system is a device that measures the...

  11. The manifold phospholipases A of Legionella pneumophila - identification, export, regulation, and their link to bacterial virulence.

    PubMed

    Banerji, Sangeeta; Aurass, Philipp; Flieger, Antje

    2008-04-01

    The intracellular lung pathogen Legionella pneumophila expresses secreted and cell-associated phospholipase A (PLA) and lysophospholipase A (LPLA) activities belonging to at least three enzyme families. The first family consists of three secreted PLA and LPLA activities displaying the amino acid signature motif 'GDSL'; PlaA, PlaC and PlaD. The second group contains the cell-associated and very potent PLA/LPLA, PlaB. The third group, the patatin-like proteins, comprises 11 members. One patatin-like protein, PatA/VipD, shows LPLA and PLA activities and interferes with vesicular trafficking when expressed in yeast and therefore is possibly involved in the intracellular infection process. Likewise, members of the first two phospholipase families have roles in bacterial virulence because phospholipases are important virulence factors that have been shown to promote bacterial survival, spread and host cell modification/damage. The GDSL enzyme PlaA detoxifies cytolytic lysophospholipids, and PlaB shows contact-dependent haemolytic activity. PlaC acylates cholesterol, a lipid present in eukaryotic hosts but not in the bacterium. Many of the L. pneumophila PLAs are exported by the type II Lsp or the type IVB Dot/Icm secretion systems involved in virulence factor export. Moreover, the regulation of lipolytic activities depends on the transcriptional regulators LetA/S and RpoS, inducing the expression of virulence traits, and on posttranscriptional activators like the zinc metalloprotease ProA.

  12. Formation and dissolution of bacterial colonies.

    PubMed

    Weber, Christoph A; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

  13. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    PubMed Central

    2010-01-01

    Background Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. Results In this study, we present the 3.3 Å crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between

  14. Novel linear polymers able to inhibit bacterial quorum sensing.

    PubMed

    Cavaleiro, Eliana; Duarte, Ana Sofia; Esteves, Ana Cristina; Correia, António; Whitcombe, Michael J; Piletska, Elena V; Piletsky, Sergey A; Chianella, Iva

    2015-05-01

    Bacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers' efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Second-Hand Cigarette Smoke Impairs Bacterial Phagocytosis in Macrophages by Modulating CFTR Dependent Lipid-Rafts

    PubMed Central

    Ni, Inzer; Ji, Changhoon; Vij, Neeraj

    2015-01-01

    Introduction First/Second-hand cigarette-smoke (FHS/SHS) exposure weakens immune defenses inducing chronic obstructive pulmonary disease (COPD) but the underlying mechanisms are not fully understood. Hence, we evaluated if SHS induced changes in membrane/lipid-raft (m-/r)-CFTR (cystic fibrosis transmembrane conductance regulator) expression/activity is a potential mechanism for impaired bacterial phagocytosis in COPD. Methods RAW264.7 murine macrophages were exposed to freshly prepared CS-extract (CSE) containing culture media and/or Pseudomonas-aeruginosa-PA01-GFP for phagocytosis (fluorescence-microscopy), bacterial survival (colony-forming-units-CFU), and immunoblotting assays. The CFTR-expression/activity and lipid-rafts were modulated by transient-transfection or inhibitors/inducers. Next, mice were exposed to acute/sub-chronic-SHS or room-air (5-days/3-weeks) and infected with PA01-GFP, followed by quantification of bacterial survival by CFU-assay. Results We investigated the effect of CSE treatment on RAW264.7 cells infected by PA01-GFP and observed that CSE treatment significantly (p<0.01) inhibits PA01-GFP phagocytosis as compared to the controls. We also verified this in murine model, exposed to acute/sub-chronic-SHS and found significant (p<0.05, p<0.02) increase in bacterial survival in the SHS-exposed lungs as compared to the room-air controls. Next, we examined the effect of impaired CFTR ion-channel-activity on PA01-GFP infection of RAW264.7 cells using CFTR172-inhibitor and found no significant change in phagocytosis. We also similarly evaluated the effect of a CFTR corrector-potentiator compound, VRT-532, and observed no significant rescue of CSE impaired PA01-GFP phagocytosis although it significantly (p<0.05) decreases CSE induced bacterial survival. Moreover, induction of CFTR expression in macrophages significantly (p<0.03) improves CSE impaired PA01-GFP phagocytosis as compared to the control. Next, we verified the link between m

  16. Role of bacterial adhesion in the microbial ecology of biofilms in cooling tower systems

    PubMed Central

    Liu, Yang; Zhang, Wei; Sileika, Tadas; Warta, Richard; Cianciotto, Nicholas P.; Packman, Aaron

    2009-01-01

    The fate of the three heterotrophic biofilm forming bacteria, Pseudomonas aeruginosa, Klebsiella pneumoniae and Flavobacterium sp. in pilot scale cooling towers was evaluated both by observing the persistence of each species in the recirculating water and the formation of biofilms on steel coupons placed in each cooling tower water reservoir. Two different cooling tower experiments were performed: a short-term study (6 days) to observe the initial bacterial colonization of the cooling tower, and a long-term study (3 months) to observe the ecological dynamics with repeated introduction of the test strains. An additional set of batch experiments (6 days) was carried out to evaluate the adhesion of each strain to steel surfaces under similar conditions to those found in the cooling tower experiments. Substantial differences were observed in the microbial communities that developed in the batch systems and cooling towers. P. aeruginosa showed a low degree of adherence to steel surfaces both in batch and in the cooling towers, but grew much faster than K. pneumoniae and Flavobacterium in mixed-species biofilms and ultimately became the dominant organism in the closed batch systems. However, the low degree of adherence caused P. aeruginosa to be rapidly washed out of the open cooling tower systems, and Flavobacterium became the dominant microorganism in the cooling towers in both the short-term and long-term experiments. These results indicate that adhesion, retention and growth on solid surfaces play important roles in the bacterial community that develops in cooling tower systems. PMID:19177226

  17. Capsule Polysaccharide Mediates Bacterial Resistance to Antimicrobial Peptides

    PubMed Central

    Campos, Miguel A.; Vargas, Miguel A.; Regueiro, Verónica; Llompart, Catalina M.; Albertí, Sebastián; Bengoechea, José A.

    2004-01-01

    The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, β-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin. PMID:15557634

  18. Bacterial lipopeptide triggers massive albuminuria in murine lupus nephritis by activating Toll-like receptor 2 at the glomerular filtration barrier

    PubMed Central

    Pawar, Rahul D; Castrezana-Lopez, Liliana; Allam, Ramanjaneyulu; Kulkarni, Onkar P; Segerer, Stephan; Radomska, Ewa; Meyer, Tobias N; Schwesinger, Catherine-Meyer; Akis, Nese; Gröne, Hermann-Josef; Anders, Hans-Joachim

    2009-01-01

    What are the molecular mechanisms of bacterial infections triggering or modulating lupus nephritis? In nephritic MRLlpr/lpr mice, transient exposure to bacterial cell wall components such as lipopeptide or lipopolysaccharide (LPS) increased splenomegaly, the production of DNA autoantibodies, and serum interleukin (IL)-6, IL-12 and tumour necrosis factor (TNF) levels, and aggravated lupus nephritis. Remarkably, bacterial lipopeptide induced massive albuminuria in nephritic but not in non-nephritic mice. This was associated with down-regulation of renal nephrin mRNA and redistribution from its normal localization at foot processes to the perinuclear podocyte area in nephritic MRLlpr/lpr mice. Bacterial lipopeptide activates Toll-like receptor 2 (TLR2), which we found to be expressed on cultured podocytes and glomerular endothelial cells. TNF and interferon (IFN)-γ induced TLR2 mRNA and receptor expression in both cell types. Albumin permeability was significantly increased in cultured podocytes and glomerular endothelial cells upon stimulation by bacterial lipopeptide. LPS also induced moderate albuminuria. In summary, bacterial lipopeptide and LPS can aggravate glomerulonephritis but only lipopeptide potently induces severe albuminuria in MRLlpr/lpr mice. PMID:19175801

  19. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.

  20. The role and regulation of catalase in respiratory tract opportunistic bacterial pathogens.

    PubMed

    Eason, Mia M; Fan, Xin

    2014-09-01

    Respiratory tract bacterial pathogens are the etiologic agents of a variety of illnesses. The ability of these bacteria to cause disease is imparted through survival within the host and avoidance of pathogen clearance by the immune system. Respiratory tract pathogens are continually bombarded by reactive oxygen species (ROS), which may be produced by competing bacteria, normal metabolic function, or host immunological responses. In order to survive and proliferate, bacteria have adapted defense mechanisms to circumvent the effects of ROS. Bacteria employ the use of anti-oxidant enzymes, catalases and catalase-peroxidases, to relieve the effects of the oxidative stressors to which they are continually exposed. The decomposition of ROS has been shown to provide favorable conditions in which respiratory tract opportunistic bacterial pathogens such as Haemophilus influenzae, Mycobacterium tuberculosis, Legionella pneumophila, and Neisseria meningitidis are able to withstand exposure to highly reactive molecules and yet survive. Bacteria possessing mutations in the catalase gene have a decreased survival rate, yet may be able to compensate for the lack of catalatic activity if peroxidatic activity is present. An incomplete knowledge of the mechanisms by which catalase and catalase-peroxidases are regulated still persists, however, in some bacterial species, a regulatory factor known as OxyR has been shown to either up-regulate or down-regulate catalase gene expression. Yet, more research is still needed to increase the knowledge base in relation to this enzyme class. As with this review, we focus on major respiratory tract opportunistic bacterial pathogens in order to elucidate the function and regulation of catalases. The importance of the research could lead to the development of novel treatments against respiratory bacterial infections. Copyright © 2014 Elsevier Ltd. All rights reserved.