Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

NASA Astrophysics Data System (ADS)

Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

2013-03-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

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.

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.

Prevention of ESKAPE pathogen biofilm formation by antimicrobial peptides WLBU2 and LL37.

PubMed

Lin, Qiao; Deslouches, Berthony; Montelaro, Ronald C; Di, Y Peter

2018-05-09

Bacterial biofilm-dependent infections (e.g., cystic fibrosis, surgical sites, and medical implants) are associated with enhanced drug-resistance and thus difficult to eradicate. The goal of this study was to systematically compare three distinct classes of antimicrobial peptides (AMPs) that include the clinically used antibiotic colistin, the natural AMP LL37, the engineered cationic-AMP WLBU2, and four commonly used antibiotics with different bactericidal mechanisms (tobramycin, ciprofloxacin, ceftazidime and vancomycin) for biofilm prevention properties. Using biofilm-prevention assays, we detected bacterial biomass post-attachment in subinhibitory concentrations (1/3 of the MIC) for each AMP, by the crystal violet method, to distinguish the commonly known bactericidal from potentially distinct mechanisms of biofilm prevention. Biofilm regulatory gene expression was assessed using RT-qPCR for correlation with biofilm growth inhibition. Commonly used antibiotics at 1x MIC showed modest ESKAPE biofilm prevention while 1/3 MIC of AMPs demonstrated up to 90% of biofilm prevention. WLBU2 was generally more effective in preventing bacterial attachment than colistin and LL37. Changes in expression of bacterial genes known to affect biofilm regulation were consistent with biofilm prevention. The data warrant further exploration of AMPs with optimized structures to fill a knowledge gap on the potential application of AMPs to difficult-to-cure bacterial biofilm-related infections. Copyright © 2018. Published by Elsevier B.V.

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.

Dynamic optimization of metabolic networks coupled with gene expression.

PubMed

Waldherr, Steffen; Oyarzún, Diego A; Bockmayr, Alexander

2015-01-21

The regulation of metabolic activity by tuning enzyme expression levels is crucial to sustain cellular growth in changing environments. Metabolic networks are often studied at steady state using constraint-based models and optimization techniques. However, metabolic adaptations driven by changes in gene expression cannot be analyzed by steady state models, as these do not account for temporal changes in biomass composition. Here we present a dynamic optimization framework that integrates the metabolic network with the dynamics of biomass production and composition. An approximation by a timescale separation leads to a coupled model of quasi-steady state constraints on the metabolic reactions, and differential equations for the substrate concentrations and biomass composition. We propose a dynamic optimization approach to determine reaction fluxes for this model, explicitly taking into account enzyme production costs and enzymatic capacity. In contrast to the established dynamic flux balance analysis, our approach allows predicting dynamic changes in both the metabolic fluxes and the biomass composition during metabolic adaptations. Discretization of the optimization problems leads to a linear program that can be efficiently solved. We applied our algorithm in two case studies: a minimal nutrient uptake network, and an abstraction of core metabolic processes in bacteria. In the minimal model, we show that the optimized uptake rates reproduce the empirical Monod growth for bacterial cultures. For the network of core metabolic processes, the dynamic optimization algorithm predicted commonly observed metabolic adaptations, such as a diauxic switch with a preference ranking for different nutrients, re-utilization of waste products after depletion of the original substrate, and metabolic adaptation to an impending nutrient depletion. These examples illustrate how dynamic adaptations of enzyme expression can be predicted solely from an optimization principle. Copyright © 2014 Elsevier Ltd. All rights reserved.

Disarming Bacterial Virulence through Chemical Inhibition of the DNA Binding Domain of an AraC-like Transcriptional Activator Protein*

PubMed Central

Yang, Ji; Hocking, Dianna M.; Cheng, Catherine; Dogovski, Con; Perugini, Matthew A.; Holien, Jessica K.; Parker, Michael W.; Hartland, Elizabeth L.; Tauschek, Marija; Robins-Browne, Roy M.

2013-01-01

The misuse of antibiotics during past decades has led to pervasive antibiotic resistance in bacteria. Hence, there is an urgent need for the development of new and alternative approaches to combat bacterial infections. In most bacterial pathogens the expression of virulence is tightly regulated at the transcriptional level. Therefore, targeting pathogens with drugs that interfere with virulence gene expression offers an effective alternative to conventional antimicrobial chemotherapy. Many Gram-negative intestinal pathogens produce AraC-like proteins that control the expression of genes required for infection. In this study we investigated the prototypical AraC-like virulence regulator, RegA, from the mouse attaching and effacing pathogen, Citrobacter rodentium, as a potential drug target. By screening a small molecule chemical library and chemical optimization, we identified two compounds that specifically inhibited the ability of RegA to activate its target promoters and thus reduced expression of a number of proteins required for virulence. Biophysical, biochemical, genetic, and computational analyses indicated that the more potent of these two compounds, which we named regacin, disrupts the DNA binding capacity of RegA by interacting with amino acid residues within a conserved region of the DNA binding domain. Oral administration of regacin to mice, commencing 15 min before or 12 h after oral inoculation with C. rodentium, caused highly significant attenuation of intestinal colonization by the mouse pathogen comparable to that of an isogenic regA-deletion mutant. These findings demonstrate that chemical inhibition of the DNA binding domains of transcriptional regulators is a viable strategy for the development of antimicrobial agents that target bacterial pathogens. PMID:24019519

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.

PubMed

Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

2017-10-01

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

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

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

Bacterial cellulose production by Gluconacetobacter sp. PKY5 in a rotary biofilm contactor.

PubMed

Kim, Yong-Jun; Kim, Jin-Nam; Wee, Young-Jung; Park, Don-Hee; Ryu, Hwa-Won

2007-04-01

A rotary biofilm contactor (RBC) inoculated with Gluconacetobacter sp. RKY5 was used as a bioreactor for improved bacterial cellulose production. The optimal number of disk for bacterial cellulose production was found to be eight, at which bacterial cellulose and cell concentrations were 5.52 and 4.98 g/L. When the aeration rate was maintained at 1.25 vvm, bacterial cellulose and cell concentrations were maximized (5.67 and 5.25 g/L, respectively). The optimal rotation speed of impeller in RBC was 15 rpm. When the culture pH in RBC was not controlled during fermentation, the maximal amount of bacterial cellulose (5.53 g/L) and cells (4.91 g/L) was obtained. Under the optimized culture conditions, bacterial cellulose and cell concentrations in RBC reached to 6.17 and 5.58 g/L, respectively.

Bacterial Cellulose Production by Gluconacetobacter sp. RKY5 in a Rotary Biofilm Contactor

NASA Astrophysics Data System (ADS)

Kim, Yong-Jun; Kim, Jin-Nam; Wee, Young-Jung; Park, Don-Hee; Ryu, Hwa-Won

A rotary biofilm contactor (RBC) inoculated with Gluconacetobacter sp. RKY5 was used as a bioreactor for improved bacterial cellulose production. The optimal number of disk for bacterial cellulose production was found to be eight, at which bacterial cellulose and cell concentrations were 5.52 and 4.98 g/L. When the aeration rate was maintained at 1.25 vvm, bacterial cellulose and cell concentrations were maximized (5.67 and 5.25 g/L, respectively). The optimal rotation speed of impeller in RBC was 15 rpm. When the culture pH in RBC was not controlled during fermentation, the maximal amount of bacterial cellulose (5.53 g/L) and cells (4.91 g/L) was obtained. Under the optimized culture conditions, bacterial cellulose and cell concentrations in RBC reached to 6.17 and 5.58 g/L, respectively.

Structure and mechanism of a molecular rheostat, an RNA thermometer that modulates immune evasion by Neisseria meningitidis

PubMed Central

Barnwal, Ravi Pratap; Loh, Edmund; Godin, Katherine S.; Yip, Jordan; Lavender, Hayley; Tang, Christoph M.; Varani, Gabriele

2016-01-01

Neisseria meningitidis causes bacterial meningitis and septicemia. It evades the host complement system by upregulating expression of immune evasion factors in response to changes in temperature. RNA thermometers within mRNAs control expression of bacterial immune evasion factors, including CssA, in the 5′-untranslated region of the operon for capsule biosynthesis. We dissect the molecular mechanisms of thermoregulation and report the structure of the CssA thermometer. We show that the RNA thermometer acts as a rheostat, whose stability is optimized to respond in a small temperature range around 37°C as occur within the upper airways during infection. Small increases in temperature gradually open up the structure to allow progressively increased access to the ribosome binding site. Even small changes in stability induced by mutations of imperfect base pairs, as in naturally occurring polymorphisms, shift the thermometer response outside of the desired temperature range, suggesting that its activity could be modulated by pharmacological intervention. PMID:27369378

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.

Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli.

PubMed

Tseng, Chi-Ling; Leng, Chih-Hsiang

2012-02-01

Bacterial lipoproteins are crucial antigens for protective immunity against bacterial pathogens. Expression of exogenous lipoproteins in Escherichia coli at high levels is thought to be an extremely difficult endeavor because it frequently results in incomplete or absent lipid modification. Previously, we identified a fusion sequence (D1) from a Neisseria meningitidis lipoprotein that induced a non-lipidated protein, E3 (the domain III of the dengue virus envelope protein), to become lipidated. However, without optimizing the growth conditions, some of the D1-fusion proteins were not lipidated. Here, we report the influence of medium components on the expression of recombinant lipoproteins in E. coli. For high-level expression of mature lipoproteins in the C43 (DE3) strain, M9 medium was better than M63 and the rich medium. Furthermore, we analyzed the influence of other media factors (including nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH) on the levels of lipoprotein expression. The results showed that excess nitrogen sources and phosphate in M9 medium could increase the amount of immature lipoproteins, and glucose was a better carbon source than glycerol for expressing mature lipoproteins. We also found that lipoproteins tended to be completely processed in the alkaline environment, even in the nutrient-rich medium. Additional constructs expressing different immunogens or lipid signal peptides as targets were also utilized, demonstrating that these targets could be expressed as completely mature lipoproteins in the M9 medium but not in the rich medium. Our results provide the useful information for expressing mature exogenous lipoproteins in E. coli.

Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene.

PubMed

Mason, H S; Haq, T A; Clements, J D; Arntzen, C J

1998-08-01

The authors have designed and constructed a plant-optimize synthetic gene encoding the Escherichia coli heat-labile enterotoxin B subunit (LT-B), for use in transgenic plants as an edible vaccine against enterotoxigenic E. coli. Expression of the synthetic LT-B gene in potato plants under the control of a constitutive promoter yielded increased accumulation of LT-B in leaves and tubers, as compared to the bacterial LT-B gene. The plant-derived LT-B assembled into native pentameric structures as evidenced by its ability to bind ganglioside. The authors demonstrated immunogenicity by feeding mice the raw tubers and comparing the anti-LT-B serum IgG and faecal IgA to that produced in mice gavaged with bacterial LT-B. Mice were fed three weekly doses of 5 g tuber tissue containing either 20 or 50 micrograms LT-B, or gavaged weekly with 5 micrograms of LT-B from recombinant E. coli. One week after the third dose, mice immunized with potato LT-B had higher levels of serum and mucosal anti-LT-B than those gavaged with bacterial LT-B. Mice were challenged by oral administration of 25 micrograms LT, and protection assessed by comparing the gut/carcass mass ratios. Although none of the mice were completely protected, the higher dose potato vaccine compared favourably with the bacterial vaccine. These findings show that an edible vaccine against E. coli LT-B is feasible.

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 transcription factor relish, which is necessary for effector gene expression. PMID:21479237

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.

Computational Investigation of Environment-Noise Interaction in Single-Cell Organisms: The Merit of Expression Stochasticity Depends on the Quality of Environmental Fluctuations.

PubMed

Lück, Anja; Klimmasch, Lukas; Großmann, Peter; Germerodt, Sebastian; Kaleta, Christoph

2018-01-10

Organisms need to adapt to changing environments and they do so by using a broad spectrum of strategies. These strategies include finding the right balance between expressing genes before or when they are needed, and adjusting the degree of noise inherent in gene expression. We investigated the interplay between different nutritional environments and the inhabiting organisms' metabolic and genetic adaptations by applying an evolutionary algorithm to an agent-based model of a concise bacterial metabolism. Our results show that constant environments and rapidly fluctuating environments produce similar adaptations in the organisms, making the predictability of the environment a major factor in determining optimal adaptation. We show that exploitation of expression noise occurs only in some types of fluctuating environment and is strongly dependent on the quality and availability of nutrients: stochasticity is generally detrimental in fluctuating environments and beneficial only at equal periods of nutrient availability and above a threshold environmental richness. Moreover, depending on the availability and nutritional value of nutrients, nutrient-dependent and stochastic expression are both strategies used to deal with environmental changes. Overall, we comprehensively characterize the interplay between the quality and periodicity of an environment and the resulting optimal deterministic and stochastic regulation strategies of nutrient-catabolizing pathways.

Autonomous Bacterial Localization and Gene Expression Based on Nearby Cell Receptor Density

DTIC Science & Technology

2013-01-22

synthesis of novel products (Kwok, 2010). In such cases, cell populations are aligned for optimal production . While Nature’s biosynthetic toolbox is vast...less commonly examined but equally innovative strategy envisions a repro- grammed cell itself or small collections of cells as the end products of...to surfaces. In Figure 2B, we depict the level of NF binding and AI-2 production as a function of coated avidin. We used a mathematical model for E

A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes.

PubMed

Mank, Nils N; Berghoff, Bork A; Klug, Gabriele

2013-03-01

Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes

PubMed Central

Mank, Nils N.; Berghoff, Bork A.; Klug, Gabriele

2013-01-01

Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail. PMID:23392242

A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors And Controls Bacterial Pathogenicity

PubMed Central

Swem, Lee R.; Swem, Danielle L.; O’Loughlin, Colleen T.; Gatmaitan, Raleene; Zhao, Bixiao; Ulrich, Scott M.; Bassler, Bonnie L.

2009-01-01

Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development. PMID:19647512

Chassis optimization as a cornerstone for the application of synthetic biology based strategies in microbial secondary metabolism.

PubMed

Beites, Tiago; Mendes, Marta V

2015-01-01

The increased number of bacterial genome sequencing projects has generated over the last years a large reservoir of genomic information. In silico analysis of this genomic data has renewed the interest in bacterial bioprospecting for bioactive compounds by unveiling novel biosynthetic gene clusters of unknown or uncharacterized metabolites. However, only a small fraction of those metabolites is produced under laboratory-controlled conditions; the remaining clusters represent a pool of novel metabolites that are waiting to be "awaken". Activation of the biosynthetic gene clusters that present reduced or no expression (known as cryptic or silent clusters) by heterologous expression has emerged as a strategy for the identification and production of novel bioactive molecules. Synthetic biology, with engineering principles at its core, provides an excellent framework for the development of efficient heterologous systems for the expression of biosynthetic gene clusters. However, a common problem in its application is the host-interference problem, i.e., the unpredictable interactions between the device and the host that can hamper the desired output. Although an effort has been made to develop orthogonal devices, the most proficient way to overcome the host-interference problem is through genome simplification. In this review we present an overview on the strategies and tools used in the development of hosts/chassis for the heterologous expression of specialized metabolites biosynthetic gene clusters. Finally, we introduce the concept of specialized host as the next step of development of expression hosts.

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

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.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

PubMed Central

Carnes, Aaron E.; Luke, Jeremy M.; Vincent, Justin M.; Anderson, Sheryl; Schukar, Angela; Hodgson, Clague P.; Williams, James A.

2010-01-01

Background For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Methods Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Results Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. Conclusions We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. PMID:20806425

Sex-specific consequences of an induced immune response on reproduction in a moth.

PubMed

Barthel, Andrea; Staudacher, Heike; Schmaltz, Antje; Heckel, David G; Groot, Astrid T

2015-12-16

Immune response induction benefits insects in combatting infection by pathogens. However, organisms have a limited amount of resources available and face the dilemma of partitioning resources between immunity and other life-history traits. Since males and females differ in their life histories, sex-specific resource investment strategies to achieve an optimal immune response following an infection can be expected. We investigated immune response induction of females and males of Heliothis virescens in response to the entomopathogenic bacterium Serratia entomophila, and its effects on mating success and the female sexual signal. We found that females had higher expression levels of immune-related genes after bacterial challenge than males. However, males maintained a higher baseline expression of immune-related genes than females. The increased investment in immunity of female moths was negatively correlated with mating success and the female sexual signal. Male mating success was unaffected by bacterial challenge. Our results show that the sexes differed in their investment strategies: females invested in immune defense after a bacterial challenge, indicating facultative immune deployment, whereas males had higher baseline immunity than females, indicating immune maintenance. Interestingly, these differences in investment were reflected in the mate choice assays. As female moths are the sexual signallers, females need to invest resources in their attractiveness. However, female moths appeared to invest in immunity at the cost of reproductive effort.

Response surface methodology for optimization of medium for decolorization of textile dye Direct Black 22 by a novel bacterial consortium.

PubMed

Mohana, Sarayu; Shrivastava, Shalini; Divecha, Jyoti; Madamwar, Datta

2008-02-01

Decolorization and degradation of polyazo dye Direct Black 22 was carried out by distillery spent wash degrading mixed bacterial consortium, DMC. Response surface methodology (RSM) involving a central composite design (CCD) in four factors was successfully employed for the study and optimization of decolorization process. The hyper activities and interactions between glucose concentration, yeast extract concentration, dye concentration and inoculum size on dye decolorization were investigated and modeled. Under optimized conditions the bacterial consortium was able to decolorize the dye almost completely (>91%) within 12h. Bacterial consortium was able to decolorize 10 different azo dyes. The optimum combination of the four variables predicted through RSM was confirmed through confirmatory experiments and hence this bacterial consortium holds potential for the treatment of industrial waste water. Dye degradation products obtained during the course of decolorization were analyzed by HPTLC.

Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa.

PubMed

Khaledi, Ariane; Schniederjans, Monika; Pohl, Sarah; Rainer, Roman; Bodenhofer, Ulrich; Xia, Boyang; Klawonn, Frank; Bruchmann, Sebastian; Preusse, Matthias; Eckweiler, Denitsa; Dötsch, Andreas; Häussler, Susanne

2016-08-01

Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa

PubMed Central

Khaledi, Ariane; Schniederjans, Monika; Pohl, Sarah; Rainer, Roman; Bodenhofer, Ulrich; Xia, Boyang; Klawonn, Frank; Bruchmann, Sebastian; Preusse, Matthias; Eckweiler, Denitsa; Dötsch, Andreas

2016-01-01

Emerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinical Pseudomonas aeruginosa isolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution. PMID:27216077

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.

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

Using Chemical Reaction Kinetics to Predict Optimal Antibiotic Treatment Strategies.

PubMed

Abel Zur Wiesch, Pia; Clarelli, Fabrizio; Cohen, Ted

2017-01-01

Identifying optimal dosing of antibiotics has proven challenging-some antibiotics are most effective when they are administered periodically at high doses, while others work best when minimizing concentration fluctuations. Mechanistic explanations for why antibiotics differ in their optimal dosing are lacking, limiting our ability to predict optimal therapy and leading to long and costly experiments. We use mathematical models that describe both bacterial growth and intracellular antibiotic-target binding to investigate the effects of fluctuating antibiotic concentrations on individual bacterial cells and bacterial populations. We show that physicochemical parameters, e.g. the rate of drug transmembrane diffusion and the antibiotic-target complex half-life are sufficient to explain which treatment strategy is most effective. If the drug-target complex dissociates rapidly, the antibiotic must be kept constantly at a concentration that prevents bacterial replication. If antibiotics cross bacterial cell envelopes slowly to reach their target, there is a delay in the onset of action that may be reduced by increasing initial antibiotic concentration. Finally, slow drug-target dissociation and slow diffusion out of cells act to prolong antibiotic effects, thereby allowing for less frequent dosing. Our model can be used as a tool in the rational design of treatment for bacterial infections. It is easily adaptable to other biological systems, e.g. HIV, malaria and cancer, where the effects of physiological fluctuations of drug concentration are also poorly understood.

Using Chemical Reaction Kinetics to Predict Optimal Antibiotic Treatment Strategies

PubMed Central

Abel zur Wiesch, Pia; Cohen, Ted

2017-01-01

Identifying optimal dosing of antibiotics has proven challenging—some antibiotics are most effective when they are administered periodically at high doses, while others work best when minimizing concentration fluctuations. Mechanistic explanations for why antibiotics differ in their optimal dosing are lacking, limiting our ability to predict optimal therapy and leading to long and costly experiments. We use mathematical models that describe both bacterial growth and intracellular antibiotic-target binding to investigate the effects of fluctuating antibiotic concentrations on individual bacterial cells and bacterial populations. We show that physicochemical parameters, e.g. the rate of drug transmembrane diffusion and the antibiotic-target complex half-life are sufficient to explain which treatment strategy is most effective. If the drug-target complex dissociates rapidly, the antibiotic must be kept constantly at a concentration that prevents bacterial replication. If antibiotics cross bacterial cell envelopes slowly to reach their target, there is a delay in the onset of action that may be reduced by increasing initial antibiotic concentration. Finally, slow drug-target dissociation and slow diffusion out of cells act to prolong antibiotic effects, thereby allowing for less frequent dosing. Our model can be used as a tool in the rational design of treatment for bacterial infections. It is easily adaptable to other biological systems, e.g. HIV, malaria and cancer, where the effects of physiological fluctuations of drug concentration are also poorly understood. PMID:28060813

Vaccine-preventable infections in Systemic Lupus Erythematosus

PubMed Central

Murdaca, Giuseppe; Orsi, Andrea; Spanò, Francesca; Faccio, Valeria; Puppo, Francesco; Durando, Paolo; Icardi, Giancarlo; Ansaldi, Filippo

2016-01-01

Systemic Lupus Erythematosus (SLE) is characterized by abnormal autoantibody production and clearance. Infections are among the most important causes of morbidity and mortality in SLE patients; they have an increased frequency of severe bacterial and viral infections possibly due to inherited genetic and immunologic defects and to immunosuppressive therapies. In addition, infectious agents can switch on lupus disease expression and activity. Among the strategies to reduce the risk of infection, vaccination can be considered the most reliable option. Most vaccines are effective and safe in SLE patients, although in certain cases immunogenicity may be sub-optimal and vaccination can trigger a flare. Although these issues are currently unresolved, the risk benefit balance is in favor for vaccination to reduce the risk of infection in SLE patients. In the present review we discuss the preventive strategies currently recommended to reduce bacterial and viral infections in SLE. PMID:26750996

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

PubMed

Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

2015-01-01

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp.

PubMed

Ausec, Luka; Črnigoj, Miha; Šnajder, Marko; Ulrih, Nataša Poklar; Mandic-Mulec, Ines

2015-12-01

Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.

Production of Phloroglucinol, a Platform Chemical, in Arabidopsis using a Bacterial Gene.

PubMed

Abdel-Ghany, Salah E; Day, Irene; Heuberger, Adam L; Broeckling, Corey D; Reddy, Anireddy S N

2016-12-07

Phloroglucinol (1,3,5-trihydroxybenzene; PG) and its derivatives are phenolic compounds that are used for various industrial applications. Current methods to synthesize PG are not sustainable due to the requirement for carbon-based precursors and co-production of toxic byproducts. Here, we describe a more sustainable production of PG using plants expressing a native bacterial or a codon-optimized synthetic PhlD targeted to either the cytosol or chloroplasts. Transgenic lines were analyzed for the production of PG using gas and liquid chromatography coupled to mass spectroscopy. Phloroglucinol was produced in all transgenic lines and the line with the highest PhlD transcript level showed the most accumulation of PG. Over 80% of the produced PG was glycosylated to phlorin. Arabidopsis leaves have the machinery to glycosylate PG to form phlorin, which can be hydrolyzed enzymatically to produce PG. Furthermore, the metabolic profile of plants with PhlD in either the cytosol or chloroplasts was altered. Our results provide evidence that plants can be engineered to produce PG using a bacterial gene. Phytoproduction of PG using a bacterial gene paves the way for further genetic manipulations to enhance the level of PG with implications for the commercial production of this important platform chemical in plants.

Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

PubMed Central

Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

2010-01-01

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments[OPEN

PubMed Central

2017-01-01

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. PMID:28619883

Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.

PubMed

Chee Wei, T; Nurul Wahida, A G; Shaharum, S

2014-12-01

Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

High density growth of T7 expression strains with auto-induction option

DOEpatents

Studier, F. William

2010-07-20

A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

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.

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.

Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

PubMed

Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

PubMed Central

Wettig, Shawn; Slavcev, Roderick A.

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

PubMed Central

Sangawa, Takeshi; Tabata, Sanae; Suzuki, Kei; Saheki, Yasushi; Tanaka, Keiji; Takagi, Junichi

2013-01-01

Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. PMID:23526492

Multiple sequence alignment using multi-objective based bacterial foraging optimization algorithm.

PubMed

Rani, R Ranjani; Ramyachitra, D

2016-12-01

Multiple sequence alignment (MSA) is a widespread approach in computational biology and bioinformatics. MSA deals with how the sequences of nucleotides and amino acids are sequenced with possible alignment and minimum number of gaps between them, which directs to the functional, evolutionary and structural relationships among the sequences. Still the computation of MSA is a challenging task to provide an efficient accuracy and statistically significant results of alignments. In this work, the Bacterial Foraging Optimization Algorithm was employed to align the biological sequences which resulted in a non-dominated optimal solution. It employs Multi-objective, such as: Maximization of Similarity, Non-gap percentage, Conserved blocks and Minimization of gap penalty. BAliBASE 3.0 benchmark database was utilized to examine the proposed algorithm against other methods In this paper, two algorithms have been proposed: Hybrid Genetic Algorithm with Artificial Bee Colony (GA-ABC) and Bacterial Foraging Optimization Algorithm. It was found that Hybrid Genetic Algorithm with Artificial Bee Colony performed better than the existing optimization algorithms. But still the conserved blocks were not obtained using GA-ABC. Then BFO was used for the alignment and the conserved blocks were obtained. The proposed Multi-Objective Bacterial Foraging Optimization Algorithm (MO-BFO) was compared with widely used MSA methods Clustal Omega, Kalign, MUSCLE, MAFFT, Genetic Algorithm (GA), Ant Colony Optimization (ACO), Artificial Bee Colony (ABC), Particle Swarm Optimization (PSO) and Hybrid Genetic Algorithm with Artificial Bee Colony (GA-ABC). The final results show that the proposed MO-BFO algorithm yields better alignment than most widely used methods. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.

PubMed

Dammeyer, Thorben; Steinwand, Miriam; Krüger, Sarah-C; Dübel, Stefan; Hust, Michael; Timmis, Kenneth N

2011-02-21

Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.

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

NrdR Transcription Regulation: Global Proteome Analysis and Its Role in Escherichia coli Viability and Virulence

PubMed Central

Naveen, Vankadari; Hsiao, Chwan-Deng

2016-01-01

Bacterial ribonucleotide reductases (RNRs) play an important role in the synthesis of dNTPs and their expression is regulated by the transcription factors, NrdR and Fur. Recent transcriptomic studies using deletion mutants have indicated a role for NrdR in bacterial chemotaxis and in the maintenance of topoisomerase levels. However, NrdR deletion alone has no effect on bacterial growth or virulence in infected flies or in human blood cells. Furthermore, transcriptomic studies are limited to the deletion strain alone, and so are inadequate for drawing biological implications when the NrdR repressor is active or abundant. Therefore, further examination is warranted of changes in the cellular proteome in response to both NrdR overexpression, as well as deletion, to better understand its functional relevance as a bacterial transcription repressor. Here, we profile bacterial fate under conditions of overexpression and deletion of NrdR in E. coli. Biochemical assays show auxiliary zinc enhances the DNA binding activity of NrdR. We also demonstrate at the physiological level that increased nrdR expression causes a significant reduction in bacterial growth and fitness even at normal temperatures, and causes lethality at elevated temperatures. Corroborating these direct effects, global proteome analysis following NrdR overexpression showed a significant decrease in global protein expression. In parallel, studies on complementary expression of downregulated essential genes polA, eno and thiL showed partial rescue of the fitness defect caused by NrdR overexpression. Deletion of downregulated non-essential genes ygfK and trxA upon NrdR overexpression resulted in diminished bacterial growth and fitness suggesting an additional role for NrdR in regulating other genes. Moreover, in comparison with NrdR deletion, E. coli cells overexpressing NrdR showed significantly diminished adherence to human epithelial cells, reflecting decreased bacterial virulence. These results suggest that elevated expression of NrdR could be a suitable means to retard bacterial growth and virulence, as its elevated expression reduces bacterial fitness and impairs host cell adhesion. PMID:27275780

Carbon dioxide sequestration by chemolithotrophic oleaginous bacteria for production and optimization of polyhydroxyalkanoate.

PubMed

Kumar, Manish; Gupta, Asmita; Thakur, Indu Shekhar

2016-08-01

The present work involved screening of a previously reported carbon concentrating oleaginous bacterial strain Serratia sp. ISTD04 for production of PHA and optimization of process parameters for enhanced PHA and biomass generation. The selected bacterial strain was screened for PHA production based on Nile red staining followed by visualization under fluorescence microscope. Spectrofluorometric measurement of Nile red fluorescence of the bacterial culture was also done. Confirmatory analysis of PHA accumulation by GC-MS revealed the presence of 3-hydroxyvalerate. Detection of characteristic peaks in the FT-IR spectrum further confirmed the production of PHA by the bacterium. Response Surface Methodology was used for optimization of pH and carbon sources' concentrations for higher PHA production. There was almost a 2 fold increase in the production of PHA following optimization as compared to un-optimized condition. The study thus establishes the production of PHA by Serratia sp. ISTD04. Copyright © 2016 Elsevier Ltd. All rights reserved.

Iterative algorithm-guided design of massive strain libraries, applied to itaconic acid production in yeast.

PubMed

Young, Eric M; Zhao, Zheng; Gielesen, Bianca E M; Wu, Liang; Benjamin Gordon, D; Roubos, Johannes A; Voigt, Christopher A

2018-05-09

Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4Mb and 32 variants, 0.6Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration. Copyright © 2018. Published by Elsevier Inc.

Approaches to achieve high-level heterologous protein production in plants.

PubMed

Streatfield, Stephen J

2007-01-01

Plants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression. Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies.

Human dendritic cell DC-SIGN and TLR-2 mediate complementary immune regulatory activities in response to Lactobacillus rhamnosus JB-1.

PubMed

Konieczna, Patrycja; Schiavi, Elisa; Ziegler, Mario; Groeger, David; Healy, Selena; Grant, Ray; O'Mahony, Liam

2015-01-01

The microbiota is required for optimal host development and ongoing immune homeostasis. Lactobacilli are common inhabitants of the mammalian large intestine and immunoregulatory effects have been described for certain, but not all, strains. The mechanisms underpinning these protective effects are beginning to be elucidated. One such protective organism is Lactobacillus rhamnosus JB-1 (Lb. rhamnosus JB-1). Lb. murinus has no such anti-inflammatory protective effects and was used as a comparator organism. Human monocyte-derived dendritic cells (MDDCs) were co-incubated with bacteria and analysed over time for bacterial adhesion and intracellular processing, costimulatory molecule expression, cytokine secretion and induction of lymphocyte polarization. Neutralising antibodies were utilized to identify the responsible MDDC receptors. Lb. rhamnosus JB-1 adhered to MDDCs, but internalization and intracellular processing was significantly delayed, compared to Lb. murinus which was rapidly internalized and processed. Lb. murinus induced CD80 and CD86 expression, accompanied by high levels of cytokine secretion, while Lb. rhamnosus JB-1 was a poor inducer of costimulatory molecule expression and cytokine secretion. Lb. rhamnosus JB-1 primed MDDCs induced Foxp3 expression in autologous lymphocytes, while Lb. murinus primed MDDCs induced Foxp3, T-bet and Ror-γt expression. DC-SIGN was required for Lb. rhamnosus JB-1 adhesion and influenced IL-12 secretion, while TLR-2 influenced IL-10 and IL-12 secretion. Here we demonstrate that the delayed kinetics of bacterial processing by MDDCs correlates with MDDC activation and stimulation of lymphocytes. Thus, inhibition or delay of intracellular processing may be a novel strategy by which certain commensals may avoid the induction of proinflammatory responses.

Human Dendritic Cell DC-SIGN and TLR-2 Mediate Complementary Immune Regulatory Activities in Response to Lactobacillus rhamnosus JB-1

PubMed Central

Konieczna, Patrycja; Schiavi, Elisa; Ziegler, Mario; Groeger, David; Healy, Selena; Grant, Ray; O’Mahony, Liam

2015-01-01

The microbiota is required for optimal host development and ongoing immune homeostasis. Lactobacilli are common inhabitants of the mammalian large intestine and immunoregulatory effects have been described for certain, but not all, strains. The mechanisms underpinning these protective effects are beginning to be elucidated. One such protective organism is Lactobacillus rhamnosus JB-1 (Lb. rhamnosus JB-1). Lb. murinus has no such anti-inflammatory protective effects and was used as a comparator organism. Human monocyte-derived dendritic cells (MDDCs) were co-incubated with bacteria and analysed over time for bacterial adhesion and intracellular processing, costimulatory molecule expression, cytokine secretion and induction of lymphocyte polarization. Neutralising antibodies were utilized to identify the responsible MDDC receptors. Lb. rhamnosus JB-1 adhered to MDDCs, but internalization and intracellular processing was significantly delayed, compared to Lb. murinus which was rapidly internalized and processed. Lb. murinus induced CD80 and CD86 expression, accompanied by high levels of cytokine secretion, while Lb. rhamnosus JB-1 was a poor inducer of costimulatory molecule expression and cytokine secretion. Lb. rhamnosus JB-1 primed MDDCs induced Foxp3 expression in autologous lymphocytes, while Lb. murinus primed MDDCs induced Foxp3, T-bet and Ror-γt expression. DC-SIGN was required for Lb. rhamnosus JB-1 adhesion and influenced IL-12 secretion, while TLR-2 influenced IL-10 and IL-12 secretion. Here we demonstrate that the delayed kinetics of bacterial processing by MDDCs correlates with MDDC activation and stimulation of lymphocytes. Thus, inhibition or delay of intracellular processing may be a novel strategy by which certain commensals may avoid the induction of proinflammatory responses. PMID:25816321

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.

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.

Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both?

PubMed Central

Sharifi, Rouhallah; Ryu, Choong-Min

2016-01-01

Biological control (biocontrol) agents act on plants via numerous mechanisms, and can be used to protect plants from pathogens. Biocontrol agents can act directly as pathogen antagonists or competitors or indirectly to promote plant induced systemic resistance (ISR). Whether a biocontrol agent acts directly or indirectly depends on the specific strain and the pathosystem type. We reported previously that bacterial volatile organic compounds (VOCs) are determinants for eliciting plant ISR. Emerging data suggest that bacterial VOCs also can directly inhibit fungal and plant growth. The aim of the current study was to differentiate direct and indirect mechanisms of bacterial VOC effects against Botrytis cinerea infection of Arabidopsis. Volatile emissions from Bacillus subtilis GB03 successfully protected Arabidopsis seedlings against B. cinerea. First, we investigated the direct effects of bacterial VOCs on symptom development and different phenological stages of B. cinerea including spore germination, mycelial attachment to the leaf surface, mycelial growth, and sporulation in vitro and in planta. Volatile emissions inhibited hyphal growth in a dose-dependent manner in vitro, and interfered with fungal attachment on the hydrophobic leaf surface. Second, the optimized bacterial concentration that did not directly inhibit fungal growth successfully protected Arabidopsis from fungal infection, which indicates that bacterial VOC-elicited plant ISR has a more important role in biocontrol than direct inhibition of fungal growth on Arabidopsis. We performed qRT-PCR to investigate the priming of the defense-related genes PR1, PDF1.2, and ChiB at 0, 12, 24, and 36 h post-infection and 14 days after the start of plant exposure to bacterial VOCs. The results indicate that bacterial VOCs potentiate expression of PR1 and PDF1.2 but not ChiB, which stimulates SA- and JA-dependent signaling pathways in plant ISR and protects plants against pathogen colonization. This study provides new evidence for bacterial VOC-elicited plant ISR that protects Arabidopsis plants from infection by the necrotrophic fungus B. cinerea. Our work reveals that bacterial VOCs primarily act via an indirect mechanism to elicit plant ISR, and have a major role in biocontrol against fungal pathogens. PMID:26941721

Treatment of bacterial meningitis: an update.

PubMed

Shin, Seon Hee; Kim, Kwang Sik

2012-10-01

The introduction of protein conjugate vaccines for Haemophilus influenzae type b (Hib), Streptococcus pneumoniae (S. pneumoniae) and Neisseria meningitidis (N. menigitidis) has changed the epidemiology of bacterial meningitis. Bacterial meningitis continues to be an important cause of mortality and morbidity, and our incomplete knowledge of its pathogenesis and emergence of antimicrobial resistant bacteria contribute to such mortality and morbidity. An early empiric antibiotic treatment is critical for the management of patients with bacterial meningitis. This article gives an overview on optimal treatment strategies of bacterial meningitis, along with considerations of new insights on epidemiology, clinical and laboratory findings supportive of bacterial meningitis, chemoprophylaxis, selection of initial antimicrobial agents for suspected bacterial meningitis, antimicrobial resistance and utility of new antibiotics, status on anti-inflammatory agents and adjunctive therapy, and pathogenesis of bacterial meningitis. Prompt treatment of bacterial meningitis with an appropriate antibiotic is essential. Optimal antimicrobial treatment of bacterial meningitis requires bactericidal agents able to penetrate the blood-brain barrier (BBB), with efficacy in cerebrospinal fluid (CSF). Several new antibiotics have been introduced for the treatment of meningitis caused by resistant bacteria, but their use in human studies has been limited. More complete understanding of the microbial and host interactions that are involved in the pathogenesis of bacterial meningitis and associated neurologic sequelae is likely to help in developing new strategies for the prevention and therapy of bacterial meningitis.

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 rights reserved.

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.

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

Catalytic Properties of Staphylococcus aureus and Bacillus Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an Escherichia coli Host▿

PubMed Central

Swartz, Talia H.; Ito, Masahiro; Ohira, Takayuki; Natsui, Shinsuke; Hicks, David B.; Krulwich, Terry A.

2007-01-01

Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary Na+(Li+)/H+ antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited Na+(Li+)/H+ antiport. A second paralogue of S. aureus (Mnh2) did not. K+, Ca2+, and Mg2+ did not support significant antiport by any of the test antiporters. All three Na+(Li+)/H+ Mrp antiporters had alkaline pH optima and apparent Km values for Na+ that are among the lowest reported for bacterial Na+/H+ antiporters. Using a fluorescent probe of the transmembrane electrical potential (ΔΨ), Mrp Na+/H+ antiport was shown to be ΔΨ consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher ΔΨ levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, ΔΨ-consuming antiporters can elicit increased capacity for ΔΨ generation in a bacterial host. PMID:17293423

A teleost CD46 is involved in the regulation of complement activation and pathogen infection.

PubMed

Li, Mo-Fei; Sui, Zhi-Hai; Sun, Li

2017-11-03

In mammals, CD46 is involved in the inactivation of complement by factor I (FI). In teleost, study on the function of CD46 is very limited. In this study, we examined the immunological property of a CD46 molecule (CsCD46) from tongue sole, a teleost species with important economic value. We found that recombinant CsCD46 (rCsCD46) interacted with FI and inhibited complement activation in an FI-dependent manner. rCsCD46 also interacted with bacterial pathogens via a different mechanism to that responsible for the FI interaction, involving different rCsCD46 sites. Cellular study showed that CsCD46 was expressed on peripheral blood leukocytes (PBL) and protected the cells against the killing effect of complement. When the CsCD46 on PBL was blocked by antibody before incubation of the cells with bacterial pathogens, cellular infection was significantly reduced. Consistently, when tongue sole were infected with bacterial pathogens in the presence of rCsCD46, tissue dissemination and survival of the pathogens were significantly inhibited. These results provide the first evidence to indicate that CD46 in teleosts negatively regulates complement activation via FI and protects host cells from complement-induced damage, and that CD46 is required for optimal bacterial infection probably by serving as a receptor for the bacteria.

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

Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta

The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agentmore » of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.« less

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

DOE PAGES

Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; ...

2015-01-01

Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

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

Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan

Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

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

Microeconomic principles explain an optimal genome size in bacteria.

PubMed

Ranea, Juan A G; Grant, Alastair; Thornton, Janet M; Orengo, Christine A

2005-01-01

Bacteria can clearly enhance their survival by expanding their genetic repertoire. However, the tight packing of the bacterial genome and the fact that the most evolved species do not necessarily have the biggest genomes suggest there are other evolutionary factors limiting their genome expansion. To clarify these restrictions on size, we studied those protein families contributing most significantly to bacterial-genome complexity. We found that all bacteria apply the same basic and ancestral 'molecular technology' to optimize their reproductive efficiency. The same microeconomics principles that define the optimum size in a factory can also explain the existence of a statistical optimum in bacterial genome size. This optimum is reached when the bacterial genome obtains the maximum metabolic complexity (revenue) for minimal regulatory genes (logistic cost).

Preparation of RNA from bacteria infected with bacteriophages: a case study from the marine unicellular Synechococcus sp. WH7803 infected by phage S-PM2.

PubMed

Shan, Jinyu; Clokie, Martha

2009-01-01

Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

PubMed

Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-08-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

PubMed Central

Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2013-01-01

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

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.

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

PubMed

Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

2017-11-17

Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

NASA Astrophysics Data System (ADS)

Close, Dan M.; Hahn, Ruth E.; Patterson, Stacey S.; Baek, Seung J.; Ripp, Steven A.; Sayler, Gary S.

2011-04-01

Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 104 cells but at the cost of increasing overall image integration time.

Band Gap Engineering of Titania Film through Cobalt Regulation for Oxidative Damage of Bacterial Respiration and Viability.

PubMed

Li, Jinhua; Wang, Jiaxing; Wang, Donghui; Guo, Geyong; Yeung, Kelvin W K; Zhang, Xianlong; Liu, Xuanyong

2017-08-23

Biomaterial-related bacterial infections cause patient suffering, mortality, and extended periods of hospitalization and impose a substantial burden on medical systems. In this context, understanding the interactions between nanomaterials and bacteria is clinically significant. Herein, TiO 2 -based heterojunctions, including Co-TiO 2 , CoO-TiO 2 , and Co 3 O 4 -TiO 2 , were first designed by optimizing magnetron sputtering to establish a platform to explore the interactions between nanomaterials and bacteria. We found that the energy band bending and band gap narrowing were effectively promoted at the contact interface of the heterojunctions, which have the ability to induce abiotic reactive oxygen species formation. Using methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, in vitro studies showed that the heterojunctions of Co-TiO 2 , CoO-TiO 2 , and especially Co 3 O 4 -TiO 2 can effectively downregulate the expression levels of bacterial respiratory genes and cause oxidative damage to bacterial membrane respiration and viability. As a result, the surfaces of the heterojunctions possess a favorable antiadherent bacterial activity. Moreover, using an osteomyelitis model, the preclinical study on rats further confirmed the favorable anti-infection effect of the elaborately designed heterojunctions (especially Co 3 O 4 -TiO 2 ). We hope this study can provide new insights into the surface antibacterial design of biomaterials using energy band engineering for both basic research and clinical needs. Meanwhile, this attempt may also contribute to expanding the biomedical applications of cobalt-based nanoparticles for the treatment of antibiotic-resistant infections.

Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection.

PubMed

Lipsky, Alexander; Joshi, Janak Raj; Carmi, Nir; Yedidia, Iris

2016-11-20

The genus Ornithogalum includes several ornamental species that suffer substantial losses from bacterial soft rot caused by Pectobacteria. The absence of effective control measures for use against soft rot bacteria led to the initiation of a project in which a small antimicrobial peptide from an Asian horseshoe crab, tachyplesin (tpnI), was introduced into two commercial cultivars: O. dubium and O. thyrsoides. Disease severity and bacterial colonization were examined in transgenic lines expressing this peptide. Disease resistance was evaluated in six lines of each species by measuring bacterial proliferation in the plant tissue. Three transgenic lines of each species were subjected to further analysis in which the expression level of the transgene was evaluated using RT-PCR and qRT-PCR. The development of disease symptoms and bacterial colonization of the plant tissue were also examined using GFP-expressing strain of P. carotovorum subsp. brasiliense Pcb3. Confocal-microscopy imaging revealed significantly reduced quantities of bacterial cells in the transgenic plant lines that had been challenged with the bacterium. The results clearly demonstrate that tpnI expression reduces bacterial proliferation, colonization and disease symptom (reduced by 95-100%) in the transgenic plant tissues. The quantity of tpnI transcripts, as measured by qRT-PCR, was negatively correlated with the protection afforded to the plants, as measured by the reduced severity of disease symptoms in the tissue. Copyright © 2016 Elsevier B.V. All rights reserved.

A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

PubMed Central

Wang, Jin Yuan; Carrasco, Jose A.; Lloyd, Scott A.; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D.; Nataro, James P.; Pasetti, Marcela F.

2014-01-01

Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. PMID:25332120

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

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

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

Determining β-lactam exposure threshold to suppress resistance development in Gram-negative bacteria.

PubMed

Tam, Vincent H; Chang, Kai-Tai; Zhou, Jian; Ledesma, Kimberly R; Phe, Kady; Gao, Song; Van Bambeke, Françoise; Sánchez-Díaz, Ana María; Zamorano, Laura; Oliver, Antonio; Cantón, Rafael

2017-05-01

β-Lactams are commonly used for nosocomial infections and resistance to these agents among Gram-negative bacteria is increasing rapidly. Optimized dosing is expected to reduce the likelihood of resistance development during antimicrobial therapy, but the target for clinical dose adjustment is not well established. We examined the likelihood that various dosing exposures would suppress resistance development in an in vitro hollow-fibre infection model. Two strains of Klebsiella pneumoniae and two strains of Pseudomonas aeruginosa (baseline inocula of ∼10 8  cfu/mL) were examined. Various dosing exposures of cefepime, ceftazidime and meropenem were simulated in the hollow-fibre infection model. Serial samples were obtained to ascertain the pharmacokinetic simulations and viable bacterial burden for up to 120 h. Drug concentrations were determined by a validated LC-MS/MS assay and the simulated exposures were expressed as C min /MIC ratios. Resistance development was detected by quantitative culture on drug-supplemented media plates (at 3× the corresponding baseline MIC). The C min /MIC breakpoint threshold to prevent bacterial regrowth was identified by classification and regression tree (CART) analysis. For all strains, the bacterial burden declined initially with the simulated exposures, but regrowth was observed in 9 out of 31 experiments. CART analysis revealed that a C min /MIC ratio ≥3.8 was significantly associated with regrowth prevention (100% versus 44%, P  = 0.001). The development of β-lactam resistance during therapy could be suppressed by an optimized dosing exposure. Validation of the proposed target in a well-designed clinical study is warranted. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

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 implemented as a Disease Risk Score in the validation group (130 children, including 23 bacterial, 28 viral, 79 indeterminate; median age 17 months, 57% males), bacterial infection was identified in all 23 microbiologically-confirmed definite bacterial patients, with a sensitivity of 100% (95% confidence interval [CI], 100 - 100), and in 1 of 28 definite viral patients, with specificity of 96.4% (95% CI, 89.3 – 100). When applied to additional validation datasets from patients with meningococcal and inflammatory diseases, bacterial infection was identified with a sensitivity of 91.7% (79.2-100) and 90.0% (70.0-100) respectively, and with specificity of 96.0% (88.0-100) and 95.8% (89.6-100). A minority of children in the indeterminate group were classified as having bacterial infection (63 of 136, 46.3%), although most received antibiotic treatment (129 of 136, 94.9%). Conclusions and Relevance This study provides preliminary data regarding test accuracy of a 2-transcript host RNA signature discriminating bacterial from viral infection in febrile children. Further studies are needed in diverse groups of patients to assess accuracy and clinical utility of this test in different clinical settings. PMID:27552617

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

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

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 limited bioinformatics experience to analyze their data and integrate next generation sequencing (NGS) technologies into the classroom. The SPARTA software and tutorial are available at sparta.readthedocs.org.

Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response▿ †

PubMed Central

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J.

2006-01-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway. PMID:16980424

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response.

PubMed

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J

2006-11-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on a HAB causing Alexandrium tamarense.

PubMed

Li, Yi; Zhu, Hong; Zhang, Huajun; Chen, Zhangran; Tian, Yun; Xu, Hong; Zheng, Tianling; Zheng, Wei

2014-08-15

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on Alexandrium tamarense were measured through studying the algicidal procedure, nuclear damage and transcription of related genes. Medium components were optimized to improve algicidal activity, and characteristics of algicidal extracts were determined. Transmission electron microscope analysis revealed that the cell structure was broken. Cell membrane integrity destruction and nuclear structure degradation were monitored using confocal laser scanning microscope, and the rbcS, hsp and proliferating cell nuclear antigen (PCNA) gene expressions were studied. Results showed that 1.0% tryptone, 0.4% glucose and 0.8% MgCl2 were the optimal nutrient sources. The algicidal extracts were heat and pH stable, non-protein and less than 1kD. Cell membrane and nuclear structure integrity were lost, and the transcription of the rbcS and PCNA genes were significantly inhibited and there was up-regulation of hsp gene expression during the exposure procedure. The algicidal extracts destroyed the cell membrane and nuclear structure integrity, inhibited related gene expression and, eventually, lead to the inhibition of algal growth. All the results may elaborate firstly the cell death process and nuclear damage in A. tamarense which was induced by algicidal extracts, and the algicidal extracts could be potentially used as bacterial control of HABs in future. Copyright © 2014 Elsevier B.V. All rights reserved.

Different nitrogen sources change the transcriptome of welan gum-producing strain Sphingomonas sp. ATCC 31555.

PubMed

Xu, Xiaopeng; Nie, Zuoming; Zheng, Zhiyong; Zhu, Li; Zhang, Hongtao; Zhan, Xiaobei

2017-09-01

To reveal effects of different nitrogen sources on the expressions and functions of genes in Sphingomonas sp. ATCC 31555, it was cultivated in medium containing inorganic nitrogen (IN), organic nitrogen (ON), or inorganic-organic combined nitrogen (CN). Welan gum production and bacterial biomass were determined, and RNA sequencing (RNA-seq) was performed. Differentially expressed genes (DEGs) between the different ATCC 31555 groups were identified, and their functions were analyzed. Welan gum production and bacterial biomass were significantly higher in the ON and CN groups compared with those in the IN group. RNA-seq produced 660 unigenes, among which 488, 731, and 844 DEGs were identified between the IN vs. ON, IN vs. CN, and ON vs. CN groups, respectively. All the DEGs were related significantly to metabolic process and signal transduction. DEGs between the IN vs. CN and ON vs. CN groups were potentially associated with bacterial chemotaxis. Real-time PCR confirmed the expressions of selected DEGs. Organic nitrogen led to higher bacterial biomass and welan gum production than inorganic nitrogen, which might reflect differences in gene expression associated with metabolic process, signal transduction, and bacterial chemotaxis induced by different nitrogen sources.

Optimization of 15 parameters influencing the long-term survival of bacteria in aquatic systems

NASA Technical Reports Server (NTRS)

Obenhuber, D. C.

1993-01-01

NASA is presently engaged in the design and development of a water reclamation system for the future space station. A major concern in processing water is the control of microbial contamination. As a means of developing an optimal microbial control strategy, studies were undertaken to determine the type and amount of contamination which could be expected in these systems under a variety of changing environmental conditions. A laboratory-based Taguchi optimization experiment was conducted to determine the ideal settings for 15 parameters which influence the survival of six bacterial species in aquatic systems. The experiment demonstrated that the bacterial survival period could be decreased significantly by optimizing environmental conditions.

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.

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

Diversity of Hindgut Bacterial Population in Subterranean Termite, Reticulitermes flavipes

Treesearch

Olanrewaju Raji; Dragica Jeremic-Nikolic; Juliet D. Tang

2017-01-01

The termite hindgut contains a bacterial community that symbiotically aids in digestion of cellulosic materials. For this paper, a species survey of bacterial hindgut symbionts in termites collected from Saucier, Mississippi was examined. Two methods were tested for optimal genetic material isolation. Genomic DNA was isolated from the hindgut luminal contents of five...

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Secreted Gaussia princeps luciferase as a reporter of Escherichia coli replication in a mouse tissue cage model of infection.

PubMed

Liu, Mingyu; Blinn, Christina; McLeod, Sarah M; Wiseman, John W; Newman, Joseph V; Fisher, Stewart L; Walkup, Grant K

2014-01-01

Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.

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.

The three-dimensional structure of CFA/I adhesion pili: traveler's diarrhea bacteria hang on by a spring.

PubMed

Mu, Xiang-Qi; Savarino, Stephen J; Bullitt, Esther

2008-02-22

To survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. Herein, we elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained samples in combination with iterative three-dimensional helical reconstruction methods for image processing, we determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to maintain attachment to the gut lining during vortex mixing and downward flow of the intestinal contents, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease.

Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity.

PubMed

Binder, Dennis; Drepper, Thomas; Jaeger, Karl-Erich; Delvigne, Frank; Wiechert, Wolfgang; Kohlheyer, Dietrich; Grünberger, Alexander

2017-07-01

In natural habitats, microbes form multispecies communities that commonly face rapidly changing and highly competitive environments. Thus, phenotypic heterogeneity has evolved as an innate and important survival strategy to gain an overall fitness advantage over cohabiting competitors. However, in defined artificial environments such as monocultures in small- to large-scale bioreactors, cell-to-cell variations are presumed to cause reduced production yields as well as process instability. Hence, engineering microbial production toward phenotypic homogeneity is a highly promising approach for synthetic biology and bioprocess optimization. In this review, we discuss recent studies that have unraveled the cell-to-cell heterogeneity observed during bacterial gene expression and metabolite production as well as the molecular mechanisms involved. In addition, current single-cell technologies are briefly reviewed with respect to their applicability in exploring cell-to-cell variations. We highlight emerging strategies and tools to reduce phenotypic heterogeneity in biotechnological expression setups. Here, strain or inducer modifications are combined with cell physiology manipulations to achieve the ultimate goal of equalizing bacterial populations. In this way, the majority of cells can be forced into high productivity, thus reducing less productive subpopulations that tend to consume valuable resources during production. Modifications in uptake systems, inducer molecules or nutrients represent valuable tools for diminishing heterogeneity. Finally, we address the challenge of transferring homogeneously responding cells into large-scale bioprocesses. Environmental heterogeneity originating from extrinsic factors such as stirring speed and pH, oxygen, temperature or nutrient distribution can significantly influence cellular physiology. We conclude that engineering microbial populations toward phenotypic homogeneity is an increasingly important task to take biotechnological productions to the next level of control. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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.

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.

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

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.

Expression of green fluorescent protein in Xylella fastidiosa is affected by passage through host plants.

PubMed

Qin, Xiaoting; Hartung, John S

2004-09-01

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierce's disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.

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

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.

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.

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.

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

PubMed Central

Brander, Søren; Mikkelsen, Jørn D.; Kepp, Kasper P.

2014-01-01

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ∼0.5–2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (KM) but to pH dependence of catalytic turnover: The kcat of B. clausii cotA was 1 s−1 at pH 6 and 5 s−1 at pH 8 in contrast to 6 s−1 at pH 6 and 2 s−1 at pH 8 for of B. subtilis cotA. Overall, kcat/KM was 10-fold higher for B. subtilis cotA at pHopt. While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500–700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ∼20 minutes half-life at 80°C, less than the ∼50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH∼8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization. PMID:24915287

Auto-induction for high level production of biologically active reteplase in Escherichia coli.

PubMed

Fathi-Roudsari, Mehrnoosh; Maghsoudi, Nader; Maghsoudi, Amirhossein; Niazi, Sepideh; Soleiman, Morvarid

2018-06-07

Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield growth of bacterial cells and higher expression of recombinant proteins. Here we have tried to optimize the auto-induction procedure for soluble and active expression of reteplase in E. coli. Results showed that using auto-induction strategy at 37 °C, Rosetta-gami (DE3) had the highest level of active and soluble reteplase production in comparison to E. coli strains BL21 (DE3), and Shuffel T7. Temperature dominantly affected the level of active reteplase production. Decreasing the temperature to 25 and 18 °C increased the level of active reteplase by 20 and 60%, respectively. The composition of auto-induction medium also dramatically changed the active production of reteplase in cytoplasm. Using higher enriched auto-induction medium, super broth base including trace elements, significantly increased biologically active reteplase by 30%. It is demonstrated here that auto-induction is a powerful method for expression of biologically active reteplase in oxidative cytoplasm of Rosetta-gami. Optimizing expression condition by decreasing temperature and using an enriched auto-induction medium resulted in at least three times higher level of active reteplase production. Production of correctly folded and active reteplase in spite of its complex structure helps for removal of inefficient and cumbersome step of refolding. Copyright © 2018. Published by Elsevier Inc.

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

Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content

PubMed Central

Crawford, Matthew A.; Tapscott, Timothy; Fitzsimmons, Liam F.; Liu, Lin; Reyes, Aníbal M.; Libby, Stephen J.; Trujillo, Madia; Fang, Ferric C.; Radi, Rafael

2016-01-01

ABSTRACT The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. PMID:27094335

Overexpression and Purification of C-terminal Fragment of the Passenger Domain of Hap Protein from Nontypeable Haemophilus influenzae in a Highly Optimized Escherichia coli Expression System

PubMed Central

Tabatabaee, Akram; Siadat, Seyed Davar; Moosavi, Seyed Fazllolah; Aghasadeghi, Mohammad Reza; Memarnejadian, Arash; Pouriayevali, Mohammad Hassan; Yavari, Neda

2013-01-01

Background Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae (H. influenzae) Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called “HapS” and a 45 kDa C-terminal translocator domain called “Hapβ”. Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain (CBD) which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. Methods To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24a-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography (IMAC) using Ni-NTA columns. Results The highest expression level of target protein was achieved when CBD expressing E. coli BL21 (DE3) cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase (OD600 nm equal to 0.6) for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than %97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Conclusion Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process. PMID:23919121

Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling.

PubMed

Barbier, Mariette; Damron, F Heath

2016-01-01

Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.

Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

PubMed

Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

2012-05-01

The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

Communication between Bacteria and Their Hosts

PubMed Central

2013-01-01

It is clear that a dialogue is occurring between microbes and their hosts and that chemical signals are the language of this interkingdom communication. Microbial endocrinology shows that, through their long coexistence with animals and plants, microorganisms have evolved sensors for detecting eukaryotic hormones, which the microbe uses to determine that they are within proximity of a suitable host and to optimally time the expression of genes needed for host colonisation. It has also been shown that some prokaryotic chemical communication signals are recognized by eukaryotes. Deciphering what is being said during the cross-talk between microbe and host is therefore important, as it could lead to new strategies for preventing or treating bacterial infections. PMID:24381789

Recent research progress with phospholipase C from Bacillus cereus.

PubMed

Lyu, Yan; Ye, Lidan; Xu, Jun; Yang, Xiaohong; Chen, Weiwei; Yu, Hongwei

2016-01-01

Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.

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.

Quorum Quenching: Enzymatic Disruption of N-Acylhomoserine Lactone-Mediated Bacterial Communication in Burkholderia thailandensis

DTIC Science & Technology

2004-10-01

lactonases failed to enhance beta-hemolytic activity. The results of this study demonstrate that heterologous expression of Bacillus sp . AiiA lactonases in...results of this study demonstrate that heterologous expression of Bacillus sp . AiiA lactonases in B. thailandensis reduced AHL accumulation, affected both...hemo- lysis, and carbon utilization by the expression of Bacillus sp . AiiA lactonases in B. thailandensis. MATERIALS AND METHODS Bacterial strains and

Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-Glucanase (CenC) from Ruminiclostridium thermocellum in a Mesophilic Host Through Various Cultivation and Induction Strategies.

PubMed

Haq, Ikram Ul; Akram, Fatima

2017-09-01

Commonly, unintentional induction and inadvertently preparing medium for engineered Escherichia coli BL21 CodonPlus (DE3)-RIPL, give poor or variable yields of heterologous proteins. Therefore, to enhance the activity and production of an industrially relevant recombinant processive endo-1,4-β-glucanase (CenC) propagated in Escherichia coli BL21 CodonPlus(DE3)-RIPL through various cultivation and induction strategies. Investigation of various growth media and induction parameters revealed that high-cell-density and optimal CenC expression were obtained in ZYBM9 medium induced either with 0.5 mM IPTG/150 mM lactose, after 6 h induction at 37 °C; and before induction, bacterial cells were given heat shock (42 °C) for 1 h when culture density (OD 600nm ) reached at 0.6. Intracellular enzyme activity was enhanced by 6.67 and 3.20-fold in ZYBM9 and 3×ZYBM9 medium, respectively, under optimal conditions. Using YNG auto-induction medium, activity was 2.5-fold increased after 10 h incubation at 37 °C. Approximately similar results were obtained by transferring the optimized process at the bioreactor level. Results showed that the effective process strategy is essential to enhance recombinant bacterial cell mass and enzyme production from small to large-scale. To the best of our knowledge, this is the first ever report on enhanced production of thermostable processive endo-1,4-β-glucanase cloned from Ruminiclostridium thermocellum, which is a suitable candidate for industrial applications. Graphical Abstract Flow Chart Summary of Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-glucanase from Ruminiclostridium thermocellum.

A network-based approach for resistance transmission in bacterial populations.

PubMed

Gehring, Ronette; Schumm, Phillip; Youssef, Mina; Scoglio, Caterina

2010-01-07

Horizontal transfer of mobile genetic elements (conjugation) is an important mechanism whereby resistance is spread through bacterial populations. The aim of our work is to develop a mathematical model that quantitatively describes this process, and to use this model to optimize antimicrobial dosage regimens to minimize resistance development. The bacterial population is conceptualized as a compartmental mathematical model to describe changes in susceptible, resistant, and transconjugant bacteria over time. This model is combined with a compartmental pharmacokinetic model to explore the effect of different plasma drug concentration profiles. An agent-based simulation tool is used to account for resistance transfer occurring when two bacteria are adjacent or in close proximity. In addition, a non-linear programming optimal control problem is introduced to minimize bacterial populations as well as the drug dose. Simulation and optimization results suggest that the rapid death of susceptible individuals in the population is pivotal in minimizing the number of transconjugants in a population. This supports the use of potent antimicrobials that rapidly kill susceptible individuals and development of dosage regimens that maintain effective antimicrobial drug concentrations for as long as needed to kill off the susceptible population. Suggestions are made for experiments to test the hypotheses generated by these simulations.

Supercritical Fluid Extraction of Bacterial and Archaeal Lipid Biomarkers from Anaerobically Digested Sludge

PubMed Central

Hanif, Muhammad; Atsuta, Yoichi; Fujie, Koichi; Daimon, Hiroyuki

2012-01-01

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile. PMID:22489140

In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

PubMed

Kämpf, Michael M; Braun, Martin; Sirena, Dominique; Ihssen, Julian; Thöny-Meyer, Linda; Ren, Qun

2015-01-23

Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

MRP8/14 Enhances Corneal Susceptibility to Pseudomonas aeruginosa Infection by Amplifying Inflammatory Responses

PubMed Central

Deng, Qiuchan; Sun, Mingxia; Yang, Kun; Zhu, Min; Chen, Kang; Yuan, Jin; Wu, Minhao; Huang, Xi

2013-01-01

Purpose. We explored the role of myeloid-related protein 8 and 14 (MRP8/14) in Pseudomonas aeruginosa (PA) keratitis. Methods. MRP8/14 mRNA levels in human corneal scrapes and mouse corneas infected by PA were tested using real-time PCR. MRP8/14 protein expression in C57BL/6 (B6) corneas was confirmed using Western blot assay and immunohistochemistry. B6 mice were injected subconjunctivally with siRNA for MRP8/14, and then infected with PA. Bacterial plate counts and myeloperoxidase assays were used to determine the bacterial load and polymorphonuclear neutrophil (PMN) infiltration in infected B6 corneas. Pro-inflammatory cytokine levels in vivo and in vitro were examined with PCR and ELISA. In murine macrophage-like RAW264.7 cells, phagocytosis and bacterial killing were assessed using plate count assays, and reactive oxygen species (ROS) and nitric oxide (NO) levels were tested with flow cytometry and Griess assay, respectively. Results. MRP8/14 expression levels were increased significantly in human corneal scrapes and B6 corneas after PA infection. Silencing of MRP8/14 in B6 corneas significantly reduced the severity of corneal disease, bacterial clearance, PMN infiltration, and pro-inflammatory cytokine expression after PA infection. In vitro studies demonstrated further that silencing of MRP8/14 suppressed pro-inflammatory cytokine production, bacterial killing, and ROS production, but not phagocytosis or NO production. Conclusions. Our study demonstrated a dual role for MRP8/14 in bacterial keratitis. Although MRP8/14 promotes bacterial clearance by enhancing ROS production, it functions more importantly as an inflammatory amplifier at the ocular surface by enhancing pro-inflammatory cytokine expression, thus contributing to the corneal susceptibility. PMID:23299480

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila

PubMed Central

Xiong, Xiao-Peng; Chang, Kung-Yen; Ren, Xingjie; Ni, Jian-Quan; Rana, Tariq M.; Zhou, Rui

2016-01-01

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity. PMID:27893816

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

PubMed

Xiong, Xiao-Peng; Kurthkoti, Krishna; Chang, Kung-Yen; Li, Jian-Liang; Ren, Xingjie; Ni, Jian-Quan; Rana, Tariq M; Zhou, Rui

2016-11-01

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

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.

A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

PubMed Central

Rao, Lang; Xue, Yanfen; Zheng, Yingying; Lu, Jian R.; Ma, Yanhe

2013-01-01

Background Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. Methodology/Principal Findings Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. Conclusion EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues. PMID:23577139

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

An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis[OPEN

PubMed Central

Sugliani, Matteo; Ke, Hang; Bouveret, Emmanuelle; Robaglia, Christophe; Caffarri, Stefano

2016-01-01

The chloroplast originated from the endosymbiosis of an ancient photosynthetic bacterium by a eukaryotic cell. Remarkably, the chloroplast has retained elements of a bacterial stress response pathway that is mediated by the signaling nucleotides guanosine penta- and tetraphosphate (ppGpp). However, an understanding of the mechanism and outcomes of ppGpp signaling in the photosynthetic eukaryotes has remained elusive. Using the model plant Arabidopsis thaliana, we show that ppGpp is a potent regulator of chloroplast gene expression in vivo that directly reduces the quantity of chloroplast transcripts and chloroplast-encoded proteins. We then go on to demonstrate that the antagonistic functions of different plant RelA SpoT homologs together modulate ppGpp levels to regulate chloroplast function and show that they are required for optimal plant growth, chloroplast volume, and chloroplast breakdown during dark-induced and developmental senescence. Therefore, our results show that ppGpp signaling is not only linked to stress responses in plants but is also an important mediator of cooperation between the chloroplast and the nucleocytoplasmic compartment during plant growth and development. PMID:26908759

Bacterial community evolutions driven by organic matter and powder activated carbon in simultaneous anammox and denitrification (SAD) process.

PubMed

Ge, Cheng-Hao; Sun, Na; Kang, Qi; Ren, Long-Fei; Ahmad, Hafiz Adeel; Ni, Shou-Qing; Wang, Zhibin

2018-03-01

A distinct shift of bacterial community driven by organic matter (OM) and powder activated carbon (PAC) was discovered in the simultaneous anammox and denitrification (SAD) process which was operated in an anti-fouling submerged anaerobic membrane bio-reactor. Based on anammox performance, optimal OM dose (50 mg/L) was advised to start up SAD process successfully. The results of qPCR and high throughput sequencing analysis indicated that OM played a key role in microbial community evolutions, impelling denitrifiers to challenge anammox's dominance. The addition of PAC not only mitigated the membrane fouling, but also stimulated the enrichment of denitrifiers, accounting for the predominant phylum changing from Planctomycetes to Proteobacteria in SAD process. Functional genes forecasts based on KEGG database and COG database showed that the expressions of full denitrification functional genes were highly promoted in R C , which demonstrated the enhanced full denitrification pathway driven by OM and PAC under low COD/N value (0.11). Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry

PubMed Central

Greis, Kenneth D.; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-01-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP. PMID:16048957

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

PubMed

Moore, Simon J; Lai, Hung-En; Kelwick, Richard J R; Chee, Soo Mei; Bell, David J; Polizzi, Karen Marie; Freemont, Paul S

2016-10-21

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.

One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer.

PubMed

Lee, Young-Tae; Gibbons, Garrett; Lee, Shirley Y; Nikolovska-Coleska, Zaneta; Dou, Yali

2015-06-01

We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted using the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirms that the reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization.

PubMed

Hinsberger, Stefan; Hüsecken, Kristina; Groh, Matthias; Negri, Matthias; Haupenthal, Jörg; Hartmann, Rolf W

2013-11-14

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.

Computational tool for optimizing the essential oils utilization in inhibiting the bacterial growth

PubMed Central

El-Attar, Noha E; Awad, Wael A

2017-01-01

Day after day, the importance of relying on nature in many fields such as food, medical, pharmaceutical industries, and others is increasing. Essential oils (EOs) are considered as one of the most significant natural products for use as antimicrobials, antioxidants, antitumorals, and anti-inflammatories. Optimizing the usage of EOs is a big challenge faced by the scientific researchers because of the complexity of chemical composition of every EO, in addition to the difficulties to determine the best in inhibiting the bacterial activity. The goal of this article is to present a new computational tool based on two methodologies: reduction by using rough sets and optimization with particle swarm optimization. The developed tool dubbed as Essential Oil Reduction and Optimization Tool is applied on 24 types of EOs that have been tested toward 17 different species of bacteria. PMID:28919787

Cloning strategy for producing brush-forming protein-based polymers.

PubMed

Henderson, Douglas B; Davis, Richey M; Ducker, William A; Van Cott, Kevin E

2005-01-01

Brush-forming polymers are being used in a variety of applications, and by using recombinant DNA technology, there exists the potential to produce protein-based polymers that incorporate unique structures and functions in these brush layers. Despite this potential, production of protein-based brush-forming polymers is not routinely performed. For the design and production of new protein-based polymers with optimal brush-forming properties, it would be desirable to have a cloning strategy that allows an iterative approach wherein the protein based-polymer product can be produced and evaluated, and then if necessary, it can be sequentially modified in a controlled manner to obtain optimal surface density and brush extension. In this work, we report on the development of a cloning strategy intended for the production of protein-based brush-forming polymers. This strategy is based on the assembly of modules of DNA that encode for blocks of protein-based polymers into a commercially available expression vector; there is no need for custom-modified vectors and no need for intermediate cloning vectors. Additionally, because the design of new protein-based biopolymers can be an iterative process, our method enables sequential modification of a protein-based polymer product. With at least 21 bacterial expression vectors and 11 yeast expression vectors compatible with this strategy, there are a number of options available for production of protein-based polymers. It is our intent that this strategy will aid in advancing the production of protein-based brush-forming polymers.

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.

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.

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

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.

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.

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

Metagenomic and metatranscriptomic analysis of saliva reveals disease-associated microbiota in patients with periodontitis and dental caries.

PubMed

Belstrøm, Daniel; Constancias, Florentin; Liu, Yang; Yang, Liang; Drautz-Moses, Daniela I; Schuster, Stephan C; Kohli, Gurjeet Singh; Jakobsen, Tim Holm; Holmstrup, Palle; Givskov, Michael

2017-01-01

The taxonomic composition of the salivary microbiota has been reported to differentiate between oral health and disease. However, information on bacterial activity and gene expression of the salivary microbiota is limited. The purpose of this study was to perform metagenomic and metatranscriptomic characterization of the salivary microbiota and test the hypothesis that salivary microbial presence and activity could be an indicator of the oral health status. Stimulated saliva samples were collected from 30 individuals (periodontitis: n  = 10, dental caries: n  = 10, oral health: n  = 10). Salivary microbiota was characterized using metagenomics and metatranscriptomics in order to compare community composition and the gene expression between the three groups. Streptococcus was the predominant bacterial genus constituting approx. 25 and 50% of all DNA and RNA reads, respectively. A significant disease-associated higher relative abundance of traditional periodontal pathogens such as Porphyromonas gingivalis and Filifactor alocis and salivary microbial activity of F . alocis was associated with periodontitis. Significantly higher relative abundance of caries-associated bacteria such as Streptococcus mutans and Lactobacillus fermentum was identified in saliva from patients with dental caries. Multiple genes involved in carbohydrate metabolism were significantly more expressed in healthy controls compared to periodontitis patients. Using metagenomics and metatranscriptomics we show that relative abundance of specific oral bacterial species and bacterial gene expression in saliva associates with periodontitis and dental caries. Further longitudinal studies are warranted to evaluate if screening of salivary microbial activity of specific oral bacterial species and metabolic gene expression can identify periodontitis and dental caries at preclinical stages.

Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

PubMed

Peng, Bingyin; Huang, Shuangcheng; Liu, Tingting; Geng, Anli

2015-05-17

Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation. Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw(-1) h(-1), and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw(-1) h(-1), much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene. This study demonstrated that XIs clustered in the mammal gut Bacteroidetes group were able to be expressed functionally in S. cerevisiae and background strain anaerobic adaptive evolution in xylose medium is essential for the screening of functional XIs. The methods outlined in this paper are instructive for the identification of novel XIs that are functional in S. cerevisiae.

Bacterial meningitis - principles of antimicrobial treatment.

PubMed

Jawień, Miroslaw; Garlicki, Aleksander M

2013-01-01

Bacterial meningitis is associated with significant morbidity and mortality despite the availability of effective antimicrobial therapy. The management approach to patients with suspected or proven bacterial meningitis includes emergent cerebrospinal fluid analysis and initiation of appropriate antimicrobial and adjunctive therapies. The choice of empirical antimicrobial therapy is based on the patient's age and underlying disease status; once the infecting pathogen is isolated, antimicrobial therapy can be modified for optimal treatment. Successful treatment of bacterial meningitis requires the knowledge on epidemiology including prevalence of antimicrobial resistant pathogens, pathogenesis of meningitis, pharmacokinetics and pharmacodynamics of antimicrobial agents. The emergence of antibiotic-resistant bacterial strains in recent years has necessitated the development of new strategies for empiric antimicrobial therapy for bacterial meningitis.

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.

The filter-feeding ciliates Colpidium striatum and Tetrahymena pyriformis display selective feeding behaviours in the presence of mixed, equally-sized, bacterial prey.

PubMed

Thurman, Jill; Parry, Jacqueline D; Hill, Philip J; Laybourn-Parry, Johanna

2010-10-01

This study examined whether two ciliates could discriminate between equally-sized bacterial prey in mixture and if so, how selectivity might benefit the ciliate population. Live Klebsiella aerogenes, K. ozaenae and Escherichia coli, expressing different coloured fluorescent proteins, were cultured in such a way as to provide populations containing equally-sized cells (to prevent size-selective grazing taking place) and these prey were fed to each ciliate in 50:50 mixtures. Colpidium striatum selected K. aerogenes over K. ozaenae which itself was selected over E. coli. Tetrahymena pyriformis showed no selectivity between K. aerogenes and E. coli but K. aerogenes was selected over K. ozaenae while E. coli was not. This apparent selection of K. aerogenes over K. ozaenae was sustained in ciliate populations with different feeding histories and when K. aerogenes comprised only 20% of the prey mixture, suggesting possible optimal foraging behaviour. The metabolic benefits for selecting K. aerogenes were identified as possibly being an increase in cell biovolume and yield for C. striatum and T. pyriformis, respectively. The mechanism by which these ciliates selected specific bacterial cells in mixture is currently unknown but the use of live fluorescent bacteria, in prey mixtures, offers an exciting avenue for further investigation of selective feeding by protozoa. Copyright 2010 Elsevier Ltd. All rights reserved.

Factors affecting use of ballistics gelatin in laboratory studies of bacterial contamination in projectile wounds.

PubMed

Evans, Jessica J; Bost, Aaron; Muci-Küchler, Karim H; DeVeaux, Linda C

2018-05-25

Ballistics gelatin is a common tissue surrogate used in bacterial contamination models for projectile wounds. Although these studies have demonstrated that bacteria are transferred from the surface of the gelatin to the wound track by a projectile, quantifiable results have been inconsistent and not repeatable in successive tests. In this study, five areas of a typical contamination model in which bacterial recovery or survival are affected were identified for optimization. The first was a contaminated "skin" surrogate, where the novel use of vacuum filtration of a bacterial culture and buffer onto filter paper was employed. The other possibly problematic areas of the bacterial distribution model included the determination of bacterial survival when the contamination model is dried, survival in solid and molten gelatin, and the effect of high-intensity lights used for recording high-speed video. Vacuum filtration of bacteria and buffer resulted in a consistent bacterial distribution and recovery. The use of phosphate buffer M9 (pH 7) aided in neutralizing the ballistics gelatin and improving bacterial survival in solid gelatin. Additionally, the use of high-intensity lights to record high-speed video and the use of a 42 ° C water bath to melt the gelatin were found to be bactericidal for gram-positive and gram-negative bacteria. Multiple areas of a typical contamination model in which bacterial survival may be impeded were identified, and methods were proposed to improve survival in each area. These methods may be used to optimize the results of bacterial contamination models for medical applications, such as understanding the progression of infection in penetrating wounds and to identify possible sources of contamination for forensic purposes.

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.

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

PubMed

Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

2014-01-01

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

PubMed

Kim, Tae-Geum; Kim, Bang-Geul; Kim, Mi-Young; Choi, Jae-Kwon; Jung, Eun-Sun; Yang, Moon-Sik

2010-01-01

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

PubMed

Zhang, Yuyao; Ma, Xiuli; Huang, Bing; Li, Yufeng; Yu, Kexiang; Li, Jianliang; Liu, Cunxia; Han, Hongyu; Cui, Yanshun

2015-04-04

To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.

PubMed

Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki

2013-10-01

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Inference of Vohradský's Models of Genetic Networks by Solving Two-Dimensional Function Optimization Problems

PubMed Central

Kimura, Shuhei; Sato, Masanao; Okada-Hatakeyama, Mariko

2013-01-01

The inference of a genetic network is a problem in which mutual interactions among genes are inferred from time-series of gene expression levels. While a number of models have been proposed to describe genetic networks, this study focuses on a mathematical model proposed by Vohradský. Because of its advantageous features, several researchers have proposed the inference methods based on Vohradský's model. When trying to analyze large-scale networks consisting of dozens of genes, however, these methods must solve high-dimensional non-linear function optimization problems. In order to resolve the difficulty of estimating the parameters of the Vohradský's model, this study proposes a new method that defines the problem as several two-dimensional function optimization problems. Through numerical experiments on artificial genetic network inference problems, we showed that, although the computation time of the proposed method is not the shortest, the method has the ability to estimate parameters of Vohradský's models more effectively with sufficiently short computation times. This study then applied the proposed method to an actual inference problem of the bacterial SOS DNA repair system, and succeeded in finding several reasonable regulations. PMID:24386175

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.

Crowd evacuation model based on bacterial foraging algorithm

NASA Astrophysics Data System (ADS)

Shibiao, Mu; Zhijun, Chen

To understand crowd evacuation, a model based on a bacterial foraging algorithm (BFA) is proposed in this paper. Considering dynamic and static factors, the probability of pedestrian movement is established using cellular automata. In addition, given walking and queue times, a target optimization function is built. At the same time, a BFA is used to optimize the objective function. Finally, through real and simulation experiments, the relationship between the parameters of evacuation time, exit width, pedestrian density, and average evacuation speed is analyzed. The results show that the model can effectively describe a real evacuation.

An optimized regulating method for composting phosphorus fractions transformation based on biochar addition and phosphate-solubilizing bacteria inoculation.

PubMed

Wei, Yuquan; Zhao, Yue; Wang, Huan; Lu, Qian; Cao, Zhenyu; Cui, Hongyang; Zhu, Longji; Wei, Zimin

2016-12-01

The study was conducted to investigate the influence of biochar and/or phosphate-solubilizing bacteria (PSB) inoculants on microbial biomass, bacterial community composition and phosphorus (P) fractions during kitchen waste composting amended with rock phosphate (RP). There were distinct differences in the physic-chemical parameters, the proportion of P fractions and bacterial diversity in different treatments. The contribution of available P fractions increased during composting especially in the treatment with the addition of PSB and biochar. Redundancy analysis showed that bacterial compositions were significantly influenced by P content, inoculation and biochar. Variance partitioning further showed that synergy of inoculated PSB and indigenous bacterial communities and the joint effect between biochar and bacteria explained the largest two proportion of the variation in P fractions. Therefore, the combined application of PSB and biochar to improve the inoculation effect and an optimized regulating method were suggested based on the distribution of P fractions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis

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

Lin, Mingqun ..; Kikuchi, Takane; Brewer, Heather M.

2011-02-17

Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular {alpha}-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins ({approx}99%) with known functions were expressed, whereas only approximately 80% of hypothetical proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in bothmore » bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.« less

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.

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.

Optimization of a new mathematical model for bacterial growth

USDA-ARS?s Scientific Manuscript database

The objective of this research is to optimize a new mathematical equation as a primary model to describe the growth of bacteria under constant temperature conditions. An optimization algorithm was used in combination with a numerical (Runge-Kutta) method to solve the differential form of the new gr...

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.

Amplified RLR signaling activation through an interferon-stimulated gene-endoplasmic reticulum stress-mitochondrial calcium uniporter protein loop

PubMed Central

Cheng, Jinbo; Liao, Yajin; Zhou, Lujun; Peng, Shengyi; Chen, Hong; Yuan, Zengqiang

2016-01-01

Type I interferon (IFN-I) is critical for a host against viral and bacterial infections via induction of hundreds of interferon-stimulated genes (ISGs), but the mechanism underlying the regulation of IFN-I remains largely unknown. In this study, we first demonstrate that ISG expression is required for optimal IFN-β levels, an effect that is further enhanced by endoplasmic reticulum (ER) stress. Furthermore, we identify mitochondrial calcium uniporter protein (MCU) as a mitochondrial antiviral signaling protein (MAVS)-interacting protein that is important for ER stress induction and amplified MAVS signaling activation. In addition, by performing an ectopic expression assay to screen a library of 117 human ISGs for effects on IFN-β levels, we found that tumor necrosis factor receptor 1 (TNFR1) significantly increases IFN-β levels independent of ER stress. Altogether, our findings suggest that MCU and TNFR1 are involved in the regulation of RIG-I-like receptors (RLR) signaling. PMID:26892273

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage.

PubMed

Garmiri, Penelope; Coles, Karen E; Humphrey, Tom J; Cogan, Tristan A

2008-04-01

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.

Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus

PubMed Central

Hieggelke, Lena; Schneiker, Bianca; Lysson, Mariola; Stoffels, Burkhard; Nuding, Sabine; Wehkamp, Jan; Kikhney, Judith; Moter, Annette; Kalff, Joerg C.

2018-01-01

Aim To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery. PMID:29652914

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 candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27. The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607.

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

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures.

PubMed

Wang, Ying; Hougaard, Anni B; Paulander, Wilhelm; Skibsted, Leif H; Ingmer, Hanne; Andersen, Mogens L

2015-09-01

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

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.

In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.

PubMed Central

Heithoff, D M; Sinsheimer, R L; Low, D A; Mahan, M J

2000-01-01

Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g. thermal, osmotic, oxygen, nutrient and acid stress). Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection. A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting. Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues. Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites. Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials. PMID:10874736

RNA interference of tubulin genes has lethal effects in Mythimna separate.

PubMed

Wang, Jin-da; Wang, Ya-Ru; Wang, Yong-Zhi; Wang, Wei-Zhong; Wang, Rong; Gao, San-Ji

2018-05-23

RNAi (RNA interference) is a technology for silencing expression of target genes via sequence-specific double-stranded RNA (dsRNA). Recently, dietary introduction of bacterially expressed dsRNA has shown great potential in the field of pest management. Identification of potential candidate genes for RNAi is the first step in this application. The oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae) is a polyphagous, migratory pest, and outbreaks have led to severe crop damage in China. In the present study, two tubulin genes were chosen as target genes because of their crucial role in insect development. Both Msα-tubulin and Msβ-tubulin genes are expressed across all life stages and are highly expressed in the head and epidermis. Feeding of bacterially expressed dsRNA of Msα-tubulin and Msβ-tubulin to third-instar larvae knocked down target mRNAs. A lethal phenotype was observed with knockdown of Msα-tubulin and Msβ-tubulin concurrent with reduction in body weight. Bacterially expressed dsRNA can be used to control M. separata, and tubulin genes could be effective candidate genes for an RNAi-based control strategy of this pest. Copyright © 2017. Published by Elsevier B.V.

Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

PubMed Central

Chella Krishnan, Karthickeyan; Mukundan, Santhosh; Landero Figueroa, Julio A.; Caruso, Joseph A.

2014-01-01

Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues 47Cys and 195His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections. PMID:24799625

Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony.

PubMed

Yan, Muxia; Li, Weidong; Zhou, Zhenwen; Peng, Hongxia; Luo, Ziyan; Xu, Ling

2017-01-01

In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 45 min with 25 μl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 10 2 and 10 4 or 10 5  CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

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...

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

PubMed

Mohammadinezhad, Rezvan; Farahmand, Hamid; Jalali, Seyed Amir Hossein; Mirvaghefi, Alireza

2018-05-01

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

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.

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.

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.

Evolution of defence cocktails: Antimicrobial peptide combinations reduce mortality and persistent infection.

PubMed

Zanchi, Caroline; Johnston, Paul R; Rolff, Jens

2017-10-01

The simultaneous expression of costly immune effectors such as multiple antimicrobial peptides is a hallmark of innate immunity of multicellular organisms, yet the adaptive advantage remains unresolved. Here, we test current hypotheses on the evolution of such defence cocktails. We use RNAi gene knock-down to explore, the effects of three highly expressed antimicrobial peptides, displaying different degrees of activity in vitro against Staphylococcus aureus, during an infection in the beetle Tenebrio molitor. We find that a defensin confers no survival benefit but reduces bacterial loads. A coleoptericin contributes to host survival without affecting bacterial loads. An attacin has no individual effect. Simultaneous knock-down of the defensin with the other AMPs results in increased mortality and elevated bacterial loads. Contrary to common expectations, the effects on host survival and bacterial load can be independent. The expression of multiple AMPs increases host survival and contributes to the control of persisting infections and tolerance. This is an emerging property that explains the adaptive benefit of defence cocktails. © 2017 John Wiley & Sons Ltd.

Antisense Treatments for Biothreat Agents

DTIC Science & Technology

2006-08-01

2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

PubMed

Cordeiro, I B; Castro, D P; Nogueira, P P O; Angelo, P C S; Nogueira, P A; Gonçalves, J F C; Pereira, A M R F; Garcia, J S; Souza, G H M F; Arruda, M A Z; Eberlin, M N; Astolfi-Filho, S; Andrade, E V; López-Lozano, J L

2013-10-29

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.

PubMed

Kulmala, Antti; Huovinen, Tuomas; Lamminmäki, Urpo

2017-06-19

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

Fight or flight? - Flight increases immune gene expression but does not help to fight an infection.

PubMed

Woestmann, L; Kvist, J; Saastamoinen, M

2017-03-01

Flight represents a key trait in most insects, being energetically extremely demanding, yet often necessary for foraging and reproduction. Additionally, dispersal via flight is especially important for species living in fragmented landscapes. Even though, based on life-history theory, a negative relationship may be expected between flight and immunity, a number of previous studies have indicated flight to induce an increased immune response. In this study, we assessed whether induced immunity (i.e. immune gene expression) in response to 15-min forced flight treatment impacts individual survival of bacterial infection in the Glanville fritillary butterfly (Melitaea cinxia). We were able to confirm previous findings of flight-induced immune gene expression, but still observed substantially stronger effects on both gene expression levels and life span due to bacterial infection compared to flight treatment. Even though gene expression levels of some immunity-related genes were elevated due to flight, these individuals did not show increased survival of bacterial infection, indicating that flight-induced immune activation does not completely protect them from the negative effects of bacterial infection. Finally, an interaction between flight and immune treatment indicated a potential trade-off: flight treatment increased immune gene expression in naïve individuals only, whereas in infected individuals no increase in immune gene expression was induced by flight. Our results suggest that the up-regulation of immune genes upon flight is based on a general stress response rather than reflecting an adaptive response to cope with potential infections during flight or in new habitats. © 2016 The Authors. Journal of Evolutionary Biology Published by John Wiley & Sons ltd on behalf of European Society for Evolutionary Biology.

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.

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

A plant EPF-type zinc-finger protein, CaPIF1, involved in defence against pathogens.

PubMed

Oh, Sang-Keun; Park, Jeong Mee; Joung, Young Hee; Lee, Sanghyeob; Chung, Eunsook; Kim, Soo-Yong; Yu, Seung Hun; Choi, Doil

2005-05-01

SUMMARY To understand better the defence responses of plants to pathogen attack, we challenged hot pepper plants with bacterial pathogens and identified transcription factor-encoding genes whose expression patterns were altered during the subsequent hypersensitive response. One of these genes, CaPIF1 (Capsicum annuum Pathogen-Induced Factor 1), was characterized further. This gene encodes a plant-specific EPF-type protein that contains two Cys(2)/His(2) zinc fingers. CaPIF1 expression was rapidly and specifically induced when pepper plants were challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generated weak CaPIF1 expression. CaPIF1 expression was also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene-releasing compound, and salicylic acid, whereas methyl jasmonate had only moderate effects. CaPIF1 localized to the nuclei of onion epidermis when expressed as a CaPIF1-smGFP fusion protein. Transgenic tobacco plants over-expressing CaPIF1 driven by the CaMV 35S promoter showed increased resistance to challenge with a tobacco-specific pathogen or non-host bacterial pathogens. These plants also showed constitutive up-regulation of multiple defence-related genes. Moreover, virus-induced silencing of the CaPIF1 orthologue in Nicotiana benthamiana enhanced susceptibility to the same host or non-host bacterial pathogens. These observations provide evidence that an EPF-type Cys(2)/His(2) zinc-finger protein plays a crucial role in the activation of the pathogen defence response in plants.

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.

Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

PubMed Central

2009-01-01

Background The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense. PMID:20025733

Effect of ceramide-1-phosphate transfer protein on intestinal bacterial translocation in severe acute pancreatitis.

PubMed

Wang, Jiang; Li, Chang; Jiang, Yingjian; Zheng, Hongmei; Li, Dehui; Liang, Yibo; Deng, Wensheng; Zhang, Dianliang

2017-02-01

The aim of the study was to investigate the effects of ceramide-1-phosphate transfer protein (CPTP) on the intestinal epithelial tight junction proteins in patients with severe acute pancreatitis (SAP). Fifty patients with SAP were classified into two groups according to the presence of bacterial translocation (BT) in the blood. Thirty healthy individuals were included in the control group. The presence of BT was analyzed by polymerase chain reaction. The expression of tight junction proteins and CPTP was determined using immunohistochemistry and western blotting. Bacterial DNA was detected in the peripheral blood of 62.0% of the patients with SAP. The expression of CPTP and tight junction proteins in SAP patients was lower than that in healthy controls. Among the patients with SAP, those positive for BT(+) showed a lower level of CPTP and occluding (OC) and zonula occludens-1 (ZO-1) expression and a higher level of IVA cPLA2 expression than BT(-) patients. Moreover, the expression of CPTP was significantly associated with ZO-1 and showed a negative correlation with expression of IVA cPLA2 in SAP-BT(+) patients. CPTP affects the expression of tight junction proteins and may protects the intestinal epithelial barrier by downregulating the expression of IVA cPLA2. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

[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. 

In Vivo Visualization of Bacterial Colonization, Antigen Expression, and Specific T-Cell Induction following Oral Administration of Live Recombinant Salmonella enterica Serovar Typhimurium

PubMed Central

Bumann, Dirk

2001-01-01

Live attenuated Salmonella strains that express a foreign antigen are promising oral vaccine candidates. Numerous genetic modifications have been empirically tested, but their effects on immunogenicity are difficult to interpret since important in vivo properties of recombinant Salmonella strains such as antigen expression and localization are incompletely characterized and the crucial early inductive events of an immune response to the foreign antigen are not fully understood. Here, methods were developed to directly localize and quantitate the in situ expression of an ovalbumin model antigen in recombinant Salmonella enterica serovar Typhimurium using two-color flow cytometry and confocal microscopy. In parallel, the in vivo activation, blast formation, and division of ovalbumin-specific CD4+ T cells were followed using a well-characterized transgenic T-cell receptor mouse model. This combined approach revealed a biphasic induction of ovalbumin-specific T cells in the Peyer's patches that followed the local ovalbumin expression of orally administered recombinant Salmonella cells in a dose- and time-dependent manner. Interestingly, intact Salmonella cells and cognate T cells seemed to remain in separate tissue compartments throughout induction, suggesting a transport of killed Salmonella cells from the colonized subepithelial dome area to the interfollicular inductive sites. The findings of this study will help to rationally optimize recombinant Salmonella strains as efficacious live antigen carriers for oral vaccination. PMID:11402006

Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean.

PubMed

Piller, Kenneth J; Clemente, Thomas E; Jun, Sang Mu; Petty, Cynthia C; Sato, Shirley; Pascual, David W; Bost, Kenneth L

2005-09-01

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4(+) T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.

MtSWEET11, a Nodule-Specific Sucrose Transporter of Medicago truncatula

DOE PAGES

Kryvoruchko, Igor S.; Sinharoy, Senjuti; Torres-Jerez, Ivone; ...

2016-03-28

Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable agriculture. Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and depends on a steady supply of carbon to both plant and bacterial cells. Here we report the functional characterization of a nodule-specific Suc transporter, MtSWEET11 from Medicago truncatula. MtSWEET11 belongs to a clade of plant SWEET proteins that are capable of transporting Suc and play critical roles in pathogen susceptibility. When expressed in mammalian cells, MtSWEET11 transported sucrose (Suc) but not glucose (Glc). The MtSWEET11 gene was found to be expressed inmore » infected root hair cells, and in the meristem, invasion zone, and vasculature of nodules. Expression of an MtSWEET11-GFP fusion protein in nodules resulted in green fluorescence associated with the plasma membrane of uninfected cells and infection thread and symbiosome membranes of infected cells. Two independent Tnt1-insertion sweet11 mutants were uncompromised in SNF. Furthermore, although MtSWEET11 appears to be involved in Suc distribution within nodules, it is not crucial for SNF, probably because other Suc transporters can fulfill its role(s).« less

Defining the bacteroides ribosomal binding site.

PubMed

Wegmann, Udo; Horn, Nikki; Carding, Simon R

2013-03-01

The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

Diagnostic value of sTREM-1 in bronchoalveolar lavage fluid in ICU patients with bacterial lung infections: a bivariate meta-analysis.

PubMed

Shi, Jia-Xin; Li, Jia-Shu; Hu, Rong; Li, Chun-Hua; Wen, Yan; Zheng, Hong; Zhang, Feng; Li, Qin

2013-01-01

The serum soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a useful biomarker in differentiating bacterial infections from others. However, the diagnostic value of sTREM-1 in bronchoalveolar lavage fluid (BALF) in lung infections has not been well established. We performed a meta-analysis to assess the accuracy of sTREM-1 in BALF for diagnosis of bacterial lung infections in intensive care unit (ICU) patients. We searched PUBMED, EMBASE and Web of Knowledge (from January 1966 to October 2012) databases for relevant studies that reported diagnostic accuracy data of BALF sTREM-1 in the diagnosis of bacterial lung infections in ICU patients. Pooled sensitivity, specificity, and positive and negative likelihood ratios were calculated by a bivariate regression analysis. Measures of accuracy and Q point value (Q*) were calculated using summary receiver operating characteristic (SROC) curve. The potential between-studies heterogeneity was explored by subgroup analysis. Nine studies were included in the present meta-analysis. Overall, the prevalence was 50.6%; the sensitivity was 0.87 (95% confidence interval (CI), 0.72-0.95); the specificity was 0.79 (95% CI, 0.56-0.92); the positive likelihood ratio (PLR) was 4.18 (95% CI, 1.78-9.86); the negative likelihood ratio (NLR) was 0.16 (95% CI, 0.07-0.36), and the diagnostic odds ratio (DOR) was 25.60 (95% CI, 7.28-89.93). The area under the SROC curve was 0.91 (95% CI, 0.88-0.93), with a Q* of 0.83. Subgroup analysis showed that the assay method and cutoff value influenced the diagnostic accuracy of sTREM-1. BALF sTREM-1 is a useful biomarker of bacterial lung infections in ICU patients. Further studies are needed to confirm the optimized cutoff value.

Validating tyrosinase homologue MelA as a photoacoustic reporter gene for imaging Escherichia coli

NASA Astrophysics Data System (ADS)

Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert; Zemp, Roger

2015-03-01

Antibiotic drug resistance is a major worldwide issue. Development of new therapies against pathogenic bacteria requires appropriate research tools for replicating and characterizing infections. Previously fluorescence and bioluminescence modalities have been used to image infectious burden in animal models but scattering significantly limits imaging depth and resolution. We hypothesize that photoacoustic imaging, which has improved depth-toresolution ratio, could be useful for visualizing MelA-expressing bacteria since MelA is a bacterial tyrosinase homologue involved in melanin production. Using an inducible expression system, E. coli expressing MelA were visibly black in liquid culture. Phosphate buffered saline (PBS), MelA-expressing bacteria (at different dilutions in PBS), and chicken embryo blood were injected in plastic tubes which were imaged using a VisualSonics Vevo LAZR system. Photoacoustic imaging at 6 different wavelengths (680, 700, 750, 800, 850 and 900nm) enabled spectral de-mixing to distinguish melanin signals from blood. The signal to noise ratio of 9x diluted MelA bacteria was 55, suggesting that ~20 bacteria cells could be detected with our system. When MelA bacteria were injected as a 100 μL bolus into a chicken embryo, photoacoustic signals from deoxy- and oxy- hemoglobin as well as MelA-expressing bacteria could be separated and overlaid on an ultrasound image, allowing visualization of the bacterial location. Photoacoustic imaging may be a useful tool for visualizing bacterial infections and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.

Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

NASA Astrophysics Data System (ADS)

Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

2012-06-01

Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

Switches in Genomic GC Content Drive Shifts of Optimal Codons under Sustained Selection on Synonymous Sites

PubMed Central

Sun, Yu; Tamarit, Daniel

2017-01-01

Abstract The major codon preference model suggests that codons read by tRNAs in high concentrations are preferentially utilized in highly expressed genes. However, the identity of the optimal codons differs between species although the forces driving such changes are poorly understood. We suggest that these questions can be tackled by placing codon usage studies in a phylogenetic framework and that bacterial genomes with extreme nucleotide composition biases provide informative model systems. Switches in the background substitution biases from GC to AT have occurred in Gardnerella vaginalis (GC = 32%), and from AT to GC in Lactobacillus delbrueckii (GC = 62%) and Lactobacillus fermentum (GC = 63%). We show that despite the large effects on codon usage patterns by these switches, all three species evolve under selection on synonymous sites. In G. vaginalis, the dramatic codon frequency changes coincide with shifts of optimal codons. In contrast, the optimal codons have not shifted in the two Lactobacillus genomes despite an increased fraction of GC-ending codons. We suggest that all three species are in different phases of an on-going shift of optimal codons, and attribute the difference to a stronger background substitution bias and/or longer time since the switch in G. vaginalis. We show that comparative and correlative methods for optimal codon identification yield conflicting results for genomes in flux and discuss possible reasons for the mispredictions. We conclude that switches in the direction of the background substitution biases can drive major shifts in codon preference patterns even under sustained selection on synonymous codon sites. PMID:27540085

An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis.

PubMed

Sugliani, Matteo; Abdelkefi, Hela; Ke, Hang; Bouveret, Emmanuelle; Robaglia, Christophe; Caffarri, Stefano; Field, Ben

2016-03-01

The chloroplast originated from the endosymbiosis of an ancient photosynthetic bacterium by a eukaryotic cell. Remarkably, the chloroplast has retained elements of a bacterial stress response pathway that is mediated by the signaling nucleotides guanosine penta- and tetraphosphate (ppGpp). However, an understanding of the mechanism and outcomes of ppGpp signaling in the photosynthetic eukaryotes has remained elusive. Using the model plant Arabidopsis thaliana, we show that ppGpp is a potent regulator of chloroplast gene expression in vivo that directly reduces the quantity of chloroplast transcripts and chloroplast-encoded proteins. We then go on to demonstrate that the antagonistic functions of different plant RelA SpoT homologs together modulate ppGpp levels to regulate chloroplast function and show that they are required for optimal plant growth, chloroplast volume, and chloroplast breakdown during dark-induced and developmental senescence. Therefore, our results show that ppGpp signaling is not only linked to stress responses in plants but is also an important mediator of cooperation between the chloroplast and the nucleocytoplasmic compartment during plant growth and development. © 2016 American Society of Plant Biologists. All rights reserved.

Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens

NASA Astrophysics Data System (ADS)

Velikova, Nadya; Fulle, Simone; Manso, Ana Sousa; Mechkarska, Milena; Finn, Paul; Conlon, J. Michael; Oggioni, Marco Rinaldo; Wells, Jerry M.; Marina, Alberto

2016-05-01

Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials.

Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

PubMed

Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

2015-06-01

Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A Simple And Rapid Minicircle DNA Vector Manufacturing System

PubMed Central

Kay, Mark A; He, Cheng-Yi; Chen, Zhi-Ying

2010-01-01

Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells/tissues. Their use has been limited by labor-intensive production. We report on a strategy for making multiple genetic modifications in E.coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, the øC31-integrase and I-SceI homing-endonuclease. This bacterial strain is capable of producing highly purified minicircle yields in the same time frame as routine plasmid DNA. It is now feasible for minicircle DNA vectors to replace routine plasmids in mammalian transgene expression studies. PMID:21102455

Development of A Flexible System for the Simultaneous Conversion of Biomass to Industrial Chemicals and the Production of Industrial Biocatalysts

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

Gao, Johnway; Hooker, Brian S.; Skeen, R S.

2002-01-01

A flexible system was developed for the simultaneous conversion of biomass to industrial chemicals and the production of industrial biocatalysts. In particular, the expression of a bacterial enzyme, beta-glucuronidase (GUS), was investigated using a genetically modified starch-degrading Saccharomyces strain in suspension cultures in starch media. Different sources of starch including corn and waste potato starch were used for yeast biomass accumulation and GUS expression studies under controls of inducible and constitutive promoters. A thermostable bacterial cellulase, Acidothermus cellulolyticus E1 endoglucanase gene was also cloned into an episomal plasmid expression vector and expressed in the starch-degrading Saccharomyces strain.

Concurrent host-pathogen gene expression in the lungs of pigs challenged with Actinobacillus pleuropneumoniae.

PubMed

Brogaard, Louise; Klitgaard, Kirstine; Heegaard, Peter M H; Hansen, Mette Sif; Jensen, Tim Kåre; Skovgaard, Kerstin

2015-05-28

Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. Host and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen. Microbial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.

Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

PubMed Central

Krasnodembskaya, Anna; Song, Yuanlin; Fang, Xiaohui; Gupta, Naveen; Serikov, Vladimir; Lee, Jae-Woo; Matthay, Michael A.

2012-01-01

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/LL-37. PMID:20945332

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.

Analysis and optimization of process parameters for production of polyhydroxyalkanoates along with wastewater treatment by Serratia sp. ISTVKR1.

PubMed

Gupta, Asmita; Kumar, Madan; Thakur, Indu Shekhar

2017-10-01

A previously reported biodegrading bacterial strain Serratia sp. ISTVKR1 was studied for polyhydroxyalkanoate (PHA) production along with wastewater contaminant removal. Nile red fluorescence, GC-MS, FT-IR, NMR and TEM confirmed the accumulation of homopolymer poly-3-hydroxyvalerate (PHV) within the bacterial cells. Analysis of culture after 72h of bacterial treatment showed maximum COD removal (8.4-fold), non-detection of organic contaminants such as 1H-Cyclopropa [a] naphthalene (R.T.=10.12) using GC-MS and increased proportion of elements like Cr, Mn, Fe, Ni, Cu, Cd and Pb in the bacterial cell pellets by SEM-EDX analysis. Optimization of process parameters for enhanced PHA production along with wastewater treatment done using Response Surface Methodology (RSM) showed 5% and 0.74% increase in the PHA production (0.3368±0.13gL -1 ) and % COD reduction (88.93±2.41) of wastewater, respectively. The study, thus established the production of PHA along with wastewater contaminant removal by Serratia sp. ISTVKR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metal resistant plants and phytoremediation of environmental contamination

DOEpatents

Meagher, Richard B.; Li, Yujing; Dhankher, Om P.

2010-04-20

The present disclosure provides a method of producing transgenic plants which are resistant to at least one metal ion by transforming the plant with a recombinant DNA comprising a nucleic acid encoding a bacterial arsenic reductase under the control of a plant expressible promoter, and a nucleic acid encoding a nucleotide sequence encoding a phytochelatin biosynthetic enzyme under the control of a plant expressible promoter. The invention also relates a method of phytoremediation of a contaminated site by growing in the site a transgenic plant expressing a nucleic acid encoding a bacterial arsenate reductase and a nucleic acid encoding a phytochelatin biosynthetic enzyme.

Nitric oxide mediates antimicrobial peptide gene expression by activating eicosanoid signaling

PubMed Central

Sadekuzzaman, Md.

2018-01-01

Nitric oxide (NO) mediates both cellular and humoral immune responses in insects. Its mediation of cellular immune responses uses eicosanoids as a downstream signal. However, the cross-talk with two immune mediators was not known in humoral immune responses. This study focuses on cross-talk between two immune mediators in inducing gene expression of anti-microbial peptides (AMPs) of a lepidopteran insect, Spodoptera exigua. Up-regulation of eight AMPs was observed in S. exigua against bacterial challenge. However, the AMP induction was suppressed by injection of an NO synthase inhibitor, L-NAME, while little expressional change was observed on injecting its enantiomer, D-NAME. The functional association between NO biosynthesis and AMP gene expression was further supported by RNA interference (RNAi) against NO synthase (SeNOS), which suppressed AMP gene expression under the immune challenge. The AMP induction was also mimicked by NO alone because injecting an NO analog, SNAP, without bacterial challenge significantly induced the AMP gene expression. Interestingly, an eicosanoid biosynthesis inhibitor, dexamethasone (DEX), suppressed the NO induction of AMP expression. The inhibitory activity of DEX was reversed by the addition of arachidonic acid, a precursor of eicosanoid biosynthesis. AMP expression of S. exigua was also controlled by the Toll/IMD signal pathway. The RNAi of Toll receptors or Relish suppressed AMP gene expression by suppressing NO levels and subsequently reducing PLA2 enzyme activity. These results suggest that eicosanoids are a downstream signal of NO mediation of AMP expression against bacterial challenge. PMID:29466449

Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

PubMed Central

Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

2010-01-01

Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

Abiotic Stresses Shift Belowground Populus-Associated Bacteria Toward a Core Stress Microbiome

PubMed Central

Carter, Kelsey R.; Carrell, Alyssa A.; Jun, Se-Ran; Jawdy, Sara S.; Vélez, Jessica M.; Gunter, Lee E.; Yang, Zamin; Nookaew, Intawat; Engle, Nancy L.; Lu, Tse-Yuan S.; Schadt, Christopher W.; Tschaplinski, Timothy J.; Tuskan, Gerald A.; Pelletier, Dale A.; Weston, David J.

2018-01-01

ABSTRACT Adverse growth conditions can lead to decreased plant growth, productivity, and survival, resulting in poor yields or failure of crops and biofeedstocks. In some cases, the microbial community associated with plants has been shown to alleviate plant stress and increase plant growth under suboptimal growing conditions. A systematic understanding of how the microbial community changes under these conditions is required to understand the contribution of the microbiome to water utilization, nutrient uptake, and ultimately yield. Using a microbiome inoculation strategy, we studied how the belowground microbiome of Populus deltoides changes in response to diverse environmental conditions, including water limitation, light limitation (shading), and metal toxicity. While plant responses to treatments in terms of growth, photosynthesis, gene expression and metabolite profiles were varied, we identified a core set of bacterial genera that change in abundance in response to host stress. The results of this study indicate substantial structure in the plant microbiome community and identify potential drivers of the phytobiome response to stress. IMPORTANCE The identification of a common “stress microbiome” indicates tightly controlled relationships between the plant host and bacterial associates and a conserved structure in bacterial communities associated with poplar trees under different growth conditions. The ability of the microbiome to buffer the plant from extreme environmental conditions coupled with the conserved stress microbiome observed in this study suggests an opportunity for future efforts aimed at predictably modulating the microbiome to optimize plant growth. PMID:29404422

Abiotic Stresses Shift Belowground Populus -Associated Bacteria Toward a Core Stress Microbiome

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

Timm, Collin M.; Carter, Kelsey R.; Carrell, Alyssa A.

Adverse growth conditions can lead to decreased plant growth, productivity, and survival, resulting in poor yields or failure of crops and biofeedstocks. In some cases, the microbial community associated with plants has been shown to alleviate plant stress and increase plant growth under suboptimal growing conditions. A systematic understanding of how the microbial community changes under these conditions is required to understand the contribution of the microbiome to water utilization, nutrient uptake, and ultimately yield. Using a microbiome inoculation strategy, we studied how the belowground microbiome ofPopulus deltoideschanges in response to diverse environmental conditions, including water limitation, light limitation (shading),more » and metal toxicity. While plant responses to treatments in terms of growth, photosynthesis, gene expression and metabolite profiles were varied, we identified a core set of bacterial genera that change in abundance in response to host stress. The results of this study indicate substantial structure in the plant microbiome community and identify potential drivers of the phytobiome response to stress.The identification of a common “stress microbiome” indicates tightly controlled relationships between the plant host and bacterial associates and a conserved structure in bacterial communities associated with poplar trees under different growth conditions. The ability of the microbiome to buffer the plant from extreme environmental conditions coupled with the conserved stress microbiome observed in this study suggests an opportunity for future efforts aimed at predictably modulating the microbiome to optimize plant growth.« less

Abiotic Stresses Shift Belowground Populus -Associated Bacteria Toward a Core Stress Microbiome

DOE PAGES

Timm, Collin M.; Carter, Kelsey R.; Carrell, Alyssa A.; ...

2018-01-23

Adverse growth conditions can lead to decreased plant growth, productivity, and survival, resulting in poor yields or failure of crops and biofeedstocks. In some cases, the microbial community associated with plants has been shown to alleviate plant stress and increase plant growth under suboptimal growing conditions. A systematic understanding of how the microbial community changes under these conditions is required to understand the contribution of the microbiome to water utilization, nutrient uptake, and ultimately yield. Using a microbiome inoculation strategy, we studied how the belowground microbiome ofPopulus deltoideschanges in response to diverse environmental conditions, including water limitation, light limitation (shading),more » and metal toxicity. While plant responses to treatments in terms of growth, photosynthesis, gene expression and metabolite profiles were varied, we identified a core set of bacterial genera that change in abundance in response to host stress. The results of this study indicate substantial structure in the plant microbiome community and identify potential drivers of the phytobiome response to stress.The identification of a common “stress microbiome” indicates tightly controlled relationships between the plant host and bacterial associates and a conserved structure in bacterial communities associated with poplar trees under different growth conditions. The ability of the microbiome to buffer the plant from extreme environmental conditions coupled with the conserved stress microbiome observed in this study suggests an opportunity for future efforts aimed at predictably modulating the microbiome to optimize plant growth.« less

Bacterial diversity at different stages of the composting process

PubMed Central

2010-01-01

Background Composting is an aerobic microbiological process that is facilitated by bacteria and fungi. Composting is also a method to produce fertilizer or soil conditioner. Tightened EU legislation now requires treatment of the continuously growing quantities of organic municipal waste before final disposal. However, some full-scale composting plants experience difficulties with the efficiency of biowaste degradation and with the emission of noxious odours. In this study we examine the bacterial species richness and community structure of an optimally working pilot-scale compost plant, as well as a full-scale composting plant experiencing typical problems. Bacterial species composition was determined by isolating total DNA followed by amplifying and sequencing the gene encoding the 16S ribosomal RNA. Results Over 1500 almost full-length 16S rRNA gene sequences were analysed and of these, over 500 were present only as singletons. Most of the sequences observed in either one or both of the composting processes studied here were similar to the bacterial species reported earlier in composts, including bacteria from the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Deinococcus-Thermus. In addition, a number of previously undetected bacterial phylotypes were observed. Statistical calculations estimated a total bacterial diversity of over 2000 different phylotypes in the studied composts. Conclusions Interestingly, locally enriched or evolved bacterial variants of familiar compost species were observed in both composts. A detailed comparison of the bacterial diversity revealed a large difference in composts at the species and strain level from the different composting plants. However, at the genus level, the difference was much smaller and illustrated a delay of the composting process in the full-scale, sub-optimally performing plants. PMID:20350306

Biomimicry of quorum sensing using bacterial lifecycle model.

PubMed

Niu, Ben; Wang, Hong; Duan, Qiqi; Li, Li

2013-01-01

Recent microbiologic studies have shown that quorum sensing mechanisms, which serve as one of the fundamental requirements for bacterial survival, exist widely in bacterial intra- and inter-species cell-cell communication. Many simulation models, inspired by the social behavior of natural organisms, are presented to provide new approaches for solving realistic optimization problems. Most of these simulation models follow population-based modelling approaches, where all the individuals are updated according to the same rules. Therefore, it is difficult to maintain the diversity of the population. In this paper, we present a computational model termed LCM-QS, which simulates the bacterial quorum-sensing (QS) mechanism using an individual-based modelling approach under the framework of Agent-Environment-Rule (AER) scheme, i.e. bacterial lifecycle model (LCM). LCM-QS model can be classified into three main sub-models: chemotaxis with QS sub-model, reproduction and elimination sub-model and migration sub-model. The proposed model is used to not only imitate the bacterial evolution process at the single-cell level, but also concentrate on the study of bacterial macroscopic behaviour. Comparative experiments under four different scenarios have been conducted in an artificial 3-D environment with nutrients and noxious distribution. Detailed study on bacterial chemotatic processes with quorum sensing and without quorum sensing are compared. By using quorum sensing mechanisms, artificial bacteria working together can find the nutrient concentration (or global optimum) quickly in the artificial environment. Biomimicry of quorum sensing mechanisms using the lifecycle model allows the artificial bacteria endowed with the communication abilities, which are essential to obtain more valuable information to guide their search cooperatively towards the preferred nutrient concentrations. It can also provide an inspiration for designing new swarm intelligence optimization algorithms, which can be used for solving the real-world problems.

Biomimicry of quorum sensing using bacterial lifecycle model

PubMed Central

2013-01-01

Background Recent microbiologic studies have shown that quorum sensing mechanisms, which serve as one of the fundamental requirements for bacterial survival, exist widely in bacterial intra- and inter-species cell-cell communication. Many simulation models, inspired by the social behavior of natural organisms, are presented to provide new approaches for solving realistic optimization problems. Most of these simulation models follow population-based modelling approaches, where all the individuals are updated according to the same rules. Therefore, it is difficult to maintain the diversity of the population. Results In this paper, we present a computational model termed LCM-QS, which simulates the bacterial quorum-sensing (QS) mechanism using an individual-based modelling approach under the framework of Agent-Environment-Rule (AER) scheme, i.e. bacterial lifecycle model (LCM). LCM-QS model can be classified into three main sub-models: chemotaxis with QS sub-model, reproduction and elimination sub-model and migration sub-model. The proposed model is used to not only imitate the bacterial evolution process at the single-cell level, but also concentrate on the study of bacterial macroscopic behaviour. Comparative experiments under four different scenarios have been conducted in an artificial 3-D environment with nutrients and noxious distribution. Detailed study on bacterial chemotatic processes with quorum sensing and without quorum sensing are compared. By using quorum sensing mechanisms, artificial bacteria working together can find the nutrient concentration (or global optimum) quickly in the artificial environment. Conclusions Biomimicry of quorum sensing mechanisms using the lifecycle model allows the artificial bacteria endowed with the communication abilities, which are essential to obtain more valuable information to guide their search cooperatively towards the preferred nutrient concentrations. It can also provide an inspiration for designing new swarm intelligence optimization algorithms, which can be used for solving the real-world problems. PMID:23815296

Automated multiplex genome-scale engineering in yeast

PubMed Central

Si, Tong; Chao, Ran; Min, Yuhao; Wu, Yuying; Ren, Wen; Zhao, Huimin

2017-01-01

Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast. PMID:28469255

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

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.

Iron regulates expression of Bacillus cereus hemolysin II via global regulator Fur.

PubMed

Sineva, Elena; Shadrin, Andrey; Rodikova, Ekaterina A; Andreeva-Kovalevskaya, Zhanna I; Protsenko, Alexey S; Mayorov, Sergey G; Galaktionova, Darya Yu; Magelky, Erica; Solonin, Alexander S

2012-07-01

The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.

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.

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

PubMed

Ravanfar, Seyed Ali; Aziz, Maheran Abdul; Saud, Halimi Mohd; Abdullah, Janna Ong

2015-11-01

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

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 for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27. The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607. PMID:23107287

Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota.

PubMed

Horng, Katti R; Ganz, Holly H; Eisen, Jonathan A; Marks, Stanley L

2018-01-01

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and -80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer ( F -value = 6.87, DF  = 3, P  < 0.001), storage temperature ( F -value=1.77, DF  = 3, P  = 0.037), and duration of sample storage ( F -value = 3.68, DF  = 3, P  < 0.001). Changes in bacterial composition were observed in samples stored in -80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations ( DF  = 8.57, P  < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

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 effects of mancozeb on diversity. PMID:25938467

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.

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.

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.

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.

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.

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

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

Phylogeographic origin of Helicobacter pylori determines host-adaptive responses upon coculture with gastric epithelial cells.

PubMed

Sheh, Alexander; Chaturvedi, Rupesh; Merrell, D Scott; Correa, Pelayo; Wilson, Keith T; Fox, James G

2013-07-01

While Helicobacter pylori infects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors in H. pylori pathogenesis, global gene expression of six H. pylori isolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factors cagA, vacA, and babB and were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin of H. pylori strains may promote increased gastric disease.

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-/r-CFTR expression and phagocytosis using methyl-β-cyclodextran (CD), as it is known to deplete CFTR from membrane lipid-rafts. We observed that CD treatment significantly (p<0.01) inhibits bacterial phagocytosis in RAW264.7 cells and adding CSE further impairs phagocytosis suggesting synergistic effect on CFTR dependent lipid-rafts. Conclusion Our data suggest that SHS impairs bacterial phagocytosis by modulating CFTR dependent lipid-rafts. PMID:25794013

Optimization of process variables for decolorization of Disperse Yellow 211 by Bacillus subtilis using Box-Behnken design.

PubMed

Sharma, Praveen; Singh, Lakhvinder; Dilbaghi, Neeraj

2009-05-30

Decolorization of textile azo dye Disperse Yellow 211 (DY 211) was carried out from simulated aqueous solution by bacterial strain Bacillus subtilis. Response surface methodology (RSM), involving Box-Behnken design matrix in three most important operating variables; temperature, pH and initial dye concentration was successfully employed for the study and optimization of decolorization process. The total 17 experiments were conducted in the study towards the construction of a quadratic model. According to analysis of variance (ANOVA) results, the proposed model can be used to navigate the design space. Under optimized conditions the bacterial strain was able to decolorize DY 211 up to 80%. Model indicated that initial dye concentration of 100 mgl(-1), pH 7 and a temperature of 32.5 degrees C were found optimum for maximum % decolorization. Very high regression coefficient between the variables and the response (R(2)=0.9930) indicated excellent evaluation of experimental data by polynomial regression model. The combination of the three variables predicted through RSM was confirmed through confirmatory experiments, hence the bacterial strain holds a great potential for the treatment of colored textile effluents.

Microarray and Real-Time PCR Analyses of the Responses of High-Arctic Soil Bacteria to Hydrocarbon Pollution and Bioremediation Treatments▿

PubMed Central

Yergeau, Etienne; Arbour, Mélanie; Brousseau, Roland; Juck, David; Lawrence, John R.; Masson, Luke; Whyte, Lyle G.; Greer, Charles W.

2009-01-01

High-Arctic soils have low nutrient availability, low moisture content, and very low temperatures and, as such, they pose a particular problem in terms of hydrocarbon bioremediation. An in-depth knowledge of the microbiology involved in this process is likely to be crucial to understand and optimize the factors most influencing bioremediation. Here, we compared two distinct large-scale field bioremediation experiments, located at the Canadian high-Arctic stations of Alert (ex situ approach) and Eureka (in situ approach). Bacterial community structure and function were assessed using microarrays targeting the 16S rRNA genes of bacteria found in cold environments and hydrocarbon degradation genes as well as quantitative reverse transcriptase PCR targeting key functional genes. The results indicated a large difference between sampling sites in terms of both soil microbiology and decontamination rates. A rapid reorganization of the bacterial community structure and functional potential as well as rapid increases in the expression of alkane monooxygenases and polyaromatic hydrocarbon-ring-hydroxylating dioxygenases were observed 1 month after the bioremediation treatment commenced in the Alert soils. In contrast, no clear changes in community structure were observed in Eureka soils, while key gene expression increased after a relatively long lag period (1 year). Such discrepancies are likely caused by differences in bioremediation treatments (i.e., ex situ versus in situ), weathering of the hydrocarbons, indigenous microbial communities, and environmental factors such as soil humidity and temperature. In addition, this study demonstrates the value of molecular tools for the monitoring of polar bacteria and their associated functions during bioremediation. PMID:19684169

Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves.

PubMed

O'brien, R D; Lindow, S E

1988-09-01

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15 Pseudomonas syringae strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at -5 or -9 degrees C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.

A sensor network based virtual beam-like structure method for fault diagnosis and monitoring of complex structures with Improved Bacterial Optimization

NASA Astrophysics Data System (ADS)

Wang, H.; Jing, X. J.

2017-02-01

This paper proposes a novel method for the fault diagnosis of complex structures based on an optimized virtual beam-like structure approach. A complex structure can be regarded as a combination of numerous virtual beam-like structures considering the vibration transmission path from vibration sources to each sensor. The structural 'virtual beam' consists of a sensor chain automatically obtained by an Improved Bacterial Optimization Algorithm (IBOA). The biologically inspired optimization method (i.e. IBOA) is proposed for solving the discrete optimization problem associated with the selection of the optimal virtual beam for fault diagnosis. This novel virtual beam-like-structure approach needs less or little prior knowledge. Neither does it require stationary response data, nor is it confined to a specific structure design. It is easy to implement within a sensor network attached to the monitored structure. The proposed fault diagnosis method has been tested on the detection of loosening screws located at varying positions in a real satellite-like model. Compared with empirical methods, the proposed virtual beam-like structure method has proved to be very effective and more reliable for fault localization.

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

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

Comparative Bacterial Proteomics: Analysis of the Core Genome Concept

PubMed Central

Callister, Stephen J.; McCue, Lee Ann; Turse, Joshua E.; Monroe, Matthew E.; Auberry, Kenneth J.; Smith, Richard D.; Adkins, Joshua N.; Lipton, Mary S.

2008-01-01

While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits. PMID:18253490

Expression of the cloned ColE1 kil gene in normal and Kilr Escherichia coli.

PubMed Central

Altieri, M; Suit, J L; Fan, M L; Luria, S E

1986-01-01

The kil gene of the ColE1 plasmid was cloned under control of the lac promoter. Its expression under this promoter gave rise to the same pattern of bacterial cell damage and lethality as that which accompanies induction of the kil gene in the colicin operon by mitomycin C. This confirms that cell damage after induction is solely due to expression of kil and is independent of the cea or imm gene products. Escherichia coli derivatives resistant to the lethal effects of kil gene expression under either the normal or the lac promoter were isolated and found to fall into several classes, some of which were altered in sensitivity to agents that affect the bacterial envelope. PMID:2946661

Requirement for STAT1 in LPS-induced gene expression in macrophages.

PubMed

Ohmori, Y; Hamilton, T A

2001-04-01

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

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, such as crustose coralline algae, biofilms or with GLW-amide neuropeptides that stimulate the entire onset of larval metamorphosis and attachment. PMID:22655067

SIGIRR, a negative regulator of TLR/IL-1R signalling promotes Microbiota dependent resistance to colonization by enteric bacterial pathogens.

PubMed

Sham, Ho Pan; Yu, Emily Yi Shan; Gulen, Muhammet F; Bhinder, Ganive; Stahl, Martin; Chan, Justin M; Brewster, Lara; Morampudi, Vijay; Gibson, Deanna L; Hughes, Michael R; McNagny, Kelly M; Li, Xiaoxia; Vallance, Bruce A

2013-01-01

Enteric bacterial pathogens such as enterohemorrhagic E. coli (EHEC) and Salmonella Typhimurium target the intestinal epithelial cells (IEC) lining the mammalian gastrointestinal tract. Despite expressing innate Toll-like receptors (TLRs), IEC are innately hypo-responsive to most bacterial products. This is thought to prevent maladaptive inflammatory responses against commensal bacteria, but it also limits antimicrobial responses by IEC to invading bacterial pathogens, potentially increasing host susceptibility to infection. One reason for the innate hypo-responsiveness of IEC is their expression of Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and TLR signaling. To address whether SIGIRR expression and the innate hypo-responsiveness of IEC impacts on enteric host defense, Sigirr deficient (-/-) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr -/- mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses but surprisingly, Sigirr -/- mice proved dramatically more susceptible to infection than wildtype mice. Through haematopoietic transplantation studies, it was determined that SIGIRR expression by non-haematopoietic cells (putative IEC) regulated these responses. Moreover, the exaggerated responses were found to be primarily dependent on IL-1R signaling. Whilst exploring the basis for their susceptibility, Sigirr -/- mice were found to be unusually susceptible to intestinal Salmonella Typhimurium colonization, developing enterocolitis without the typical requirement for antibiotic based removal of competing commensal microbes. Strikingly, the exaggerated antimicrobial responses seen in Sigirr -/- mice were found to cause a rapid and dramatic loss of commensal microbes from the infected intestine. This depletion appears to reduce the ability of the microbiota to compete for space and nutrients (colonization resistance) with the invading pathogens, leaving the intestine highly susceptible to pathogen colonization. Thus, SIGIRR expression by IEC reflects a strategy that sacrifices maximal innate responsiveness by IEC in order to promote commensal microbe based colonization resistance against bacterial pathogens.

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

PubMed

Yim, W J; Kim, K Y; Lee, Y W; Sundaram, S P; Lee, Y; Sa, T M

2014-07-15

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato. Copyright © 2014 Elsevier GmbH. All rights reserved.

GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

PubMed

Goyal, Megha; Chaudhuri, Tapan K

2015-07-01

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

Widespread genetic switches and toxicity resistance proteins for fluoride.

PubMed

Baker, Jenny L; Sudarsan, Narasimhan; Weinberg, Zasha; Roth, Adam; Stockbridge, Randy B; Breaker, Ronald R

2012-01-13

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride

PubMed Central

Weinberg, Zasha; Roth, Adam; Stockbridge, Randy B.; Breaker, Ronald R.

2014-01-01

Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion. PMID:22194412

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

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.

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 crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.

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 biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology. PMID:29515604

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.

Post-capture immune gene expression studies in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus acclimatized to atmospheric pressure.

PubMed

Barros, Inês; Divya, Baby; Martins, Inês; Vandeperre, Frederic; Santos, Ricardo Serrão; Bettencourt, Raul

2015-01-01

Deep-sea hydrothermal vents are extreme habitats that are distributed worldwide in association with volcanic and tectonic events, resulting thus in the establishment of particular environmental conditions, in which high pressure, steep temperature gradients, and potentially toxic concentrations of sulfur, methane and heavy metals constitute driving factors for the foundation of chemosynthetic-based ecosystems. Of all the different macroorganisms found at deep-sea hydrothermal vents, the mussel Bathymodiolus azoricus is the most abundant species inhabiting the vent ecosystems from the Mid-Atlantic Ridge (MAR). In the present study, the effect of long term acclimatization at atmospheric pressure on host-symbiotic associations were studied in light of the ensuing physiological adaptations from which the immune and endosymbiont gene expressions were concomitantly quantified by means of real-time PCR. The expression of immune genes at 0 h, 12 h, 24 h, 36 h, 48 h, 72 h, 1 week and 3 weeks post-capture acclimatization was investigated and their profiles compared across the samples tested. The gene signal distribution for host immune and bacterial genes followed phasic changes in gene expression at 24 h, 1 week and 3 weeks acclimatization when compared to other time points tested during this temporal expression study. Analyses of the bacterial gene expression also suggested that both bacterial density and activity could contribute to shaping the intricate association between endosymbionts and host immune genes whose expression patterns seem to be concomitant at 1 week acclimatization. Fluorescence in situ hybridization was used to assess the distribution and prevalence of endosymbiont bacteria within gill tissues confirming the gradual loss of sulfur-oxidizing (SOX) and methane-oxidizing (MOX) bacteria during acclimatization. The present study addresses the deep-sea vent mussel B. azoricus as a model organism to study how acclimatization in aquaria and the prevalence of symbiotic bacteria are driving the expression of host immune genes. Tight associations, unseen thus far, suggest that host immune and bacterial gene expression patterns reflect distinct physiological responses over the course of acclimatization under aquarium conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kynetic resazurin assay (KRA) for bacterial quantification of foodborne pathogens

NASA Astrophysics Data System (ADS)

Arenas, Yaxal; Mandel, Arkady; Lilge, Lothar

2012-03-01

Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.

Bacterial consortium for copper extraction from sulphide ore consisting mainly of chalcopyrite

PubMed Central

Romo, E.; Weinacker, D.F.; Zepeda, A.B.; Figueroa, C.A.; Chavez-Crooker, P.; Farias, J.G.

2013-01-01

The mining industry is looking forward for bacterial consortia for economic extraction of copper from low-grade ores. The main objective was to determine an optimal bacterial consortium from several bacterial strains to obtain copper from the leach of chalcopyrite. The major native bacterial species involved in the bioleaching of sulphide ore (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans and Leptospirillum ferriphilum) were isolated and the assays were performed with individual bacteria and in combination with At. thiooxidans. In conclusion, it was found that the consortium integrated by At. ferrooxidans and At. thiooxidans removed 70% of copper in 35 days from the selected ore, showing significant differences with the other consortia, which removed only 35% of copper in 35 days. To validate the assays was done an escalation in columns, where the bacterial consortium achieved a higher percentage of copper extraction regarding to control. PMID:24294251

DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B cells and Idiotype Positive Marginal Zone B cells Before and After Immunization with Streptococcus pneumoniae 1

PubMed Central

Liu, Jiabin; Behrens, Timothy W.; Kearney, John F.

2014-01-01

Marginal Zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and Follicular (FO) B cells were sort-purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. 99 genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, idiotype positive and negative MZ B cells were sort-purified before (0 hour) or after (1 hour) i.v. immunization with heat killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up regulated or down regulated at 1 hour following immunization in the idiotype positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs. FO B cells and the specific regulation of gene expression in antigen-specific MZ B cells following interaction with antigen. PMID:18453586

Lipopolysaccharide and cAMP modify placental calcitriol biosynthesis reducing antimicrobial peptides gene expression.

PubMed

Olmos-Ortiz, Andrea; García-Quiroz, Janice; Avila, Euclides; Caldiño-Soto, Felipe; Halhali, Ali; Larrea, Fernando; Díaz, Lorenza

2018-06-01

Calcitriol, the hormonal form of vitamin D 3 (VD), stimulates placental antimicrobial peptides expression; nonetheless, the regulation of calcitriol biosynthesis in the presence of bacterial products and its consequence on placental innate immunity have scarcely been addressed. We investigated how some bacterial products modify placental VD metabolism and its ability to induce antimicrobial peptides gene expression. Cultured human trophoblasts biosynthesized calcitriol only in the presence of its precursor calcidiol, a process that was inhibited by cyclic-AMP but stimulated by lipopolysaccharide (LPS). Intracrine calcitriol upregulated cathelicidin, S100A9, and β-defensins (HBDs) gene expression, while LPS further stimulated HBD2 and S100A9. Unexpectedly, LPS significantly repressed cathelicidin basal mRNA levels and drastically diminished calcidiol ability to induce it. Meanwhile, cyclic-AMP, which is used by many microbes to avoid host defenses, suppressed calcitriol biosynthesis, resulting in significant inhibition of most VD-dependent microbicidal peptides gene expression. While LPS stimulated calcitriol biosynthesis, cyclic-AMP inhibited it. LPS downregulated cathelicidin mRNA expression, whereas cyclic-AMP antagonized VD-dependent-upregulation of most antimicrobial peptides. These findings reveal LPS and cyclic-AMP involvement in dampening placental innate immunity, highlighting the importance of cyclic-AMP in the context of placental infection and suggesting its participation to facilitate bacterial survival. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

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.

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

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.

Artemin protects cells and proteins against oxidative and salt stress.

PubMed

Takalloo, Zeinab; Sajedi, Reza H; Hosseinkhani, Saman; Moazzenzade, Taghi

2017-02-01

Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H 2 O 2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

A comparative study of RNA and DNA as internal gene expression controls early in the developmental cycle of Chlamydia pneumoniae.

PubMed

Engström, Patrik; Bailey, Leslie; Onskog, Thomas; Bergström, Sven; Johansson, Jörgen

2010-03-01

Many microbial pathogens invade and proliferate within host cells and the molecular mechanism underlying this behavior is currently being revealed for several bacterial species. Testing clinically relevant antibacterial compounds and elucidating their effects on gene expression requires adequate controls, especially when studying genetically intractable organisms such as Chlamydia spp., for which various gene fusions cannot be constructed. Until now, relative mRNA levels in Chlamydia have been measured using different internal gene expression controls, including 16S rRNA, mRNAs, and DNA. Here, we compared the advantages and disadvantages of various internal expression controls during the early phase of Chlamydia pneumoniae development. The relative abundance of target mRNAs varied using the different internal control RNAs. This was partly due to variation in the transcript stability of the RNA species. Also, seven out of nine of the analyzed RNAs increased fivefold or more between 2 and 14 h postinfection, while the amount of DNA and number of cells remained essentially unaltered. Our results suggest that RNA should not be used as a gene expression control during the early phase of Chlamydia development, and that intrinsic bacterial DNA is preferable for that purpose because it is stable, abundant, and its relative amount is generally correlated with bacterial numbers.

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.

Efficacy of contact lens disinfecting solutions against Staphylococcus aureus and Pseudomonas aeruginosa.

PubMed

Manuj, Kapur; Gunderson, Charlise; Troupe, John; Huber, Mary E

2006-07-01

To evaluate the disinfection properties of multipurpose contact lens disinfection solutions, based on the International Organization for Standardization (ISO) 14729 guidelines. ReNu with MoistureLoc Multi-Purpose Solution, OPTI-FREE Express with Aldox Multi-Purpose Solution, Betadine 5% sterile ophthalmic preparation solution (povidone iodine), and 0.9% normal saline solution were inoculated with strains of Staphylococcus aureus and Pseudomonas aeruginosa. Surviving bacteria were quantified at specified times. ReNu with MoistureLoc, OPTI-FREE Express, and 5% ophthalmic povidone iodine were effective in achieving a 5-log reduction in bacterial count. Additionally, all three products maintained their effectivity at 72 hours. However, ReNu with MoistureLoc and povidone iodine resulted in the greatest reduction in bacterial colonization. ReNu with MoistureLoc, OPTI-FREE Express, and 5% ophthalmic povidone iodine meet the ISO 14729 guidelines for standalone contact lens solutions. However, ReNu with MoistureLoc and 5% ophthalmic povidone iodine are most efficient in reducing and maintaining low bacterial count for a period of 72 hours.

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.

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.

Screening host proteins required for bacterial adherence after H9N2 virus infection.

PubMed

Ma, Li-Li; Sun, Zhen-Hong; Xu, Yu-Lin; Wang, Shu-Juan; Wang, Hui-Ning; Zhang, Hao; Hu, Li-Ping; Sun, Xiao-Mei; Zhu, Lin; Shang, Hong-Qi; Zhu, Rui-Liang; Wei, Kai

2018-01-01

H9N2 subtype low pathogenic avian influenza virus (LPAIV) is distributed worldwide and causes great economic losses in the poultry industry, especially when complicated with other bacterial infections. Tissue damages caused by virus infection provide an opportunity for bacteria invasion, but this mechanism is not sufficient for low pathogenic strains. Moreover, although H9N2 virus infection was demonstrated to promote bacterial infection in several studies, its mechanism remained unclear. In this study, infection experiments in vivo and in vitro demonstrated that the adhesion of Escherichia coli (E. coli) to host cells significantly increased after H9N2 virus infection, and this increase was not caused by pathological damages. Subsequently, we constructed a late chicken embryo infection model and used proteomics techniques to analyze the expression of proteins associated with bacterial adhesion after H9N2 virus infection. A total of 279 significantly differential expressed proteins were detected through isobaric tags for relative and absolute quantitation (iTRAQ) coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed proteins were enriched in host innate immunity; cell proliferation, differentiation, and apoptosis; and pathogenicity-related signaling pathways. Finally, we screened out several proteins, such as TGF-β1, integrins, cortactin, E-cadherin, vinculin, and fibromodulin, which were probably associated with bacterial adhesion. The study analyzed the mechanism of secondary bacterial infection induced by H9N2 virus infection from a novel perspective, which provided theoretical and data support for investigating the synergistic infection mechanism between the H9N2 virus and bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

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/.

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 different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

The transcriptional response of Escherichia coli to recombinant protein insolubility.

PubMed

Smith, Harold E

2007-03-01

Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

Prophage Lysin Ply30 Protects Mice from Streptococcus suis and Streptococcus equi subsp. zooepidemicus Infections

PubMed Central

Tang, Fang; Li, Dezhi; Wang, Haojin; Ma, Zhe; Lu, Chengping

2015-01-01

Streptococcus suis and Streptococcus equi subsp. zooepidemicus are capable of infecting humans and various animals, causing significant problems for the worldwide swine industry. As antibiotic resistance has increased, lysosomal enzymes encoded by phages have shown potential for use against pathogenic bacteria. In this study, a novel bacteriophage lysin, Ply30, encoded by the S. suis prophage phi30c, was recombinantly expressed and purified. Ply30 showed high bacteriolysis activity on S. suis and S. equi subsp. zooepidemicus in vitro. The ratio of the optical density at 600 nm (OD600) with treatment versus the OD600 with no treatment for most tested S. suis and S. equi subsp. zooepidemicus strains decreased from 1 to <0.3 and <0.5, respectively, within 1 h. The results of plate viability assays showed that treated bacteria suffered a 1- to 2-log decrease in CFU within 1 h. The optimal concentration of Ply30 was 50 μg/ml, and the optimal pH was 7. Moreover, Ply30 maintained high activity over a wide pH range (pH 6 to 10). The MICs of Ply30 against Streptococcus strains ranged from 16 to 512 μg/ml. In vivo, a 2-mg dose of Ply30 protected 90% (9/10 mice) of mice from infection with S. equi subsp. zooepidemicus and 80% (8/10 mice) of mice from infection with S. suis. Seven days after lysin Ply30 treatment, bacterial loads were significantly decreased in all tested organs and blood compared with those at 1 h postinfection without Ply30 treatment. Ply30 showed in vitro and in vivo antimicrobial efficiency and protected mice against two kinds of bacterial infections, indicating that Ply30 may be an effective therapeutic against streptococci. PMID:26253669

Fundamental CRISPR-Cas9 tools and current applications in microbial systems.

PubMed

Tian, Pingfang; Wang, Jia; Shen, Xiaolin; Rey, Justin Forrest; Yuan, Qipeng; Yan, Yajun

2017-09-01

Derived from the bacterial adaptive immune system, CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering. In this review, we outline the fundamental CRISPR tools that have been employed for strain optimization. These tools include CRISPR editing, CRISPR interference, CRISPR activation and protein imaging. To further characterize the CRISPR technology, we present current applications of these tools in microbial systems, including model- and non-model industrial microorganisms. Specially, we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation. To address these challenges, we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering. Lastly, multiple promising research directions were proposed, mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.

Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

NASA Technical Reports Server (NTRS)

Wilson, James W.; Ramamurthy, Rajee; Porwollik, Steffen; McClelland, Michael; Hammond, Timothy; Allen, Pat; Ott, C. Mark; Pierson, Duane L.; Nickerson, Cheryl A.

2002-01-01

The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 x g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies.

High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate.

PubMed

Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P; Snell, Kristi D

2011-04-01

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions

PubMed Central

Becherelli, Marco; Manetti, Andrea G O; Buccato, Scilla; Viciani, Elisa; Ciucchi, Laura; Mollica, Giulia; Grandi, Guido; Margarit, Imma

2012-01-01

Summary Gram-positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT-1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in-frame deletion mutants, Lactococcus expressing heterologous FCT-1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter-bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three-dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection. PMID:22320452

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

PubMed

Chen, Linxu; Xu, Qilong; Tu, Jiagang; Ge, Yihe; Liu, Jun; Liang, Fang Ting

2013-01-01

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

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.

Antibiotic therapy of community respiratory tract infections: strategies for optimal outcomes and minimized resistance emergence.

PubMed

Ball, P; Baquero, F; Cars, O; File, T; Garau, J; Klugman, K; Low, D E; Rubinstein, E; Wise, R

2002-01-01

Widespread, increasing antibiotic resistance amongst the major respiratory pathogens has compromised traditional therapy of the major infective respiratory syndromes, including bacterial pneumonia and acute exacerbations of chronic bronchitis. Guidelines for antibiotic prescribing dating from the 1980s to 1990s, which attempted to address such problems, were commonly too prescriptive and difficult to apply, and took little account of end-user practice or locally prevalent resistance levels. Further confusion was caused by conflicting recommendations emanating from differing specialty groups. The evidence that such guidelines benefited either clinical outcomes or treatment costs has been disputed. They have probably had little effect on resistance emergence. We report the recommendations of an independent, multi-national, inter-disciplinary group, which met to identify principles underlying prescribing and guideline formulation in an age of increasing bacterial resistance. Unnecessary prescribing was recognized as the major factor in influencing resistance and costs. Antibiotic therapy must be limited to syndromes in which bacterial infection is the predominant cause and should attempt maximal reduction in bacterial load, with the ultimate aim of bacterial eradication. It should be appropriate in type and context of local resistance prevalence, and optimal in dosage for the pathogen(s) involved. Prescribing should be based on pharmacodynamic principles that predict efficacy, bacterial eradication and prevention of resistance emergence. Pharmacoeconomic analyses confirm that bacteriologically more effective antibiotics can reduce overall management costs, particularly with respect to consequential morbidity and hospital admission. Application of these principles should positively benefit therapeutic outcomes, resistance avoidance and management costs and will more accurately guide antibiotic choices by both individuals and formulary/guideline committees.

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

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge.

PubMed

Gao, Qiong; Liao, Meijie; Wang, Yingeng; Li, Bin; Zhang, Zheng; Rong, Xiaojun; Chen, Guiping; Wang, Lan

2015-07-17

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge

PubMed Central

Gao, Qiong; Liao, Meijie; Wang, Yingeng; Li, Bin; Zhang, Zheng; Rong, Xiaojun; Chen, Guiping; Wang, Lan

2015-01-01

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway. PMID:26193268

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.

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

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.

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

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.

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.

Immunogenicity of a plant-derived edible chimeric EspA, Intimin and Tir of Escherichia coli O157:H7 in mice.

PubMed

Amani, Jafar; Mousavi, Seyed Latif; Rafati, Sima; Salmanian, Ali Hatef

2011-04-01

Transgenic plants offer the possibility to produce and deliver an oral immunogen on a large-scale with low production costs and minimal purification or enrichment. Cattles are important reservoirs of Escherichia coli O157:H7 and developing a specific immunity in animals would be invaluable. Intimin, Tir, and EspA proteins are the virulence factors expressed by LEE locus of enterohemorrhagic E. coli. We hypothesized that the chimeric recombinant forms of these effectors delivered as an edible-base vaccine would reduce colonization of bacteria in mice. A synthetic gene (eit) composed of espA (e), eae (i) and tir (t) attached by linkers was constructed. The gene was codon optimized and cloned into plant expression vectors adjacent to CaMV35S and FAE promoters for expression in tobacco and canola plants. Of total soluble protein 0.2% and 0.3% (in average) were detected in transgenic tobacco leaves and canola seeds respectively. Mice immunized either subcutaneously or orally with recombinant EIT and challenged with E. coli O157:H7 significantly exhibited reduced bacterial shedding. Application of transgenic plants containing trivalent immunogen is an effective tool for protection against E. coli O157:H7. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Immunization of pigs with an attenuated pseudorabies virus recombinant expressing the haemagglutinin of pandemic swine origin H1N1 influenza A virus.

PubMed

Klingbeil, Katharina; Lange, Elke; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

2014-04-01

Pigs can be severely harmed by influenza, and represent important reservoir hosts, in which new human pathogens such as the recent pandemic swine-origin H1N1 influenza A virus can arise by mutation and reassortment of genome segments. To obtain novel, safe influenza vaccines for pigs, and to investigate the antigen-specific immune response, we modified an established live-virus vaccine against Aujeszky's disease of swine, pseudorabies virus (PrV) strain Bartha (PrV-Ba), to serve as vector for the expression of haemagglutinin (HA) of swine-origin H1N1 virus. To facilitate transgene insertion, the genome of PrV-Ba was cloned as a bacterial artificial chromosome. HA expression occurred under control of the human or murine cytomegalovirus immediate early promoters (P-HCMV, P-MCMV), but could be substantially enhanced by synthetic introns and adaptation of the codon usage to that of PrV. However, despite abundant expression, the heterologous glycoprotein was not detectably incorporated into mature PrV particles. Replication of HA-expressing PrV in cell culture was only slightly affected compared to that of the parental virus strain. A single immunization of pigs with the PrV vector expressing the codon-optimized HA gene under control of P-MCMV induced high levels of HA-specific antibodies. The vaccinated animals were protected from clinical signs after challenge with a related swine-origin H1N1 influenza A virus, and challenge virus shedding was significantly reduced.

Impact of Solar Radiation on Gene Expression in Bacteria

PubMed Central

Matallana-Surget, Sabine; Wattiez, Ruddy

2013-01-01

Microorganisms often regulate their gene expression at the level of transcription and/or translation in response to solar radiation. In this review, we present the use of both transcriptomics and proteomics to advance knowledge in the field of bacterial response to damaging radiation. Those studies pertain to diverse application areas such as fundamental microbiology, water treatment, microbial ecology and astrobiology. Even though it has been demonstrated that mRNA abundance is not always consistent with the protein regulation, we present here an exhaustive review on how bacteria regulate their gene expression at both transcription and translation levels to enable biomarkers identification and comparison of gene regulation from one bacterial species to another. PMID:28250399

Low-dose radiation induces Drosophila innate immunity through Toll pathway activation.

PubMed

Seong, Ki Moon; Kim, Cha Soon; Lee, Byung-Sub; Nam, Seon Young; Yang, Kwang Hee; Kim, Ji-Young; Park, Joong-Jean; Min, Kyung-Jin; Jin, Young-Woo

2012-01-01

Numerous studies report that exposing certain organisms to low-dose radiation induces beneficial effects on lifespan, tumorigenesis, and immunity. By analyzing survival after bacterial infection and antimicrobial peptide gene expression in irradiated flies, we demonstrate that low-dose irradiation of Drosophila enhances innate immunity. Low-dose irradiation of flies significantly increased resistance against gram-positive and gram-negative bacterial infections, as well as expression of several antimicrobial peptide genes. Additionally, low-dose irradiation also resulted in a specific increase in expression of key proteins of the Toll signaling pathway and phosphorylated forms of p38 and JNK. These results indicate that innate immunity is activated after low-dose irradiation through Toll signaling pathway in Drosophila.

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

A Novel Platform for Evaluating the Environmental Impacts on Bacterial Cellulose Production.

PubMed

Basu, Anindya; Vadanan, Sundaravadanam Vishnu; Lim, Sierin

2018-04-10

Bacterial cellulose (BC) is a biocompatible material with versatile applications. However, its large-scale production is challenged by the limited biological knowledge of the bacteria. The advent of synthetic biology has lead the way to the development of BC producing microbes as a novel chassis. Hence, investigation on optimal growth conditions for BC production and understanding of the fundamental biological processes are imperative. In this study, we report a novel analytical platform that can be used for studying the biology and optimizing growth conditions of cellulose producing bacteria. The platform is based on surface growth pattern of the organism and allows us to confirm that cellulose fibrils produced by the bacteria play a pivotal role towards their chemotaxis. The platform efficiently determines the impacts of different growth conditions on cellulose production and is translatable to static culture conditions. The analytical platform provides a means for fundamental biological studies of bacteria chemotaxis as well as systematic approach towards rational design and development of scalable bioprocessing strategies for industrial production of bacterial cellulose.

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.

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

PubMed

Kochi, Shinsuke; Yamashiro, Keisuke; Hongo, Shoichi; Yamamoto, Tadashi; Ugawa, Yuki; Shimoe, Masayuki; Kawamura, Mari; Hirata-Yoshihara, Chiaki; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

2017-12-01

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, β4, and β6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, β4, and β6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Cathelicidin Signaling via the Toll-Like Receptor Protects Against Colitis in Mice

PubMed Central

Koon, Hon Wai; Shih, David Quan; Chen, Jeremy; Bakirtzi, Kyriaki; Hing, Tressia C; Law, Ivy; Ho, Samantha; Ichikawa, Ryan; Zhao, Dezheng; Xu, Hua; Gallo, Richard; Dempsey, Paul; Cheng, Genhong; Targan, Stephan R; Pothoulakis, Charalabos

2011-01-01

Background & Aims Cathelicidin (encoded by Camp) is an anti-microbial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease; we investigated its signaling mechanisms. Methods Quantitative, real-time, reverse transcription PCR, bacterial 16S PCR, immunofluorescence, and small interfering (si)RNA analyses were performed. Colitis was induced in mice using sodium dextran sulfate (DSS); levels of cathelicidin were measured in human primary monocytes. Results Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis, compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to E coli DNA induced expression of Camp mRNA, which required signaling by ERK; expression was reduced by siRNAs against toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9 −/− mice with DSS-induced colitis. Compared with wild-type mice, Camp −/− mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. Conclusions Cathelicidin protects against colitis induction in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9–ERK signaling by bacterial DNA. This pathway might be involved in pathogenesis of ulcerative colitis. PMID:21762664

R software package based statistical optimization of process components to simultaneously enhance the bacterial growth, laccase production and textile dye decolorization with cytotoxicity study

PubMed Central

Dudhagara, Pravin; Tank, Shantilal

2018-01-01

The thermophilic bacterium, Bacillus licheniformis U1 is used for the optimization of bacterial growth (R1), laccase production (R2) and synthetic disperse blue DBR textile dye decolorization (R3) in the present study. Preliminary optimization has been performed by one variable at time (OVAT) approach using four media components viz., dye concentration, copper sulphate concentration, pH, and inoculum size. Based on OVAT result further statistical optimization of R1, R2 and R3 performed by Box–Behnken design (BBD) using response surface methodology (RSM) in R software with R Commander package. The total 29 experimental runs conducted in the experimental design study towards the construction of a quadratic model. The model indicated that dye concentration 110 ppm, copper sulphate 0.2 mM, pH 7.5 and inoculum size 6% v/v were found to be optimum to maximize the laccase production and bacterial growth. Whereas, maximum dye decolorization achieved in media containing dye concentration 110 ppm, copper sulphate 0.6 mM, pH 6 and inoculum size 6% v/v. R package predicted R2 of R1, R2 and R3 were 0.9917, 0.9831 and 0.9703 respectively; likened to Design-Expert (Stat-Ease) (DOE) predicted R2 of R1, R2, and R3 were 0.9893, 0.9822 and 0.8442 respectively. The values obtained by R software were more precise, reliable and reproducible, compared to the DOE model. The laccase production was 1.80 fold increased, and 2.24 fold enhancement in dye decolorization was achieved using optimized medium than initial experiments. Moreover, the laccase-treated sample demonstrated the less cytotoxic effect on L132 and MCF-7 cell lines compared to untreated sample using MTT assay. Higher cell viability and lower cytotoxicity observed in a laccase-treated sample suggest the impending application of bacterial laccase in the reduction of toxicity of dye to design rapid biodegradation process. PMID:29718934

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 Se in broiler chickens.

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-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.« less

Post-Weaning Diet Affects Faecal Microbial Composition but Not Selected Adipose Gene Expression in the Cat (Felis catus)

PubMed Central

Bermingham, Emma N.; Kittelmann, Sandra; Young, Wayne; Kerr, Katherine R.; Swanson, Kelly S.; Roy, Nicole C.; Thomas, David G.

2013-01-01

The effects of pre- (i.e., gestation and during lactation) and post-weaning diet on the composition of faecal bacterial communities and adipose expression of key genes in the glucose and insulin pathways were investigated in the cat. Queens were maintained on a moderate protein:fat:carbohydrate kibbled (“Diet A”; 35:20:28% DM; n  =  4) or high protein:fat:carbohydrate canned (“Diet B”; 45:37:2% DM; n = 3) diet throughout pregnancy and lactation. Offspring were weaned onto these diets in a nested design (n  =  5 per treatment). Faecal samples were collected at wk 8 and 17 of age. DNA was isolated from faeces and bacterial 16S rRNA gene amplicons were analysed by pyrosequencing. RNA was extracted from blood (wk 18) and adipose tissue and ovarian/testicular tissues (wk 24) and gene expression levels determined using RT-qPCR. Differences (P<0.05) in composition of faecal bacteria were observed between pregnant queens fed Diet A or B. However, pre-weaning diet had little effect on faecal bacterial composition in weaned kittens. In contrast, post-weaning diet altered bacterial population profiles in the kittens. Increased (P<0.05) abundance of Firmicutes (77% vs 52% of total reads) and Actinobacteria (0.8% vs 0.2% of total reads), and decreased (P<0.05) abundance of Fusobacteria (1.6% vs 18.4% of total reads) were observed for kittens fed the Diet A compared to those fed Diet B post-weaning. Feeding Diet B pre-weaning increased (P<0.05) the expression levels of INRS, LEPT, PAI-1 and tended to increase GLUT1, while the expression levels of IRS-1 in blood increased in kittens fed Diet A pre-weaning. Post-weaning diet had no effect on expression levels of target genes. Correlations between the expression levels of genes involved in glucose and insulin pathways and faecal Bacteriodetes and Firmicutes phyla were identified. The reasons for why post-weaning diet affects microbial populations and not gene expression levels are of interest. PMID:24312255

Optimal vitamin D plasma levels are associated with lower bacterial DNA translocation in HIV/hepatitis c virus coinfected patients.

PubMed

García-Álvarez, Mónica; Berenguer, Juan; Jiménez-Sousa, Maria Ángeles; Vázquez-Morón, Sonia; Carrero, Ana; Gutiérrez-Rivas, Mónica; Aldámiz-Echevarría, Teresa; López, Juan Carlos; García-Broncano, Pilar; Resino, Salvador

2016-04-24

Vitamin D has been linked to the immune response modulation and the integrity of the intestinal mucosal barrier. Therefore, vitamin D might be involved in bacterial translocation related to HIV infection. Our major aim was to analyze the association between plasma levels of 25-hydroxy-vitamin D [25(OH)D] and bacterial 16S ribosomal DNA (bactDNA) in 120 HIV/hepatitis c virus (HCV) coinfected patients. Cross-sectional study. Plasma 25(OH)D levels were quantified by enzyme immunoassay. The vitamin D status was defined as deficient (<25 nmol/l), insufficient (25-74 nmol/l), and optimal (≥75 nmol/l) plasma levels. Plasma bactDNA levels were measured by quantitative real-time PCR. For bactDNA levels the cutoffs used were as follows: low [p75th). Eighteen (15%) patients had 25(OH)D deficiency, 93 (77.5%) had insufficiency and nine (7.5%) had 25(OH)D optimal values. The bactDNA levels were lower in patients with 25(OH)D at least 75 nmol/l [37 copies/μl] than in patients with 25(OH)D insufficiency [84.2 copies/μl; P = 0.042]. Conversely, low bactDNA levels (

Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.

PubMed

Fogal, Stefano; Carotti, Marcello; Giaretta, Laura; Lanciai, Federico; Nogara, Leonardo; Bubacco, Luigi; Bergantino, Elisabetta

2015-01-01

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.

cis-antisense RNA, another level of gene regulation in bacteria.

PubMed

Georg, Jens; Hess, Wolfgang R

2011-06-01

A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology.

cis-Antisense RNA, Another Level of Gene Regulation in Bacteria

PubMed Central

Georg, Jens; Hess, Wolfgang R.

2011-01-01

Summary: A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology. PMID:21646430

The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis

PubMed Central

Kuipers, Annemarie; Stapels, Daphne A. C.; Weerwind, Lleroy T.; Ko, Ya-Ping; Ruyken, Maartje; Lee, Jean C.; van Kessel, Kok P. M.

2016-01-01

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance. PMID:27112346

The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis.

PubMed

Kuipers, Annemarie; Stapels, Daphne A C; Weerwind, Lleroy T; Ko, Ya-Ping; Ruyken, Maartje; Lee, Jean C; van Kessel, Kok P M; Rooijakkers, Suzan H M

2016-07-01

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance.

Strain Selection and Optimization of Mixed Culture Conditions for Lactobacillus pentosus K1-23 with Antibacterial Activity and Aureobasidium pullulans NRRL 58012 Producing Immune-Enhancing β-Glucan.

PubMed

Sekar, Ashokkumar; Kim, Myoungjin; Jeong, Hyeong Chul; Kim, Keun

2018-05-28

Lactobacillus pentosus K1-23 was selected from among 25 lactic acid bacterial strains owing to its high inhibitory activity against several pathogenic bacteria, including Escherichia coli , Salmonella typhimurium , S. gallinarum , Staphylococcus aureus , Pseudomonas aeruginosa , Clostridium perfringens , and Listeria monocytogenes . Additionally, among 13 strains of Aureobasidium spp., A. pullulans NRRL 58012 was shown to produce the highest amount of β-glucan (15.45 ± 0.07%) and was selected. Next, the optimal conditions for a solid-phase mixed culture with these two different microorganisms (one bacterium and one yeast) were determined. The optimal inoculum sizes for L. pentosus and A. pullulans were 1% and 5%, respectively. The appropriate inoculation time for L. pentosus K1-23 was 3 days after the inoculation of A. pullulans to initiate fermentation. The addition of 0.5% corn steep powder and 0.1% FeSO₄ to the basal medium resulted in the increased production of lactic acid bacterial cells and β-glucan. The following optimal conditions for solid-phase mixed culture were also statistically determined by using the response surface method: 37.84°C, pH 5.25, moisture content of 60.82%, and culture time of 6.08 days for L. pentosus ; and 24.11°C, pH 5.65, moisture content of 60.08%, and culture time of 5.71 days for A. pullulans. Using the predicted optimal conditions, the experimental production values of L. pentosus cells and β-glucan were 3.15 ± 0.10 × 10⁸ CFU/g and 13.41 ± 0.04%, respectively. This mixed culture may function as a highly efficient antibiotic substitute based on the combined action of its anti-pathogenic bacterial and immune-enhancing activities.

Interplay between the gut microbiota and immune responses of ayu (Plecoglossus altivelis) during Vibrio anguillarum infection.

PubMed

Nie, Li; Zhou, Qian-Jin; Qiao, Yan; Chen, Jiong

2017-09-01

Gut microbiota plays fundamental roles in protection against pathogen invasion. However, the mechanism and extent of responses of gut microbiota to pathogenic infection are poorly understood. This study investigated the gut bacterial communities and immune responses of ayu (Plecoglossus altivelis) upon exposure to Vibrio anguillarum. The succession of V. anguillarum infection was evidenced by increased expression of immune genes and bacterial loads in ayu tissues, which in turn altered the composition and predicted functions of gut bacterial community. The dynamics of gut bacterial diversity and evenness were temporally stable in control ayu but were reduced in infected subjects, particularly at the late stages of infection. Variations in the gut microbiota were significantly associated with the expression levels of TNF-α (P = 0.019) and IL-1 β (P = 0.013). The profiles of certain gut bacterial taxa were indicative of V. anguillarum infection. Compared with healthy controls, the ayu infected with V. anguillarum possessed less complex, fewer connected, and lower cooperative gut bacterial interspecies interaction, coinciding with significant shifts in keystone species. These findings imply that V. anguillarum infection substantially disrupted the compositions and interspecies interaction of ayu gut bacterial community, thereby altering gut microbial-mediated functions and inducing host immune responses. This study provides an integrated overview on the interaction between the gut microbiota and host immune responses to pathogen infection from an ecological perspective. Copyright © 2017 Elsevier Ltd. All rights reserved.

QTLs for Resistance to Major Rice Diseases Exacerbated by Global Warming: Brown Spot, Bacterial Seedling Rot, and Bacterial Grain Rot.

PubMed

Mizobuchi, Ritsuko; Fukuoka, Shuichi; Tsushima, Seiya; Yano, Masahiro; Sato, Hiroyuki

2016-12-01

In rice (Oryza sativa L.), damage from diseases such as brown spot, caused by Bipolaris oryzae, and bacterial seedling rot and bacterial grain rot, caused by Burkholderia glumae, has increased under global warming because the optimal temperature ranges for growth of these pathogens are relatively high (around 30 °C). Therefore, the need for cultivars carrying genes for resistance to these diseases is increasing to ensure sustainable rice production. In contrast to the situation for other important rice diseases such as blast and bacterial blight, no genes for complete resistance to brown spot, bacterial seedling rot or bacterial grain rot have yet been discovered. Thus, rice breeders have to use partial resistance, which is largely influenced by environmental conditions. Recent progress in molecular genetics and improvement of evaluation methods for disease resistance have facilitated detection of quantitative trait loci (QTLs) associated with resistance. In this review, we summarize the results of worldwide screening for cultivars with resistance to brown spot, bacterial seedling rot and bacterial grain rot and we discuss the identification of QTLs conferring resistance to these diseases in order to provide useful information for rice breeding programs.

The use of dried cerebrospinal fluid filter paper spots as a substrate for PCR diagnosis of the aetiology of bacterial meningitis in the Lao PDR

PubMed Central

Elliott, I; Dittrich, S; Paris, D; Sengduanphachanh, A; Phoumin, P; Newton, P N

2013-01-01

We investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro Card™ (Whatman, Maidstone, UK) was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median (IQR; range) CSF S. pneumoniae bacterial load was 1.1 × 104 GE/μL (1.2 × 105; 1 to 6.1 × 106 DNA GE/μL). Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies. PMID:23738720

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

Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

PubMed Central

Vargas-Bautista, Carol; Rahlwes, Kathryn

2014-01-01

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis. PMID:24187085

Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis.

PubMed

Vargas-Bautista, Carol; Rahlwes, Kathryn; Straight, Paul

2014-02-01

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305-310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis.

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 effective and adaptive processes to meet the increasing demand of r-protein industry.

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

USDA-ARS?s Scientific Manuscript database

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophi...

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.

Evaluation of isolation methods for bacterial RNA quantitation in Dickeya dadantii

USDA-ARS?s Scientific Manuscript database

Dickeya dadantii is a difficult source for RNA of a sufficient quality for real-time qRT-PCR analysis of gene expression. Three RNA isolation methods were evaluated for their ability to produce high-quality RNA from this bacterium. Bacterial lysis with Trizol using standard protocols consistently ga...

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 activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the prtA gene. Moreover, we reveal that PrtA expression favors the ability of S. marcescens to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of S. marcescens on abiotic surfaces and in bacterial host colonization capacity. Copyright © 2018 American Society for Microbiology.

Quorum Sensing in a Methane-Oxidizing Bacterium.

PubMed

Puri, Aaron W; Schaefer, Amy L; Fu, Yanfen; Beck, David A C; Greenberg, E Peter; Lidstrom, Mary E

2017-03-01

Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum , a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N -3-hydroxydecanoyl-l-homoserine lactone (3-OH-C 10 -HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. IMPORTANCE Aerobic methanotrophs are critical for sequestering carbon from the potent greenhouse gas methane in the environment, yet the mechanistic details of chemical interactions in methane-oxidizing bacterial communities are not well understood. Understanding these interactions is important in order to maintain, and potentially optimize, the functional potential of the bacteria that perform this vital ecosystem function. In this work, we identify a quorum sensing system in the aerobic methanotroph Methylobacter tundripaludum and use both chemical and genetic methods to characterize this system at the molecular level. Copyright © 2017 American Society for Microbiology.

Evolution and functional diversification of fructose bisphosphate aldolase genes in photosynthetic marine diatoms.

PubMed

Allen, Andrew E; Moustafa, Ahmed; Montsant, Anton; Eckert, Angelika; Kroth, Peter G; Bowler, Chris

2012-01-01

Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.

Evolution and Functional Diversification of Fructose Bisphosphate Aldolase Genes in Photosynthetic Marine Diatoms

PubMed Central

Allen, Andrew E.; Moustafa, Ahmed; Montsant, Anton; Eckert, Angelika; Kroth, Peter G.; Bowler, Chris

2012-01-01

Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively. PMID:21903677

Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria

PubMed Central

Chahales, Peter; Thanassi, David G.

2015-01-01

Bacteria assemble a wide range of adhesive proteins, termed adhesins, to mediate binding to receptors and colonization of surfaces. For pathogenic bacteria, adhesins are critical for early stages of infection, allowing the bacteria to initiate contact with host cells, colonize different tissues, and establish a foothold within the host. The adhesins expressed by a pathogen are also critical for bacterial-bacterial interactions and the formation of bacterial communities such as biofilms. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where the flow of urine functions to maintain sterility by washing away non-adherent pathogens. Adhesins vary from monomeric proteins that are directly anchored to the bacterial surface to polymeric, hairlike fibers that extend out from the cell surface. These latter fibers are termed pili or fimbriae, and were among the first identified virulence factors of uropathogenic Escherichia coli. Studies since then have identified a range of both pilus and non-pilus adhesins that contribute to bacterial colonization of the urinary tract, and have revealed molecular details of the structures, assembly pathways, and functions of these adhesive organelles. In this review, we describe the different types of adhesins expressed by both Gram-negative and Gram-positive uropathogens, what is known about their structures, how they are assembled on the bacterial surface, and the functions of specific adhesins in the pathogenesis of urinary tract infections. PMID:26542038

Influenza-induced type I interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice.

PubMed

Lee, Benjamin; Robinson, Keven M; McHugh, Kevin J; Scheller, Erich V; Mandalapu, Sivanarayana; Chen, Chen; Di, Y Peter; Clay, Michelle E; Enelow, Richard I; Dubin, Patricia J; Alcorn, John F

2015-07-15

Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection. Copyright © 2015 the American Physiological Society.

Effects of Extended Freezer Storage on the Integrity of Human Milk.

PubMed

Ahrabi, Ali Faraghi; Handa, Deepali; Codipilly, Champa N; Shah, Syed; Williams, Janet E; McGuire, Mark A; Potak, Debra; Aharon, Grace Golda; Schanler, Richard J

2016-10-01

To examine the integrity (pH, bacterial counts, host defense factors, nutrient contents, and osmolality) of freshly expressed and previously refrigerated human milk subjected to long-term freezer storage. Mothers donated 100 mL of freshly expressed milk. Samples were divided into baseline, storage at -20°C (fresh frozen) for 1, 3, 6, and 9 months, and prior storage at +4°C for 72 hours (refrigerated frozen) before storage at -20°C for 1 to 9 months. Samples were analyzed for pH, total bacterial colony count, gram-positive and gram-negative colony counts, and concentrations of total protein, fat, nonesterified fatty acids, lactoferrin, secretory IgA, and osmolality. Milk pH, total bacterial colony count, and Gram-positive colony counts decreased significantly with freezer storage (P < .001); bacterial counts decreased most rapidly in the refrigerated frozen group. The gram-negative colony count decreased significantly over time (P < .001). Nonesterified fatty acid concentrations increased significantly with time in storage (P < .001). Freezing for up to 9 months did not affect total protein, fat, lactoferrin, secretory IgA, or osmolality in either group. Freezer storage of human milk for 9 months at -20°C is associated with decreasing pH and bacterial counts, but preservation of key macronutrients and immunoactive components, with or without prior refrigeration for 72 hours. These data support current guidelines for freezer storage of human milk for up to 9 months for both freshly expressed and refrigerated milk. Copyright © 2016 Elsevier Inc. All rights reserved.

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 sponge cells and regulating the symbiotic relationship.

Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

PubMed Central

Fink, Doran L.; St. Geme III, Joseph W.

2003-01-01

The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hapβ and a 110-kDa extracellular passenger domain called HapS. All adhesive activity resides within HapS, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap. PMID:12591878

Hexavalent chromium reduction by bacterial consortia and pure strains from an alkaline industrial effluent.

PubMed

Piñón-Castillo, H A; Brito, E M S; Goñi-Urriza, M; Guyoneaud, R; Duran, R; Nevarez-Moorillon, G V; Gutiérrez-Corona, J F; Caretta, C A; Reyna-López, G E

2010-12-01

To characterize the bacterial consortia and isolates selected for their role in hexavalent chromium removal by adsorption and reduction. Bacterial consortia from industrial wastes revealed significant Cr(VI) removal after 15 days when incubated in medium M9 at pH 6·5 and 8·0. The results suggested chromium reduction. The bacterial consortia diversity (T-RFLP based on 16S rRNA gene) indicated a highest number of operational taxonomic units in an alkaline carbonate medium mimicking in situ conditions. However, incubations under such conditions revealed low Cr(VI) removal. Genomic libraries were obtained for the consortia exhibiting optimal Cr(VI) removal (M9 medium at pH 6·5 and 8·0). They revealed the dominance of 16S rRNA gene sequences related to the genera Pseudomonas/Stenotrophomonas or Enterobacter/Halomonas, respectively. Isolates related to Pseudomonas fluorescens and Enterobacter aerogenes were efficient in Cr(VI) reduction and adsorption to the biomass. Cr(VI) reduction was better at neutral pH rather than under in situ conditions (alkaline pH with carbonate). Isolated strains exhibited significant capacity for Cr(VI) reduction and adsorption. Bacterial communities from chromium-contaminated industrial wastes as well as isolates were able to remove Cr(VI). The results suggest a good potential for bioremediation of industrial wastes when optimal conditions are applied. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology. No claim to Mexican Government works.

Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

PubMed

Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

2009-08-01

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

Combined biomarkers discriminate a low likelihood of bacterial infection among surgical intensive care unit patients with suspected sepsis

PubMed Central

Kelly, Brendan J.; Lautenbach, Ebbing; Nachamkin, Irving; Coffin, Susan E.; Gerber, Jeffrey S.; Fuchs, Barry D.; Garrigan, Charles; Han, Xiaoyan; Bilker, Warren B.; Wise, Jacqueleen; Tolomeo, Pam; Han, Jennifer H.

2016-01-01

Among surgical intensive care unit (SICU) patients, it is difficult to distinguish bacterial sepsis from other causes of systemic inflammatory response syndrome (SIRS). Biomarkers have proven useful to identify the presence of bacterial infection. We enrolled a prospective cohort of 69 SICU patients with suspected sepsis and assayed the concentrations of nine biomarkers (α-2 macroglobulin (A2M), C-reactive protein, ferritin, fibrinogen, haptoglobin, procalcitonin (PCT), serum amyloid A, serum amyloid P, and tissue plasminogen activator) at baseline, 24-, 48-, and 72-hours. 42 patients (61%) had bacterial sepsis by chart review. A2M concentrations were significantly lower and PCT concentrations significantly higher in subjects with bacterial sepsis at three of four timepoints. Using optimal cutoff values, the combination of baseline A2M and 72-hour PCT achieved a negative predictive value of 75% (95% CI, 54%–96%). The combination of A2M and PCT discriminated bacterial sepsis from other SIRS among SICU patients with suspected sepsis. PMID:26971636

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection.

PubMed

Schmidt, M; Hourfar, M K; Wahl, A; Nicol, S-B; Montag, T; Roth, W K; Seifried, E

2006-05-01

The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.

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.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Disposable bioluminescence-based biosensor for detection of bacterial count in food.

PubMed

Luo, Jinping; Liu, Xiaohong; Tian, Qing; Yue, Weiwei; Zeng, Jing; Chen, Guangquan; Cai, Xinxia

2009-11-01

A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10(3) to 10(8) colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n=22) within 5min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.

Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content.

PubMed

Fazi, Stefano; Amalfitano, Stefano; Pizzetti, Ilaria; Pernthaler, Jakob

2007-09-01

We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.

Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

PubMed

Jia, Kun; Ionescu, Rodica Elena

2016-01-01

: Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

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 might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. PMID:25673765

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 might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria.

PubMed

Li, Mo-Fei; Zhang, Min; Wang, Chun-Lin; Sun, Li

2012-02-01

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn(2+)-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Diagnostic values of plasma CD64 and sTREM-1 for pediatric pneumonia].

PubMed

Zhong, Mei-Feng; Zhao, Jian-Mei

2016-07-01

To determine the diagnostic values of plasma CD64 and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in children with pneumonia. Sixty children with pneumonia between August 2014 and October 2015 were classified into bacterial pneumonia group (25 cases), viral pneumonia group (17 cases), and Mycoplasma pneumonia group (18 cases) according to their clinical manifestations, pathogen cultures, and X-ray findings. Another 30 healthy children who underwent physical examination during the same period were selected as the control group. The concentrations of CD64 and sTREM-1 in blood samples were determined using ELISA. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic sensitivity and specificity of plasma CD64 and/or sTREM-1 for bacterial pneumonia. The expression of CD64 and sTREM-1 in the bacterial pneumonia group was significantly higher than that in the viral pneumonia, Mycoplasma pneumonia, and control groups (P<0.05). The areas under the ROC curves of CD64, sTREM-1, and a combination of the two markers for diagnosing bacterial pneumonia were 0.878, 0.805, and 0.956, respectively. The sensitivity and specificity of CD64 for diagnosing bacterial pneumonia were 81.30% and 92.32%, respectively, when the cut-off value was 641 pg/mL. The sensitivity and specificity of sTREM-1 for diagnosing bacterial pneumonia were 78.65% and 84.67%, respectively, when the cut-off value was 1 479 pg/mL. The sensitivity and specificity of a combination of the two markers for diagnosing bacterial pneumonia were 93.15% and 91.54%, respectively. Plasma CD64 and sTREM-1 can be used as markers for diagnosing pediatric bacterial pneumonia, and a combination of the two markers results in better diagnosis.

Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria.

PubMed

Pavankumar, Asalapuram R; Ayyappasamy, Sudalaiyadum Perumal; Sankaran, Krishnan

2012-03-01

Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.

Atomic resolution mechanistic studies of ribocil: A highly selective unnatural ligand mimic of the E. coli FMN riboswitch

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

Howe, John A.; Xiao, Li; Fischmann, Thierry O.

2016-08-02

Bacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway. Although ribocil ismore » structurally distinct from FMN, ribocil functions as a potent and highly selective synthetic mimic of the natural ligand to repress riboswitch-mediated ribB gene expression and inhibit bacterial growth both in vitro and in vivo. Herein, we expand our analysis of ribocil; including mode of binding in the FMN binding pocket of the riboswitch, mechanisms of resistance and structure-activity relationship guided efforts to generate more potent analogs.« less

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

PubMed Central

Pinzon, Neissa M.; Aukema, Kelly G.; Gralnick, Jeffrey A.; Wackett, Lawrence P.

2011-01-01

ABSTRACT 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 production 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. PMID:21712420

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.

Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD.

PubMed

Pierantonelli, Irene; Rychlicki, Chiara; Agostinelli, Laura; Giordano, Debora Maria; Gaggini, Melania; Fraumene, Cristina; Saponaro, Chiara; Manghina, Valeria; Sartini, Loris; Mingarelli, Eleonora; Pinto, Claudio; Buzzigoli, Emma; Trozzi, Luciano; Giordano, Antonio; Marzioni, Marco; Minicis, Samuele De; Uzzau, Sergio; Cinti, Saverio; Gastaldelli, Amalia; Svegliati-Baroni, Gianluca

2017-09-22

Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A "multi-hit" theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3 -/- and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3 -/- -HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3 -/- -HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy.

Influence of milk processing temperature on growth performance, nitrogen retention, and hindgut's inflammatory status and bacterial populations in a calf model.

PubMed

Bach, Alex; Aris, Anna; Vidal, Maria; Fàbregas, Francesc; Terré, Marta

2017-08-01

This research communication describes a study aimed at evaluating the effects of heat treatment of milk on growth performance, N retention, and hindgut's inflammatory status and bacterial populations using young dairy calves as a model. Twenty-one Holstein calves were randomly allocated to one of three treatments: raw milk (RM), pasteurised milk (PAST), or UHT milk (UHT). Calves were submitted to a N balance study, and a biopsy from the distal colon and a faecal sample were obtained from 5 animals per treatment to determine expression of several genes and potential changes in the hindgut's bacterial population. Milk furosine content was 33-fold greater in UHT than in RM and PAST milks. Calves receiving RM grew more than those fed UHT, and urinary N excretion was greatest in calves fed UHT. Quantification of Lactobacillus was lower in calves consuming PAST or UHT, and Gram negative bacteria were greater in UHT than in PAST calves. The expression of IL-8 in the hindgut's mucosa was lowest and that of IL-10 tended to be lowest in RM calves, and expression of claudin-4 tended to be greatest in UHT calves. In conclusion, the nutritional value of UHT-treated milk may be hampered because it compromises growth and increases N excretion in young calves and may have deleterious effects on the gut's bacterial population and inflammation status.

[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.

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.

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.

Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus subtilis to Gold Surfaces

PubMed Central

Kesel, S.; Mader, A.; Seeberger, P. H.; Lieleg, O.

2014-01-01

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion—the first step in colonization and biofilm formation—is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future. PMID:25038098

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.

Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome.

PubMed

Strutton, Benjamin; Jaffé, Stephen R P; Pandhal, Jagroop; Wright, Phillip C

2018-01-01

Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%. Copyright © 2017 Elsevier Inc. All rights reserved.

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

PubMed

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

A transcription activator-like effector induction system mediated by proteolysis

PubMed Central

Copeland, Matthew F.; Politz, Mark C.; Johnson, Charles B.; Markley, Andrew L.; Pfleger, Brian F.

2016-01-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. In this work we expand the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic. PMID:26854666

Design of a Comprehensive Biochemistry and Molecular Biology Experiment: Phase Variation Caused by Recombinational Regulation of Bacterial Gene Expression

ERIC Educational Resources Information Center

Sheng, Xiumei; Xu, Shungao; Lu, Renyun; Isaac, Dadzie; Zhang, Xueyi; Zhang, Haifang; Wang, Huifang; Qiao, Zheng; Huang, Xinxiang

2014-01-01

Scientific experiments are indispensable parts of Biochemistry and Molecular Biology. In this study, a comprehensive Biochemistry and Molecular Biology experiment about "Salmonella enterica" serovar Typhi Flagellar phase variation has been designed. It consisted of three parts, namely, inducement of bacterial Flagellar phase variation,…

In silico Evolution of Lysis-Lysogeny Strategies Reproduces Observed Lysogeny Propensities in Temperate Bacteriophages

PubMed Central

Sinha, Vaibhhav; Goyal, Akshit; Svenningsen, Sine L.; Semsey, Szabolcs; Krishna, Sandeep

2017-01-01

Bacteriophages are the most abundant organisms on the planet and both lytic and temperate phages play key roles as shapers of ecosystems and drivers of bacterial evolution. Temperate phages can choose between (i) lysis: exploiting their bacterial hosts by producing multiple phage particles and releasing them by lysing the host cell, and (ii) lysogeny: establishing a potentially mutually beneficial relationship with the host by integrating their chromosome into the host cell's genome. Temperate phages exhibit lysogeny propensities in the curiously narrow range of 5–15%. For some temperate phages, the propensity is further regulated by the multiplicity of infection, such that single infections go predominantly lytic while multiple infections go predominantly lysogenic. We ask whether these observations can be explained by selection pressures in environments where multiple phage variants compete for the same host. Our models of pairwise competition, between phage variants that differ only in their propensity to lysogenize, predict the optimal lysogeny propensity to fall within the experimentally observed range. This prediction is robust to large variation in parameters such as the phage infection rate, burst size, decision rate, as well as bacterial growth rate, and initial phage to bacteria ratio. When we compete phage variants whose lysogeny strategies are allowed to depend upon multiplicity of infection, we find that the optimal strategy is one which switches from full lysis for single infections to full lysogeny for multiple infections. Previous attempts to explain lysogeny propensity have argued for bet-hedging that optimizes the response to fluctuating environmental conditions. Our results suggest that there is an additional selection pressure for lysogeny propensity within phage populations infecting a bacterial host, independent of environmental conditions. PMID:28798729

Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

PubMed

Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

2017-01-01

Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

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

Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

PubMed Central

Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

2016-01-01

ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

Tailoring the Immune Response via Customization of Pathogen Gene Expression.

PubMed

Runco, Lisa M; Stauft, Charles B; Coleman, J Robert

2014-01-01

The majority of studies focused on the construction and reengineering of bacterial pathogens have mainly relied on the knocking out of virulence factors or deletion/mutation of amino acid residues to then observe the microbe's phenotype and the resulting effect on the host immune response. These knockout bacterial strains have also been proposed as vaccines to combat bacterial disease. Theoretically, knockout strains would be unable to cause disease since their virulence factors have been removed, yet they could induce a protective memory response. While knockout strains have been valuable tools to discern the role of virulence factors in host immunity and bacterial pathogenesis, they have been unable to yield clinically relevant vaccines. The advent of synthetic biology and enhanced user-directed gene customization has altered this binary process of knockout, followed by observation. Recent studies have shown that a researcher can now tailor and customize a given microbe's gene expression to produce a desired immune response. In this commentary, we highlight these studies as a new avenue for controlling the inflammatory response as well as vaccine development.

Tailoring the Immune Response via Customization of Pathogen Gene Expression

PubMed Central

Runco, Lisa M.; Stauft, Charles B.

2014-01-01

The majority of studies focused on the construction and reengineering of bacterial pathogens have mainly relied on the knocking out of virulence factors or deletion/mutation of amino acid residues to then observe the microbe's phenotype and the resulting effect on the host immune response. These knockout bacterial strains have also been proposed as vaccines to combat bacterial disease. Theoretically, knockout strains would be unable to cause disease since their virulence factors have been removed, yet they could induce a protective memory response. While knockout strains have been valuable tools to discern the role of virulence factors in host immunity and bacterial pathogenesis, they have been unable to yield clinically relevant vaccines. The advent of synthetic biology and enhanced user-directed gene customization has altered this binary process of knockout, followed by observation. Recent studies have shown that a researcher can now tailor and customize a given microbe's gene expression to produce a desired immune response. In this commentary, we highlight these studies as a new avenue for controlling the inflammatory response as well as vaccine development. PMID:24719769

Genome expression analyses revealing the modulation of the Salmonella Rcs regulon by the attenuator IgaA.

PubMed

Mariscotti, Javier F; García-del Portillo, Francisco

2009-03-01

Intracellular growth attenuator A (IgaA) was identified as a Salmonella enterica regulator limiting bacterial growth inside fibroblasts. Genetic evidence further linked IgaA to repression of the RcsCDB regulatory system, which responds to envelope stress. How IgaA attenuates this system is unknown. Here, we present genome expression profiling data of S. enterica serovar Typhimurium igaA mutants grown at high osmolarity and displaying exacerbated Rcs responses. Transcriptome data revealed that IgaA attenuates gene expression changes requiring phosphorylated RcsB (RcsB~P) activity. Some RcsB-regulated genes, yciGFE and STM1862 (pagO)-STM1863-STM1864, were equally expressed in wild-type and igaA strains, suggesting a maximal expression at low levels of RcsB ~P. Other genes, such as metB, ypeC, ygaC, glnK, glnP, napA, glpA, and nirB, were shown for the first time and by independent methods to be regulated by the RcsCDB system. Interestingly, IgaA-deficient strains with reduced RcsC or RcsD levels exhibited different Rcs responses and distinct virulence properties. spv virulence genes were differentially expressed in most of the analyzed strains. spvA expression required RcsB and IgaA but, unexpectedly, was also impaired upon stimulation of the RcsC-->RcsD-->RcsB phosphorelay. Overproduction of either RcsB(+) or a nonphosphorylatable RcsB(D56Q) variant in strains displaying low spvA expression unveiled that both dephosphorylated RcsB and RcsB~P are required for optimal spvA expression. Taken together, our data support a model with IgaA attenuating the RcsCDB system by favoring the switch of RcsB~P to the dephosphorylated state. This role of IgaA in constantly fine-tuning the RcsB~P/RcsB ratio may ensure the proper expression of important virulence factors, such as the Spv proteins.

Transcriptomic Analysis and the Expression of Disease-Resistant Genes in Oryza meyeriana under Native Condition

PubMed Central

He, Bin; Tao, Xiang; Gu, Yinghong; Wei, Changhe; Cheng, Xiaojie; Xiao, Suqin; Cheng, Zaiquan; Zhang, Yizheng

2015-01-01

Oryza meyeriana (O. meyeriana), with a GG genome type (2n = 24), accumulated plentiful excellent characteristics with respect to resistance to many diseases such as rice shade and blast, even immunity to bacterial blight. It is very important to know if the diseases-resistant genes exist and express in this wild rice under native conditions. However, limited genomic or transcriptomic data of O. meyeriana are currently available. In this study, we present the first comprehensive characterization of the O. meyeriana transcriptome using RNA-seq and obtained 185,323 contigs with an average length of 1,692 bp and an N50 of 2,391 bp. Through differential expression analysis, it was found that there were most tissue-specifically expressed genes in roots, and next to stems and leaves. By similarity search against protein databases, 146,450 had at least a significant alignment to existed gene models. Comparison with the Oryza sativa (japonica-type Nipponbare and indica-type 93–11) genomes revealed that 13% of the O. meyeriana contigs had not been detected in O. sativa. Many diseases-resistant genes, such as bacterial blight resistant, blast resistant, rust resistant, fusarium resistant, cyst nematode resistant and downy mildew gene, were mined from the transcriptomic database. There are two kinds of rice bacterial blight-resistant genes (Xa1 and Xa26) differentially or specifically expressed in O. meyeriana. The 4 Xa1 contigs were all only expressed in root, while three of Xa26 contigs have the highest expression level in leaves, two of Xa26 contigs have the highest expression profile in stems and one of Xa26 contigs was expressed dominantly in roots. The transcriptomic database of O. meyeriana has been constructed and many diseases-resistant genes were found to express under native condition, which provides a foundation for future discovery of a number of novel genes and provides a basis for studying the molecular mechanisms associated with disease resistance in O. meyeriana. PMID:26640944

Growth-rate dependent global effects on gene expression in bacteria

PubMed Central

Klumpp, Stefan; Zhang, Zhongge; Hwa, Terence

2010-01-01

Summary Bacterial gene expression depends not only on specific regulations but also directly on bacterial growth, because important global parameters such as the abundance of RNA polymerases and ribosomes are all growth-rate dependent. Understanding these global effects is necessary for a quantitative understanding of gene regulation and for the robust design of synthetic genetic circuits. The observed growth-rate dependence of constitutive gene expression can be explained by a simple model using the measured growth-rate dependence of the relevant cellular parameters. More complex growth dependences for genetic circuits involving activators, repressors and feedback control were analyzed, and salient features were verified experimentally using synthetic circuits. The results suggest a novel feedback mechanism mediated by general growth-dependent effects and not requiring explicit gene regulation, if the expressed protein affects cell growth. This mechanism can lead to growth bistability and promote the acquisition of important physiological functions such as antibiotic resistance and tolerance (persistence). PMID:20064380

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 farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions that R. solanacearum expresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen's dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further, R. solanacearum was highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

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.

Heat shock protein 83 plays pleiotropic roles in embryogenesis, longevity, and fecundity of the pea aphid Acyrthosiphon pisum.

PubMed

Will, Torsten; Schmidtberg, Henrike; Skaljac, Marisa; Vilcinskas, Andreas

2017-01-01

Heat shock protein 83 (HSP83) is homologous to the chaperone HSP90. It has pleiotropic functions in Drosophila melanogaster, including the control of longevity and fecundity, and facilitates morphological evolution by buffering cryptic deleterious mutations in wild populations. In the pea aphid Acyrthosiphon pisum, HSP83 expression is moderately induced by bacterial infection but upregulated more strongly in response to heat stress and fungal infection. Stress-inducible heat shock proteins are of considerable evolutionary and ecological importance because they are known to buffer environmental variation and to influence fitness under non-optimal conditions. To investigate the functions of HSP83 in viviparous aphids, we used RNA interference to attenuate its expression and studied the impact on complex parameters. The RNA interference (RNAi)-mediated depletion of HSP83 expression in A. pisum reduced both longevity and fecundity, suggesting this chaperone has an evolutionarily conserved function in insects. Surprisingly, HSP83 depletion reduced the number of viviparous offspring while simultaneously increasing the number of premature nymphs developing in the ovaries, suggesting an unexpected role in aphid embryogenesis and eclosion. The present study indicates that reduced HSP83 expression in A. pisum reveals both functional similarities and differences compared with its reported roles in holometabolous insects. Its impact on aphid lifespan, fecundity, and embryogenesis suggests a function that determines their fitness. This could be achieved by targeting different client proteins, recruiting distinct co-chaperones or transposon activation.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

PubMed

Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

2012-10-01

To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

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.

Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines

PubMed Central

Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.

2014-01-01

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

PubMed

Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

2017-12-01

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

Compositions and Methods for the Treatment of Pierce's Disease

DOEpatents

Gupta, Goutam

2008-10-07

Chimeric anti-microbial proteins, compositions, and methods for the therapeutic and prophylactic treatment of plant diseases caused by the bacterial pathogen Xylella fastidiosa are provided. The anti-microbial proteins of the invention generally comprise a surface recognition domain polypeptide, capable of binding to a bacterial membrane component, fused to a bacterial lysis domain polypeptide, capable of affecting lysis or rupture of the bacterial membrane, typically via a fused polypeptide linker. In particular, methods and compositions for the treatment or prevention of Pierce's disease of grapevines are provided. Methods for the generation of transgenic Vitus vinefera plants expressing xylem-secreted anti-microbial chimeras are also provided.

Bacterial growth laws reflect the evolutionary importance of energy efficiency.

PubMed

Maitra, Arijit; Dill, Ken A

2015-01-13

We are interested in the balance of energy and protein synthesis in bacterial growth. How has evolution optimized this balance? We describe an analytical model that leverages extensive literature data on growth laws to infer the underlying fitness landscape and to draw inferences about what evolution has optimized in Escherichia coli. Is E. coli optimized for growth speed, energy efficiency, or some other property? Experimental data show that at its replication speed limit, E. coli produces about four mass equivalents of nonribosomal proteins for every mass equivalent of ribosomes. This ratio can be explained if the cell's fitness function is the the energy efficiency of cells under fast growth conditions, indicating a tradeoff between the high energy costs of ribosomes under fast growth and the high energy costs of turning over nonribosomal proteins under slow growth. This model gives insight into some of the complex nonlinear relationships between energy utilization and ribosomal and nonribosomal production as a function of cell growth conditions.

Bacterial Diversity and Nitrogen Utilization Strategies in the Upper Layer of the Northwestern Pacific Ocean.

PubMed

Li, Yuan-Yuan; Chen, Xiao-Huang; Xie, Zhang-Xian; Li, Dong-Xu; Wu, Peng-Fei; Kong, Ling-Fen; Lin, Lin; Kao, Shuh-Ji; Wang, Da-Zhi

2018-01-01

Nitrogen (N) is a primary limiting nutrient for bacterial growth and productivity in the ocean. To better understand bacterial community and their N utilization strategy in different N regimes of the ocean, we examined bacterial diversity, diazotrophic diversity, and N utilization gene expressions in the northwestern Pacific Ocean (NWPO) using a combination of high-throughput sequencing and real-time qPCR methods. 521 and 204 different operational taxonomic units (OTUs) were identified in the 16s rRNA and nifH libraries from nine surface samples. Of the 16s rRNA gene OTUs, 11.9% were observed in all samples while 3.5 and 15.9% were detected only in N-sufficient and N-deficient samples. Proteobacteria, Cyanobacteria and Bacteroidetes dominated the bacterial community. Prochlorococcus and Pseudoalteromonas were the most abundant at the genus level in N-deficient regimes, while SAR86, Synechococcus and SAR92 were predominant in the Kuroshio-Oyashio confluence region. The distribution of the nifH gene presented great divergence among sampling stations: Cyanobacterium_UCYN-A dominated the N-deficient stations, while clusters related to the Alpha-, Beta- , and Gamma-Proteobacteria were abundant in other stations. Temperature was the main factor that determined bacterial community structure and diversity while concentration of NO X -N was significantly correlated with structure and distribution of N 2 -fixing microorganisms. Expression of the ammonium transporter was much higher than that of urea transporter subunit A ( urtA ) and ferredoxin-nitrate reductase , while urtA had an increased expression in N-deficient surface water. The predicted ammonium transporter and ammonium assimilation enzymes were most abundant in surface samples while urease and nitrogenase were more abundant in the N-deficient regions. These findings underscore the fact that marine bacteria have evolved diverse N utilization strategies to adapt to different N habitats, and that urea metabolism is of vital ecological importance in N-deficient regimes.

Statistical optimization of medium composition for bacterial cellulose production by Gluconacetobacter hansenii UAC09 using coffee cherry husk extract--an agro-industry waste.

PubMed

Rani, Mahadevaswamy Usha; Rastogi, Navin K; Appaiah, K A Anu

2011-07-01

During the production of grape wine, the formation of thick leathery pellicle/bacterial cellulose (BC) at the airliquid interface was due to the bacterium, which was isolated and identified as Gluconacetobacter hansenii UAC09. Cultural conditions for bacterial cellulose production from G. hansenii UAC09 were optimized by central composite rotatable experimental design. To economize the BC production, coffee cherry husk (CCH) extract and corn steep liquor (CSL) were used as less expensive sources of carbon and nitrogen, respectively. CCH and CSL are byproducts from the coffee processing and starch processing industry, respectively. The interactions between pH (4.5- 8.5), CSL (2-10%), alcohol (0.5-2%), acetic acid (0.5- 2%), and water dilution rate to CCH ratio (1:1 to 1:5) were studied using response surface methodology. The optimum conditions for maximum BC production were pH (6.64), CSL (10%), alcohol (0.5%), acetic acid (1.13%), and water to CCH ratio (1:1). After 2 weeks of fermentation, the amount of BC produced was 6.24 g/l. This yield was comparable to the predicted value of 6.09 g/l. This is the first report on the optimization of the fermentation medium by RSM using CCH extract as the carbon source for BC production by G. hansenii UAC09.

Allochthonous bioaugmentation in ex situ treatment of crude oil-polluted sediments in the presence of an effective degrading indigenous microbiome.

PubMed

Fodelianakis, S; Antoniou, E; Mapelli, F; Magagnini, M; Nikolopoulou, M; Marasco, R; Barbato, M; Tsiola, A; Tsikopoulou, I; Giaccaglia, L; Mahjoubi, M; Jaouani, A; Amer, R; Hussein, E; Al-Horani, F A; Benzha, F; Blaghen, M; Malkawi, H I; Abdel-Fattah, Y; Cherif, A; Daffonchio, D; Kalogerakis, N

2015-04-28

Oil-polluted sediment bioremediation depends on both physicochemical and biological parameters, but the effect of the latter cannot be evaluated without the optimization of the former. We aimed in optimizing the physicochemical parameters related to biodegradation by applying an ex-situ landfarming set-up combined with biostimulation to oil-polluted sediment, in order to determine the added effect of bioaugmentation by four allochthonous oil-degrading bacterial consortia in relation to the degradation efficiency of the indigenous community. We monitored hydrocarbon degradation, sediment ecotoxicity and hydrolytic activity, bacterial population sizes and bacterial community dynamics, characterizing the dominant taxa through time and at each treatment. We observed no significant differences in total degradation, but increased ecotoxicity between the different treatments receiving both biostimulation and bioaugmentation and the biostimulated-only control. Moreover, the added allochthonous bacteria quickly perished and were rarely detected, their addition inducing minimal shifts in community structure although it altered the distribution of the residual hydrocarbons in two treatments. Therefore, we concluded that biodegradation was mostly performed by the autochthonous populations while bioaugmentation, in contrast to biostimulation, did not enhance the remediation process. Our results indicate that when environmental conditions are optimized, the indigenous microbiome at a polluted site will likely outperform any allochthonous consortium. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.

PubMed

Baumschlager, Armin; Aoki, Stephanie K; Khammash, Mustafa

2017-11-17

Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.

Programming stress-induced altruistic death in engineered bacteria

PubMed Central

Tanouchi, Yu; Pai, Anand; Buchler, Nicolas E; You, Lingchong

2012-01-01

Programmed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is ‘altruistic': the killing of some cells can benefit the survivors through release of ‘public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, we determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. We further predicted that altruistic death could generate the ‘Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, we experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. Our findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment. PMID:23169002

[Optimization of labeling and localizing bacterial membrane and nucleus with FM4-64 and Hoechst dyes].

PubMed

Wang, Jing; Han, Yanping; Yang, Ruifu; Zhao, Xingxu

2015-08-04

To observe cell membrane and nucleus in bacteria for subcellular localization. FM4-64 and Hoechst were dyed that can label cell membrane and nucleus, respectively. Both dyes were used to co-stain the membranes and nucleus of eight bacterial strains ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Yersinia pestis, Legionella pneumonia, Vibrio cholerae and Bacillus anthracis). E. coli was dyed with different dye concentrations and times and then observed by confocal fluorescence microscopic imaging. Fluorescence intensity of cell membrane and nucleus is affected by dye concentrations and times. The optimal conditions were determined as follows: staining cell membrane with 20 μg/mL FM4-64 for 1 min and cell nucleus with 20 μg/mL Hoechst for 20 min. Gram-negative bacteria were dyed better than gram-positive bacteria with FM4-64dye. FM4-64 and Hoechst can be used to stain membrane and nucleus in different types of bacteria. Co-staining bacterial membrane and nucleus provides the reference to observe cell structure in prokaryotes for studying subcellular localization.

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

PubMed Central

Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

2010-01-01

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

Hemozoin Regulates iNOS Expression by Modulating the Transcription Factor NF-κB in Macrophages.

PubMed

Ranjan, Ravi; Karpurapu, Manjula; Rani, Asha; Chishti, Athar H; Christman, John W

2016-01-01

Hemozoin (Hz) is released from ruptured erythrocytes during malaria infection caused by Plasmodium sp., in addition the malaria infected individuals are prone to bacterial sepsis. The molecular interactions between Hz, bacterial components and macrophages remains poorly investigated. In this report, we investigated the combinatorial immune-modulatory effects of phagocytosed Hz, Interferon gamma (IFNγ) or lipopolysaccharide (LPS) in macrophages. Macrophages were treated with various concentrations of commercial synthetic Hz, and surprisingly it did not result in inducible nitric oxide synthase (iNOS) expression. However, when macrophages were pretreated with Hz and then challenged with IFNγ or LPS, there was a differential impact on iNOS expression. There was an increase in iNOS expression when macrophages were pre-treated with Hz and subsequently treated with IFNγ when compared to IFNγ alone. Whereas iNOS expression was reduced when Hz phagocytosed macrophages were stimulated with LPS compared to LPS alone. Furthermore, there was an increased activation of NF-κB in Hz phagocytosed macrophages that were challenged with IFNγ. The interaction between Hz and macrophages has an impact on iNOS expression.

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

Improved Escherichia coli Bactofection and Cytotoxicity by Heterologous Expression of Bacteriophage ΦX174 Lysis Gene E.

PubMed

Chung, Tai-Chun; Jones, Charles H; Gollakota, Akhila; Kamal Ahmadi, Mahmoud; Rane, Snehal; Zhang, Guojian; Pfeifer, Blaine A

2015-05-04

Bactofection offers a gene delivery option particularly useful in the context of immune modulation. The bacterial host naturally attracts recognition and cellular uptake by antigen presenting cells (APCs) as the initial step in triggering an immune response. Moreover, depending on the bacterial vector, molecular biology tools are available to influence and/or overcome additional steps and barriers to effective antigen presentation. In this work, molecular engineering was applied using Escherichia coli as a bactofection vector. In particular, the bacteriophage ΦX174 lysis E (LyE) gene was designed for variable expression across strains containing different levels of lysteriolysin O (LLO). The objective was to generate a bacterial vector with improved attenuation and delivery characteristics. The resulting strains exhibited enhanced gene and protein release and inducible cellular death. In addition, the new vectors demonstrated improved gene delivery and cytotoxicity profiles to RAW264.7 macrophage APCs.

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Shi, Xiaoli; Shi, Limei; Liu, Jinlin; Stone, Victoria; Kong, Fanxiang; Kitten, Todd; Xu, Ping

2016-01-01

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation. PMID:26950587

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.

Bacterial antisense RNAs are mainly the product of transcriptional noise

PubMed Central

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

2016-01-01

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. PMID:26973873

Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

PubMed Central

Wilson, James W.; Ramamurthy, Rajee; Porwollik, Steffen; McClelland, Michael; Hammond, Timothy; Allen, Pat; Ott, C. Mark; Pierson, Duane L.; Nickerson, Cheryl A.

2002-01-01

The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 × g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies. PMID:12370447

Serum Lipoproteins Are Critical for Pulmonary Innate Defense against Staphylococcus aureus Quorum Sensing.

PubMed

Manifold-Wheeler, Brett C; Elmore, Bradley O; Triplett, Kathleen D; Castleman, Moriah J; Otto, Michael; Hall, Pamela R

2016-01-01

Hyperlipidemia has been extensively studied in the context of atherosclerosis, whereas the potential health consequences of the opposite extreme, hypolipidemia, remain largely uninvestigated. Circulating lipoproteins are essential carriers of insoluble lipid molecules and are increasingly recognized as innate immune effectors. Importantly, severe hypolipidemia, which may occur with trauma or critical illness, is clinically associated with bacterial pneumonia. To test the hypothesis that circulating lipoproteins are essential for optimal host innate defense in the lung, we used lipoprotein-deficient mice and a mouse model of Staphylococcus aureus pneumonia in which invasive infection requires virulence factor expression controlled by the accessory gene regulator (agr) operon. Activation of agr and subsequent virulence factor expression is inhibited by apolipoprotein B, the structural protein of low-density lipoprotein, which binds and sequesters the secreted agr-signaling peptide (AIP). In this article, we report that lipoprotein deficiency impairs early pulmonary innate defense against S. aureus quorum-sensing-dependent pathogenesis. Specifically, apolipoprotein B levels in the lung early postinfection are significantly reduced with lipoprotein deficiency, coinciding with impaired host control of S. aureus agr-signaling and increased agr-dependent morbidity (weight loss) and inflammation. Given that lipoproteins also inhibit LTA- and LPS-mediated inflammation, these results suggest that hypolipidemia may broadly impact posttrauma pneumonia susceptibility to both Gram-positive and -negative pathogens. Together with previous reports demonstrating that hyperlipidemia also impairs lung innate defense, these results suggest that maintenance of normal serum lipoprotein levels is necessary for optimal host innate defense in the lung. Copyright © 2015 by The American Association of Immunologists, Inc.

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate1[C][OA

PubMed Central

Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P.; Snell, Kristi D.

2011-01-01

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5′ end by the host plant’s psbA coding sequence and at the 3′ end by the host plant’s 3′ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated. PMID:21325565

RiboMaker: computational design of conformation-based riboregulation.

PubMed

Rodrigo, Guillermo; Jaramillo, Alfonso

2014-09-01

The ability to engineer control systems of gene expression is instrumental for synthetic biology. Thus, bioinformatic methods that assist such engineering are appealing because they can guide the sequence design and prevent costly experimental screening. In particular, RNA is an ideal substrate to de novo design regulators of protein expression by following sequence-to-function models. We have implemented a novel algorithm, RiboMaker, aimed at the computational, automated design of bacterial riboregulation. RiboMaker reads the sequence and structure specifications, which codify for a gene regulatory behaviour, and optimizes the sequences of a small regulatory RNA and a 5'-untranslated region for an efficient intermolecular interaction. To this end, it implements an evolutionary design strategy, where random mutations are selected according to a physicochemical model based on free energies. The resulting sequences can then be tested experimentally, providing a new tool for synthetic biology, and also for investigating the riboregulation principles in natural systems. Web server is available at http://ribomaker.jaramillolab.org/. Source code, instructions and examples are freely available for download at http://sourceforge.net/projects/ribomaker/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Comparison of expression and enzymatic properties of Aspergillus oryzae lysine aminopeptidases ApsA and ApsB.

PubMed

Marui, Junichiro; Matsushita-Morita, Mayumi; Tada, Sawaki; Hattori, Ryota; Suzuki, Satoshi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei; Takeuchi, Michio; Kusumoto, Ken-Ichi

2012-08-01

The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.

Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

NASA Astrophysics Data System (ADS)

Zhu, Banghe; Robinson, Holly; Wilganowski, Nathaniel; Nobles, Christopher L.; Sevick-Muraca, Eva; Maresso, Anthony

2012-03-01

B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.

The Novel Bacterial N-Demethylase PdmAB Is Responsible for the Initial Step of N,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

PubMed Central

Gu, Tao; Zhou, Chaoyang; Sørensen, Sebastian R.; Zhang, Ji; He, Jian; Yu, Peiwen; Li, Shunpeng

2013-01-01

The environmental fate of phenylurea herbicides has received considerable attention in recent decades. The microbial metabolism of N,N-dimethyl-substituted phenylurea herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. The pdmAB genes in Sphingobium sp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted phenylurea herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genes pdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity of pdmAB. Without its cognate electron transport components, expression of PdmAB in Escherichia coli, Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp. strain RW1 greatly enhanced the catalytic activity of PdmAB in E. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved in O- or N-demethylation reactions, indicating that PdmAB is a distinct bacterial RO N-demethylase. PMID:24123738

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum.

PubMed

Tomás-Cortázar, Julen; Plaza-Vinuesa, Laura; de Las Rivas, Blanca; Lavín, José Luis; Barriales, Diego; Abecia, Leticia; Mancheño, José Miguel; Aransay, Ana M; Muñoz, Rosario; Anguita, Juan; Rodríguez, Héctor

2018-02-26

Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds. We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanB Fnp ). TanB Fnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanB Fnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanB Fnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanB Fnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanB Fnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications. We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanB Fnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.

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.

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

NASA Technical Reports Server (NTRS)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

PubMed Central

Ojanen, Markus J. T.; Turpeinen, Hannu; Cordova, Zuzet M.; Hammarén, Milka M.; Harjula, Sanna-Kaisa E.; Parikka, Mataleena; Rämet, Mika

2015-01-01

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection. PMID:25624351

Staphylococcus aureus Promotes Smed-PGRP-2/Smed-setd8-1 Methyltransferase Signalling in Planarian Neoblasts to Sensitize Anti-bacterial Gene Responses During Re-infection.

PubMed

Torre, Cedric; Abnave, Prasad; Tsoumtsa, Landry Laure; Mottola, Giovanna; Lepolard, Catherine; Trouplin, Virginie; Gimenez, Gregory; Desrousseaux, Julie; Gempp, Stephanie; Levasseur, Anthony; Padovani, Laetitia; Lemichez, Emmanuel; Ghigo, Eric

2017-06-01

Little is known about how organisms exposed to recurrent infections adapt their innate immune responses. Here, we report that planarians display a form of instructed immunity to primo-infection by Staphylococcus aureus that consists of a transient state of heightened resistance to re-infection that persists for approximately 30days after primo-infection. We established the involvement of stem cell-like neoblasts in this instructed immunity using the complementary approaches of RNA-interference-mediated cell depletion and tissue grafting-mediated gain of function. Mechanistically, primo-infection leads to expression of the peptidoglycan receptor Smed-PGRP-2, which in turn promotes Smed-setd8-1 histone methyltransferase expression and increases levels of lysine methylation in neoblasts. Depletion of neoblasts did not affect S. aureus clearance in primo-infection but, in re-infection, abrogated the heightened elimination of bacteria and reduced Smed-PGRP-2 and Smed-setd8-1 expression. Smed-PGRP-2 and Smed-setd8-1 sensitize animals to heightened expression of Smed-p38 MAPK and Smed-morn2, which are downstream components of anti-bacterial responses. Our study reveals a central role of neoblasts in innate immunity against S. aureus to establish a resistance state facilitating Smed-sted8-1-dependent expression of anti-bacterial genes during re-infection. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of P limitation and molecules from peanut root exudates on pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia sp. S119.

PubMed

Ludueña, Liliana M; Anzuay, Maria S; Magallanes-Noguera, Cynthia; Tonelli, Maria L; Ibañez, Fernando J; Angelini, Jorge G; Fabra, Adriana; McIntosh, Matthew; Taurian, Tania

2017-10-01

The mineral phosphate-solubilizing phenotype in bacteria is attributed predominantly to secretion of gluconic acid produced by oxidation of glucose by the glucose dehydrogenase enzyme and its cofactor, pyrroloquinoline quinone. This study analyzes pqqE gene expression and pqq promoter activity in the native phosphate-solubilizing bacterium Serratia sp S119 growing under P-limitation, and in the presence of root exudates obtained from peanut plants, also growing under P-limitation. Results indicated that Serratia sp. S119 contains a pqq operon composed of six genes (pqqA,B,C,D,E,F) and two promoters, one upstream of pqqA and other between pqqA and pqqB. PqqE gene expression and pqq promoter activity increased under P-limiting growth conditions and not under N-deficient conditions. In the plant-bacteria interaction assay, the activity of the bacterial pqq promoter region varied depending on the concentration and type of root exudates and on the bacterial growth phase. Root exudates from peanut plants growing under P-available and P-limiting conditions showed differences in their composition. It is concluded from this study that the response of Serratia sp. S119 to phosphorus limitation involves an increase in expression of pqq genes, and that molecules exuded by peanut roots modify expression of these phosphate-solubilizing bacterial genes during plant-bacteria interactions. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis.

PubMed

Singh, Vishal; Yeoh, Beng San; Chassaing, Benoit; Zhang, Benyue; Saha, Piu; Xiao, Xia; Awasthi, Deepika; Shashidharamurthy, Rangaiah; Dikshit, Madhu; Gewirtz, Andrew; Vijay-Kumar, Matam

2016-07-01

Lipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD. Lcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. WT and Lcn2 knockout ( Lcn2 KO) mice were analyzed for gut bacterial load, composition by 16S rRNA gene pyrosequencing and, their colitogenic potential by co-housing with Il-10 KO mice. Acute (dextran sodium sulfate) and chronic (IL-10R neutralization and T-cell adoptive transfer) colitis was induced in WT and Lcn2 KO mice with or without antibiotics. Lcn2 expression was dramatically induced upon inflammation and was dependent upon presence of a gut microbiota and MyD88 signaling. Use of bone-marrow chimeric mice revealed non-immune cells are the major contributors of circulating Lcn2. Lcn2 KO mice exhibited elevated levels of entA -expressing gut bacteria burden and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2 KO mice developed highly colitogenic T-cells and exhibited exacerbated colitis upon exposure to DSS or neutralization of IL-10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2 KO mice, via cohousing, resulted in severe colitis in Il-10 KO mice. Lcn2 is a bacterially-induced, MyD88-dependent, protein that play an important role in gut homeostasis and a pivotal role upon challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota.

Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

PubMed

Doerflinger, Sylvie Y; Throop, Andrea L; Herbst-Kralovetz, Melissa M

2014-06-15

Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Artemisia princeps Pamp. Essential oil and its constituents eucalyptol and α-terpineol ameliorate bacterial vaginosis and vulvovaginal candidiasis in mice by inhibiting bacterial growth and NF-κB activation.

PubMed

Trinh, Hien-Trung; Lee, In-Ah; Hyun, Yang-Jin; Kim, Dong-Hyun

2011-12-01

To investigate the inhibitory effects of Artemisia princeps Pamp. (family Asteraceae) essential oil (APEO) and its main constituents against bacterial vaginosis and vulvovaginal candidiasis, their antimicrobial activities against Gardnerella vaginalis and Candida albicans in vitro and their anti-inflammatory effects against G. vaginalis-induced vaginosis and vulvovaginal candidiasis were examined in mice. APEO and its constituents eucalyptol and α-terpineol were found to inhibit microbe growths. α-Terpineol most potently inhibited the growths of G. vaginalis and C. albicans with MIC values of 0.06 and 0.125 % (v/v), respectively. The antimicrobial activity of α-terpineol was found to be comparable to that of clotrimazole. Intravaginal treatment with APEO, eucalyptol, or α-terpineol significantly decreased viable G. vaginalis and C. albicans numbers in the vaginal cavity and myeloperoxidase activity in mouse vaginal tissues compared with controls. These agents also inhibited the expressions of proinflammatory cytokines (IL-1 β, IL-6, TNF- α), COX-2, iNOS, and the activation of NF- κB and increased expression of the anti-inflammatory cytokine IL-10. In addition, they inhibited the expressions of proinflammatory cytokines and the activation of NF- κB in lipopolysaccharide-stimulated peritoneal macrophages, and α-terpineol most potently inhibited the expressions of proinflammatory cytokines and NF- κB activation. Based on these findings, APEO and its constituents, particularly α-terpineol, ameliorate bacterial vaginosis and vulvovaginal candidiasis by inhibiting the growths of vaginal pathogens and the activation of NF- κB. © Georg Thieme Verlag KG Stuttgart · New York.

Isolation of bacterial metabolites as natural inducers for larval settlement in the marine polychaete Hydroides elegans (Haswell).

PubMed

Harder, Tilmann; Lau, Stanley Chun Kwan; Dahms, Hans-Uwe; Qian, Pei-Yuan

2002-10-01

The bacterial component of marine biofilms plays an important role in the induction of larval settlement in the polychaete Hydroides elegans. In this study, we provide experimental evidence that bacterial metabolites comprise the chemical signal for larval settlement. Bacteria were isolated from biofilms, purified and cultured according to standard procedures. Bacterial metabolites were isolated from spent culture broth by chloroform extraction as well as by closed-loop stripping and adsorption of volatile components on surface-modified silica gel. A pronounced biological activity was exclusively observed when concentrated metabolites were adsorbed on activated charcoal. Larvae did not respond to waterbome metabolites when prevented from contacting the bacterial film surface. These results indicate that an association of the chemical signal with a sorbent-like substratum may be an essential cofactor for the expression of biological activity. The functional role of bacterial exopolymers as an adsorptive matrix for larval settlement signals is discussed.

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

DOE PAGES

Peterson, Joseph R.; Thor, ShengShee; Kohler, Lars; ...

2016-11-16

Here, while a few studies on the variations in mRNA expression and half-lives measured under different growth conditions have been used to predict patterns of regulation in bacterial organisms, the extent to which this information can also play a role in defining metabolic phenotypes has yet to be examined systematically. Here we present the first comprehensive study for a model methanogen. As a result, we use expression and half-life data for the methanogen Methanosarcina acetivorans growing on fast- and slow-growth substrates to examine the regulation of its genes. Unlike Escherichia coli where only small shifts in half-lives were observed, wemore » found that most mRNA have significantly longer half-lives for slow growth on acetate compared to fast growth on methanol or trimethylamine. Interestingly, half-life shifts are not uniform across functional classes of enzymes, suggesting the existence of a selective stabilization mechanism for mRNAs. Using the transcriptomics data we determined whether transcription or degradation rate controls the change in transcript abundance. Degradation was found to control abundance for about half of the metabolic genes underscoring its role in regulating metabolism. Genes involved in half of the metabolic reactions were found to be differentially expressed among the substrates suggesting the existence of drastically different metabolic phenotypes that extend beyond just the methanogenesis pathways. By integrating expression data with an updated metabolic model of the organism (iST807) significant differences in pathway flux and production of metabolites were predicted for the three growth substrates. In conclusion, this study provides the first global picture of differential expression and half-lives for a class II methanogen, as well as provides the first evidence in a single organism that drastic genome-wide shifts in RNA half-lives can be modulated by growth substrate. We determined which genes in each metabolic pathway control the flux and classified them as regulated by transcription (e.g. transcription factor) or degradation (e.g. post-transcriptional modification). We found that more than half of genes in metabolism were controlled by degradation. Our results suggest that M. acetivorans employs extensive post-transcriptional regulation to optimize key metabolic steps, and more generally that degradation could play a much greater role in optimizing an organism’s metabolism than previously thought.« less

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

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

Peterson, Joseph R.; Thor, ShengShee; Kohler, Lars

Here, while a few studies on the variations in mRNA expression and half-lives measured under different growth conditions have been used to predict patterns of regulation in bacterial organisms, the extent to which this information can also play a role in defining metabolic phenotypes has yet to be examined systematically. Here we present the first comprehensive study for a model methanogen. As a result, we use expression and half-life data for the methanogen Methanosarcina acetivorans growing on fast- and slow-growth substrates to examine the regulation of its genes. Unlike Escherichia coli where only small shifts in half-lives were observed, wemore » found that most mRNA have significantly longer half-lives for slow growth on acetate compared to fast growth on methanol or trimethylamine. Interestingly, half-life shifts are not uniform across functional classes of enzymes, suggesting the existence of a selective stabilization mechanism for mRNAs. Using the transcriptomics data we determined whether transcription or degradation rate controls the change in transcript abundance. Degradation was found to control abundance for about half of the metabolic genes underscoring its role in regulating metabolism. Genes involved in half of the metabolic reactions were found to be differentially expressed among the substrates suggesting the existence of drastically different metabolic phenotypes that extend beyond just the methanogenesis pathways. By integrating expression data with an updated metabolic model of the organism (iST807) significant differences in pathway flux and production of metabolites were predicted for the three growth substrates. In conclusion, this study provides the first global picture of differential expression and half-lives for a class II methanogen, as well as provides the first evidence in a single organism that drastic genome-wide shifts in RNA half-lives can be modulated by growth substrate. We determined which genes in each metabolic pathway control the flux and classified them as regulated by transcription (e.g. transcription factor) or degradation (e.g. post-transcriptional modification). We found that more than half of genes in metabolism were controlled by degradation. Our results suggest that M. acetivorans employs extensive post-transcriptional regulation to optimize key metabolic steps, and more generally that degradation could play a much greater role in optimizing an organism’s metabolism than previously thought.« less

Visualisation and quantification of intracellular interactions of Neisseria meningitidis and human α-actinin by confocal imaging.

PubMed

Murillo, Isabel; Virji, Mumtaz

2010-10-24

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors(1). This process leads to bacterial invasion of endothelial cells(2). More recently, we observed an interaction of Opc with a 100 kDa protein found in whole cell lysates of human cells(3). We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as α-actinin. As no surface expressed α-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and α-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with α-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

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 RcsC. PMID:22291596

The inheritance of resistance to bacterial leaf spot of lettuce caused by Xanthomonas campestris pv. vitians in three lettuce cultivars

USDA-ARS?s Scientific Manuscript database

Lettuce yields can be reduced by the disease bacterial leaf spot (BLS) caused by the pathogen Xanthomonas campestris pv. vitians (Xcv) and host resistance is the most feasible method to reduce disease losses. The cultivars La Brillante, Pavane, and Little Gem express an incompatible host-pathogen in...

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.

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

Association analysis of bacterial leaf spot resistance and SNP markers derived from expressed sequence tags (ESTs) in lettuce (Lactuca sativa L.)

USDA-ARS?s Scientific Manuscript database

Bacterial leaf spot of lettuce, caused by Xanthomonas campestris pv. vitians, is a devastating disease of lettuce worldwide. Since there are no chemicals available for effective control of the disease, host-plant resistance is highly desirable to protect lettuce production. A total of 179 lettuce ge...

A New Hybrid BFOA-PSO Optimization Technique for Decoupling and Robust Control of Two-Coupled Distillation Column Process.

PubMed

Abdelkarim, Noha; Mohamed, Amr E; El-Garhy, Ahmed M; Dorrah, Hassen T

2016-01-01

The two-coupled distillation column process is a physically complicated system in many aspects. Specifically, the nested interrelationship between system inputs and outputs constitutes one of the significant challenges in system control design. Mostly, such a process is to be decoupled into several input/output pairings (loops), so that a single controller can be assigned for each loop. In the frame of this research, the Brain Emotional Learning Based Intelligent Controller (BELBIC) forms the control structure for each decoupled loop. The paper's main objective is to develop a parameterization technique for decoupling and control schemes, which ensures robust control behavior. In this regard, the novel optimization technique Bacterial Swarm Optimization (BSO) is utilized for the minimization of summation of the integral time-weighted squared errors (ITSEs) for all control loops. This optimization technique constitutes a hybrid between two techniques, which are the Particle Swarm and Bacterial Foraging algorithms. According to the simulation results, this hybridized technique ensures low mathematical burdens and high decoupling and control accuracy. Moreover, the behavior analysis of the proposed BELBIC shows a remarkable improvement in the time domain behavior and robustness over the conventional PID controller.

Spiral bacterial foraging optimization method: Algorithm, evaluation and convergence analysis

NASA Astrophysics Data System (ADS)

Kasaiezadeh, Alireza; Khajepour, Amir; Waslander, Steven L.

2014-04-01

A biologically-inspired algorithm called Spiral Bacterial Foraging Optimization (SBFO) is investigated in this article. SBFO, previously proposed by the same authors, is a multi-agent, gradient-based algorithm that minimizes both the main objective function (local cost) and the distance between each agent and a temporary central point (global cost). A random jump is included normal to the connecting line of each agent to the central point, which produces a vortex around the temporary central point. This random jump is also suitable to cope with premature convergence, which is a feature of swarm-based optimization methods. The most important advantages of this algorithm are as follows: First, this algorithm involves a stochastic type of search with a deterministic convergence. Second, as gradient-based methods are employed, faster convergence is demonstrated over GA, DE, BFO, etc. Third, the algorithm can be implemented in a parallel fashion in order to decentralize large-scale computation. Fourth, the algorithm has a limited number of tunable parameters, and finally SBFO has a strong certainty of convergence which is rare in existing global optimization algorithms. A detailed convergence analysis of SBFO for continuously differentiable objective functions has also been investigated in this article.

A New Hybrid BFOA-PSO Optimization Technique for Decoupling and Robust Control of Two-Coupled Distillation Column Process

PubMed Central

Mohamed, Amr E.; Dorrah, Hassen T.

2016-01-01

The two-coupled distillation column process is a physically complicated system in many aspects. Specifically, the nested interrelationship between system inputs and outputs constitutes one of the significant challenges in system control design. Mostly, such a process is to be decoupled into several input/output pairings (loops), so that a single controller can be assigned for each loop. In the frame of this research, the Brain Emotional Learning Based Intelligent Controller (BELBIC) forms the control structure for each decoupled loop. The paper's main objective is to develop a parameterization technique for decoupling and control schemes, which ensures robust control behavior. In this regard, the novel optimization technique Bacterial Swarm Optimization (BSO) is utilized for the minimization of summation of the integral time-weighted squared errors (ITSEs) for all control loops. This optimization technique constitutes a hybrid between two techniques, which are the Particle Swarm and Bacterial Foraging algorithms. According to the simulation results, this hybridized technique ensures low mathematical burdens and high decoupling and control accuracy. Moreover, the behavior analysis of the proposed BELBIC shows a remarkable improvement in the time domain behavior and robustness over the conventional PID controller. PMID:27807444

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 efflux, the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for industrial purposes. We propose that this novel mechanism for sensing and regulating cysteine levels is a safety valve enabling adaptation to sudden changes in intra- or extracellular cysteine levels in bacteria. Our findings provide important insights into optimizing the production of cysteine and related biomaterials by P. ananatis and also a deep understanding of sulfur/cysteine metabolism and regulation in this plant pathogen and related bacteria. PMID:26883827

Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterization

Treesearch

Ines Pisanelli; Magdalena Kujawa; Oliver Spadiut; Roman Kittl; Petr Halada; Jindrich Volc; Michael D. Mozuch; Philip Kersten; Dietmar Haltrich; Clemens Peterbauer

2009-01-01

The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in...

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 endotoxin LPS in asthma. In addition, there was a TLR 4 expression on CD4(+) T-lymphocytes of normals and to a lesser extent in asthmatics but this TLR 4 expression could not be found on CD8(+) T cells of both groups. In conclusion, there may be an impaired concentration-dependent LPS-induced T(H)1 rather than a T(H)2 response in allergic adult asthmatics compared with normal volunteers. One reason for this could be a reduced TLR 4 expression on CD4(+) T-lymphocytes of asthmatic subjects.

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.

Autodisplay: Development of an Efficacious System for Surface Display of Antigenic Determinants in Salmonella Vaccine Strains

PubMed Central

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B.; Lattemann, Claus T.

2003-01-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp6074-86), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp6074-86 was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp6074-86-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-γ secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains. PMID:12654812

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains.

PubMed

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B; Lattemann, Claus T

2003-04-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp60(74-86)), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp60(74-86) was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp60(74-86)-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-gamma secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains.

Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease.

PubMed

Matsui, Takeshi; Takita, Eiji; Sato, Toshio; Aizawa, Michie; Ki, Misa; Kadoyama, Yumiko; Hirano, Kenji; Kinjo, Satoko; Asao, Hiroshi; Kawamoto, Keiko; Kariya, Haruko; Makino, Sou-Ichi; Hamabata, Takashi; Sawada, Kazutoshi; Kato, Ko

2011-08-01

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E. coli produces Shiga toxin 2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80 mg per 100 g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.

The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma

PubMed Central

Klüter, Tim; Fitschen-Oestern, Stefanie; Lippross, Sebastian; Weuster, Matthias; Pufe, Thomas; Tohidnezhad, Mersedeh; Beyer, Andreas; Seekamp, Andreas; Varoga, Deike

2014-01-01

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction. PMID:25258475

Isolation of bacterial cellulose nanocrystalline from pineapple peel waste: Optimization of acid concentration in the hydrolysis method

NASA Astrophysics Data System (ADS)

Anwar, Budiman; Rosyid, Nurul Huda; Effendi, Devi Bentia; Nandiyanto, Asep Bayu Dani; Mudzakir, Ahmad; Hidayat, Topik

2016-02-01

Isolation of needle-shaped bacterial cellulose nanocrystalline with a diameter of 16-64 nm, a fiber length of 258-806 nm, and a degree of crystallinity of 64% from pineapple peel waste using an acid hydrolysis process was investigated. Experimental showed that selective concentration of acid played important roles in isolating the bacterial cellulose nanocrystalline from the cellulose source. To achieve the successful isolation of bacterial cellulose nanocrystalline, various acid concentrations were tested. To confirm the effect of acid concentration on the successful isolation process, the reaction conditions were fixed at a temperature of 50°C, a hydrolysis time of 30 minutes, and a bacterial cellulose-to-acid ratio of 1:50. Pineapple peel waste was used as a model for a cellulose source because to the best of our knowledge, there is no report on the use of this raw material for producing bacterial cellulose nanocrystalline. In fact, this material can be used as an alternative for ecofriendly and cost-free cellulose sources. Therefore, understanding in how to isolate bacterial cellulose nanocrystalline from pineapple peel waste has the potential for large-scale production of inexpensive cellulose nanocrystalline.

Interactome of E. piscicida and grouper liver proteins reveals strategies of bacterial infection and host immune response.

PubMed

Li, Hui; Zhu, Qing-Feng; Peng, Xuan-Xian; Peng, Bo

2017-01-03

The occurrence of infectious diseases is related to heterogeneous protein interactions between a host and a microbe. Therefore, elucidating the host-pathogen interplay is essential. We previously revealed the protein interactome between Edwardsiella piscicida and fish gill cells, and the present study identified the protein interactome between E. piscicida and E. drummondhayi liver cells. E. drummondhayi liver cells and bacterial pull-down approaches were used to identify E. piscicida outer membrane proteins that bind to liver cells and fish liver cell proteins that interact with bacterial cells, respectively. Eight bacterial proteins and 11 fish proteins were characterized. Heterogeneous protein-protein interactions between these bacterial cells and fish liver cells were investigated through far-Western blotting and co-immunoprecipitation. A network was constructed based on 42 heterogeneous protein-protein interactions between seven bacterial proteins and 10 fish proteins. A comparison of the new interactome with the previously reported interactome showed that four bacterial proteins overlapped, whereas all of the identified fish proteins were new, suggesting a difference between bacterial tricks for evading host immunity and the host strategy for combating bacterial infection. Furthermore, these bacterial proteins were found to regulate the expression of host innate immune-related proteins. These findings indicate that the interactome contributes to bacterial infection and host immunity.

Combined biomarkers discriminate a low likelihood of bacterial infection among surgical intensive care unit patients with suspected sepsis.

PubMed

Kelly, Brendan J; Lautenbach, Ebbing; Nachamkin, Irving; Coffin, Susan E; Gerber, Jeffrey S; Fuchs, Barry D; Garrigan, Charles; Han, Xiaoyan; Bilker, Warren B; Wise, Jacqueleen; Tolomeo, Pam; Han, Jennifer H

2016-05-01

Among surgical intensive care unit (SICU) patients, it is difficult to distinguish bacterial sepsis from other causes of systemic inflammatory response syndrome (SIRS). Biomarkers have proven useful to identify the presence of bacterial infection. We enrolled a prospective cohort of 69 SICU patients with suspected sepsis and assayed the concentrations of 9 biomarkers (α-2 macroglobulin [A2M], C-reactive protein, ferritin, fibrinogen, haptoglobin, procalcitonin [PCT], serum amyloid A, serum amyloid P, and tissue plasminogen activator) at baseline, 24, 48, and 72hours. Forty-two patients (61%) had bacterial sepsis by chart review. A2M concentrations were significantly lower, and PCT concentrations were significantly higher in subjects with bacterial sepsis at 3 of 4 time points. Using optimal cutoff values, the combination of baseline A2M and 72-hour PCT achieved a negative predictive value of 75% (95% confidence interval, 54-96%). The combination of A2M and PCT discriminated bacterial sepsis from other SIRS among SICU patients with suspected sepsis. Copyright © 2016 Elsevier Inc. All rights reserved.

Common themes in microbial pathogenicity revisited.

PubMed Central

Finlay, B B; Falkow, S

1997-01-01

Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics. PMID:9184008

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

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

PubMed Central

Olsen, Katja N.; Budde, Birgitte B.; Siegumfeldt, Henrik; Rechinger, K. Björn; Jakobsen, Mogens; Ingmer, Hanne

2002-01-01

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments. PMID:12147523

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

PubMed Central

Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes.

PubMed

Telford, William G; Shcherbakova, Daria M; Buschke, David; Hawley, Teresa S; Verkhusha, Vladislav V

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.

Bacterial discrimination by Dictyostelid amoebae reveals the complexity of ancient interspecies interactions

PubMed Central

Nasser, Waleed; Santhanam, Balaji; Miranda, Edward Roshan; Parikh, Anup; Juneja, Kavina; Rot, Gregor; Dinh, Chris; Chen, Rui; Zupan, Blaz; Shaulsky, Gad; Kuspa, Adam

2014-01-01

Background Amoebae and bacteria interact within predator/prey and host/pathogen relationships, but the general response of amoeba to bacteria is not well understood. The amoeba Dictyostelium discoideum feeds on, and is colonized by diverse bacterial species including Gram-positive [Gram(+)] and Gram-negative [Gram(−)] bacteria, two major groups of bacteria that differ in structure and macromolecular composition. Results Transcriptional profiling of D. discoideum revealed sets of genes whose expression is enriched in amoebae interacting with different species of bacteria, including sets that appear specific to amoebae interacting with Gram(+), or with Gram(−) bacteria. In a genetic screen utilizing the growth of mutant amoebae on a variety of bacteria as a phenotypic readout, we identified amoebal genes that are only required for growth on Gram(+) bacteria, including one that encodes the cell surface protein gp130, as well as several genes that are only required for growth on Gram(−) bacteria including one that encodes a putative lysozyme, AlyL. These genes are required for parts of the transcriptional response of wild-type amoebae, and this allowed their classification into potential response pathways. Conclusions We have defined genes that are critical for amoebal survival during feeding on Gram(+), or Gram(−), bacteria which we propose form part of a regulatory network that allows D. discoideum to elicit specific cellular responses to different species of bacteria in order to optimize survival. PMID:23664307

Optimization of a bioremediation system of soluble uranium based on the biostimulation of an indigenous bacterial community.

PubMed

Maleke, Maleke; Williams, Peter; Castillo, Julio; Botes, Elsabe; Ojo, Abidemi; DeFlaun, Mary; van Heerden, Esta

2015-06-01

High concentrations of uranium(VI) in the Witwatersrand Basin, South Africa from mining leachate is a serious environmental concern. Treatment systems are often ineffective. Therefore, optimization of a bioremediation system that facilitates the bioreduction of U(VI) based on biostimulation of indigenous bacterial communities can be a viable alternative. Tolerance of the indigenous bacteria to high concentrations of U and the amount of citric acid required for U removal was optimized. Two bioreactor studies which showed effective U(VI) removal more than 99 % from low (0.0037 mg L(-1)) and high (10 mg L(-1)) concentrations of U to below the limit allowed by South African National Standards for drinking water (0.0015 mg L(-1)). The second bioreactor was able to successfully adapt even with increasing levels of U(VI) feed water up to 10 mg L(-1), provided that enough electron donor was available. Molecular biology analyses identified Desulfovibrio sp. and Geobacter sp. among known species, which are known to reduce U(VI). The mineralogical analysis determined that part of the uranium precipitated intracellularly, which meant that the remaining U(VI) was precipitated as U(IV) oxides and TEM-EDS also confirmed this analysis. This was predicted with the geochemical model from the chemical data, which demonstrated that the treated drainage was supersaturated with respect to uraninite > U4O9 > U3O8 > UO2(am). Therefore, the tolerance of the indigenous bacterial community could be optimized to remediate up to 10 mg L(-1), and the system can thus be upscaled and employed for remediation of U(VI) impacted sites.

Evaluating Monitoring Strategies to Detect Precipitation-Induced Microbial Contamination Events in Karstic Springs Used for Drinking Water

PubMed Central

Besmer, Michael D.; Hammes, Frederik; Sigrist, Jürg A.; Ort, Christoph

2017-01-01

Monitoring of microbial drinking water quality is a key component for ensuring safety and understanding risk, but conventional monitoring strategies are typically based on low sampling frequencies (e.g., quarterly or monthly). This is of concern because many drinking water sources, such as karstic springs are often subject to changes in bacterial concentrations on much shorter time scales (e.g., hours to days), for example after precipitation events. Microbial contamination events are crucial from a risk assessment perspective and should therefore be targeted by monitoring strategies to establish both the frequency of their occurrence and the magnitude of bacterial peak concentrations. In this study we used monitoring data from two specific karstic springs. We assessed the performance of conventional monitoring based on historical records and tested a number of alternative strategies based on a high-resolution data set of bacterial concentrations in spring water collected with online flow cytometry (FCM). We quantified the effect of increasing sampling frequency and found that for the specific case studied, at least bi-weekly sampling would be needed to detect precipitation events with a probability of >90%. We then proposed an optimized monitoring strategy with three targeted samples per event, triggered by precipitation measurements. This approach is more effective and efficient than simply increasing overall sampling frequency. It would enable the water utility to (1) analyze any relevant event and (2) limit median underestimation of peak concentrations to approximately 10%. We conclude with a generalized perspective on sampling optimization and argue that the assessment of short-term dynamics causing microbial peak loads initially requires increased sampling/analysis efforts, but can be optimized subsequently to account for limited resources. This offers water utilities and public health authorities systematic ways to evaluate and optimize their current monitoring strategies. PMID:29213255

Evaluating Monitoring Strategies to Detect Precipitation-Induced Microbial Contamination Events in Karstic Springs Used for Drinking Water.

PubMed

Besmer, Michael D; Hammes, Frederik; Sigrist, Jürg A; Ort, Christoph

2017-01-01

Monitoring of microbial drinking water quality is a key component for ensuring safety and understanding risk, but conventional monitoring strategies are typically based on low sampling frequencies (e.g., quarterly or monthly). This is of concern because many drinking water sources, such as karstic springs are often subject to changes in bacterial concentrations on much shorter time scales (e.g., hours to days), for example after precipitation events. Microbial contamination events are crucial from a risk assessment perspective and should therefore be targeted by monitoring strategies to establish both the frequency of their occurrence and the magnitude of bacterial peak concentrations. In this study we used monitoring data from two specific karstic springs. We assessed the performance of conventional monitoring based on historical records and tested a number of alternative strategies based on a high-resolution data set of bacterial concentrations in spring water collected with online flow cytometry (FCM). We quantified the effect of increasing sampling frequency and found that for the specific case studied, at least bi-weekly sampling would be needed to detect precipitation events with a probability of >90%. We then proposed an optimized monitoring strategy with three targeted samples per event, triggered by precipitation measurements. This approach is more effective and efficient than simply increasing overall sampling frequency. It would enable the water utility to (1) analyze any relevant event and (2) limit median underestimation of peak concentrations to approximately 10%. We conclude with a generalized perspective on sampling optimization and argue that the assessment of short-term dynamics causing microbial peak loads initially requires increased sampling/analysis efforts, but can be optimized subsequently to account for limited resources. This offers water utilities and public health authorities systematic ways to evaluate and optimize their current monitoring strategies.

Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

PubMed Central

Fu, Yutao; O'Kelly, Charles; Sieracki, Michael; Distel, Daniel L.

2003-01-01

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells. PMID:14602649

Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure

NASA Astrophysics Data System (ADS)

Hwang, Geelsu; Liu, Yuan; Kim, Dongyeop; Sun, Victor; Aviles-Reyes, Alejandro; Kajfasz, Jessica K.; Lemos, Jose A.; Koo, Hyun

2016-09-01

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

The coral immune response facilitates protection against microbes during tissue regeneration.

PubMed

van de Water, Jeroen A J M; Ainsworth, Tracy D; Leggat, William; Bourne, David G; Willis, Bette L; van Oppen, Madeleine J H

2015-07-01

Increasing physical damage on coral reefs from predation, storms and anthropogenic disturbances highlights the need to understand the impact of injury on the coral immune system. In this study, we examined the regulation of the coral immune response over 10 days following physical trauma artificially inflicted on in situ colonies of the coral Acropora aspera, simultaneously with bacterial colonization of the lesions. Corals responded to injury by increasing the expression of immune system-related genes involved in the Toll-like and NOD-like receptor signalling pathways and the lectin-complement system in three phases (<2, 4 and 10 days post-injury). Phenoloxidase activity was also significantly upregulated in two phases (<3 and 10 days post-injury), as were levels of non-fluorescent chromoprotein. In addition, green fluorescent protein expression was upregulated in response to injury from 4 days post-injury, while cyan fluorescent protein expression was reduced. No shifts in the composition of coral-associated bacterial communities were evident following injury based on 16S rRNA gene amplicon pyrosequencing. Bacteria-specific fluorescence in situ hybridization also showed no evidence of bacterial colonization of the wound or regenerating tissues. Coral tissues showed near-complete regeneration of lesions within 10 days. This study demonstrates that corals exhibit immune responses that support rapid recovery following physical injury, maintain coral microbial homeostasis and prevent bacterial infestation that may compromise coral fitness. © 2015 John Wiley & Sons Ltd.

Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella.

PubMed

Aye, Tin Tin; Shim, Jae-Kyoung; Rhee, In-Koo; Lee, Kyeong-Yeoll

2008-08-01

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in naïve insects and in insects manipulated with bacterial and hormonal treatments.

A virus vector based on Canine Herpesvirus for vaccine applications in canids.

PubMed

Strive, T; Hardy, C M; Wright, J; Reubel, G H

2007-01-31

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.

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

OptSSeq: High-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

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

Ghosh, Indro Neil; Landick, Robert

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate tomore » ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe 2+-dependent AdhB and Zn 2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

OptSSeq: High-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

DOE PAGES

Ghosh, Indro Neil; Landick, Robert

2016-07-16

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate tomore » ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe 2+-dependent AdhB and Zn 2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

Transpositional inactivation of gadW enhances curli production and biofilm formation in Enterohemorrhagic Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been shown to produce variants that either express or are repressed in the expression of curli fimbriae promoting bacterial attachment, aggregation, and biofilm formation. The variant expression of curli fimbriae in some instances could result fr...

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

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

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

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

NASA Astrophysics Data System (ADS)

Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.; Olsen, John C.; Johnson, Larry G.; Yankaskas, James R.; Goldberg, Joanna B.

1996-01-01

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Stable zymomonas mobilis xylose and arabinose fermenting strains

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

2008-04-08

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

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.

Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens

PubMed Central

Martínez, Isidoro; Oliveros, Juan C.; Cuesta, Isabel; de la Barrera, Jorge; Ausina, Vicente; Casals, Cristina; de Lorenzo, Alba; García, Ernesto; García-Fojeda, Belén; Garmendia, Junkal; González-Nicolau, Mar; Lacoma, Alicia; Menéndez, Margarita; Moranta, David; Nieto, Amelia; Ortín, Juan; Pérez-González, Alicia; Prat, Cristina; Ramos-Sevillano, Elisa; Regueiro, Verónica; Rodriguez-Frandsen, Ariel; Solís, Dolores; Yuste, José; Bengoechea, José A.; Melero, José A.

2017-01-01

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here. PMID:28298903

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.

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

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.

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.

Relationship between bacterial diversity and environmental parameters during composting of different raw materials.

PubMed

Wang, Xueqin; Cui, Hongyang; Shi, Jianhong; Zhao, Xinyu; Zhao, Yue; Wei, Zimin

2015-12-01

The aim of this study was to compare the bacterial structure of seven different composts. The primary environmental factors affecting bacterial species were identified, and a strategy to enhance the abundance of uncultured bacteria through controlling relevant environmental parameters was proposed. The results showed that the physical-chemical parameters of each different pile changed in its own manner during composting, which affected the structure and succession of bacteria in different ways. DGGE profiles showed that there were 10 prominent species during composting. Among them, four species existed in all compost types, two species existed in several piles and four species were detected in a single material. Redundancy analysis results showed that bacterial species compositions were significantly influenced by C/N and moisture (p<0.05). The optimal range of C/N was 14-27. Based on these results, the primary environmental factors affecting a certain species were further identified as a potential control of bacterial diversity. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Kinetics of Coinfection with Influenza A Virus and Streptococcus pneumoniae

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

Smith, Amber M.; Adler, Frederick R.; Ribeiro, Ruy M.

Secondary bacterial infections are a leading cause of illness and death during epidemic and pandemic influenza. Experimental studies suggest a lethal synergism between influenza and certain bacteria, particularly Streptococcus pneumoniae, but the precise processes involved are unclear. In this paper, to address the mechanisms and determine the influences of pathogen dose and strain on disease, we infected groups of mice with either the H1N1 subtype influenza A virus A/Puerto Rico/8/34 (PR8) or a version expressing the 1918 PB1-F2 protein (PR8-PB1-F2(1918)), followed seven days later with one of two S. pneumoniae strains, type 2 D39 or type 3 A66.1. We determinedmore » that, following bacterial infection, viral titers initially rebound and then decline slowly. Bacterial titers rapidly rise to high levels and remain elevated. We used a kinetic model to explore the coupled interactions and study the dominant controlling mechanisms. We hypothesize that viral titers rebound in the presence of bacteria due to enhanced viral release from infected cells, and that bacterial titers increase due to alveolar macrophage impairment. Dynamics are affected by initial bacterial dose but not by the expression of the influenza 1918 PB1-F2 protein. Finally, our model provides a framework to investigate pathogen interaction during coinfections and to uncover dynamical differences based on inoculum size and strain.« less

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.

Kinetics of Coinfection with Influenza A Virus and Streptococcus pneumoniae

DOE PAGES

Smith, Amber M.; Adler, Frederick R.; Ribeiro, Ruy M.; ...

2013-03-21

Secondary bacterial infections are a leading cause of illness and death during epidemic and pandemic influenza. Experimental studies suggest a lethal synergism between influenza and certain bacteria, particularly Streptococcus pneumoniae, but the precise processes involved are unclear. In this paper, to address the mechanisms and determine the influences of pathogen dose and strain on disease, we infected groups of mice with either the H1N1 subtype influenza A virus A/Puerto Rico/8/34 (PR8) or a version expressing the 1918 PB1-F2 protein (PR8-PB1-F2(1918)), followed seven days later with one of two S. pneumoniae strains, type 2 D39 or type 3 A66.1. We determinedmore » that, following bacterial infection, viral titers initially rebound and then decline slowly. Bacterial titers rapidly rise to high levels and remain elevated. We used a kinetic model to explore the coupled interactions and study the dominant controlling mechanisms. We hypothesize that viral titers rebound in the presence of bacteria due to enhanced viral release from infected cells, and that bacterial titers increase due to alveolar macrophage impairment. Dynamics are affected by initial bacterial dose but not by the expression of the influenza 1918 PB1-F2 protein. Finally, our model provides a framework to investigate pathogen interaction during coinfections and to uncover dynamical differences based on inoculum size and strain.« less

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

Analysis of bacterial fatty acids by flow modulated comprehensive two-dimensional gas chromatography with parallel flame ionization detector/mass spectrometry.

PubMed

Gu, Qun; David, Frank; Lynen, Frédéric; Rumpel, Klaus; Xu, Guowang; De Vos, Paul; Sandra, Pat

2010-06-25

Comprehensive two-dimensional gas chromatography (GCxGC) offers an interesting tool for profiling bacterial fatty acids. Flow modulated GCxGC using a commercially available system was evaluated, different parameters such as column flows and modulation time were optimized. The method was tested on bacterial fatty acid methyl esters (BAMEs) from Stenotrophomonas maltophilia LMG 958T by using parallel flame ionization detector (FID)/mass spectrometry (MS). The results are compared to data obtained using a thermal modulated GCxGC system. The data show that flow modulated GCxGC-FID/MS method can be applied in a routine environment and offers interesting perspectives for chemotaxonomy of bacteria.

The Synthetic Peroxisome Proliferator-Activated Receptor-γ Agonist Ciglitazone Attenuates Neuroinflammation and Accelerates Encapsulation in Bacterial Brain Abscesses1

PubMed Central

Kielian, Tammy; Md. Syed, Mohsin; Liu, Shuliang; Phulwani, Nirmal K.; Phillips, Napoleon; Wagoner, Gail; Drew, Paul D.; Esen, Nilufer

2008-01-01

Brain abscesses result from a pyogenic parenchymal infection commonly initiated by Gram-positive bacteria such as Staphylococcus aureus. Although the host immune response elicited following infection is essential for effective bacterial containment, this response also contributes to the significant loss of brain parenchyma by necrosis that may be reduced by modulating the inflammatory response. Ciglitazone, a PPAR-γ agonist with anti-inflammatory properties, was evaluated for its ability to influence the course of brain abscess development when treatment was initiated 3 days following infection. Interestingly, abscess-associated bacterial burdens were significantly lower following ciglitazone administration, which could be explained, in part, by the finding that ciglitazone enhanced S. aureus phagocytosis by microglia. In addition, ciglitazone attenuated the expression of select inflammatory mediators during brain abscess development including inducible NO synthase, TNF-α, IL-1β, CXCL2, and CCL3. Unexpectedly, ciglitazone also accelerated brain abscess encapsulation, which was typified by the heightened expression of fibronectin and α-smooth muscle actin-positive myofibroblasts. Collectively, through its ability to attenuate excessive inflammation and accelerate abscess encapsulation, ciglitazone may effectively sequester brain abscesses and limit bacterial dissemination. PMID:18354226

Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood.

PubMed

Askarian, Fatemeh; Uchiyama, Satoshi; Valderrama, J Andrés; Ajayi, Clement; Sollid, Johanna U E; van Sorge, Nina M; Nizet, Victor; van Strijp, Jos A G; Johannessen, Mona

2017-01-01

Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. Copyright © 2016 Askarian et al.

Toll-like receptor 22 of gilthead seabream, Sparus aurata: molecular cloning, expression profiles and post-transcriptional regulation.

PubMed

Muñoz, Iciar; Sepulcre, María Pilar; Meseguer, José; Mulero, Victoriano

2014-05-01

TLR22 is a fish-specific TLR that recognizes dsRNAs. In the present study, a TLR22 homologue gene from gilthead seabream (sbTLR22) was identified and characterized. The full coding sequence contained a single open-reading frame of 2895 nucleotides encoding a predicted protein of 964 amino acids in length. Its 3'-UTR was relatively long, 1380 nucleotides, and contained three AU-rich sequences frequently associated with mRNA instability. Functional studies showed that the sbTLR22 transcript had a short half-life, although the three AU-rich sequences in its 3'-UTR did not seem to be related with this fact. The sbTLR22 was highly expressed in the spleen, thymus and gills of healthy fish. After Vibrio anguillarum infection, the mRNA levels of sbTLR22 increased greatly in head kidney, blood and peritoneal exudate, but were only moderately induced in spleen and liver, suggesting the involvement of sbTLR22 in the immune response against bacterial infections. In addition, acidophilic granulocytes and macrophages, both considered professional phagocytes in seabream, displayed cell-type-specific sbTLR22 expression profiles when stimulated with different pathogen-associated molecular patterns (PAMPs). Although acidophilic granulocytes expressed sbTLR22, polyinosinic:polycytidylic acid (poly I:C) was unable to up-regulate the expression of this receptor. In contrast, poly I:C induced the expression of sbTLR22 in macrophages, in a process that was partially endosome-dependent. Taken together, our results suggest that sbTLR22 is involved in bacterial infection and might sense bacterial PAMPs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

PubMed Central

Chen, Jiandong

2016-01-01

ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells.

PubMed

Kim, Youngsook; Jo, Ah-ram; Jang, Da Hyun; Cho, Yong-Joon; Chun, Jongsik; Min, Byung-Moo; Choi, Youngnim

2012-07-01

Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.

Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease

PubMed Central

Hu, Yang; Zhang, Junli; Jia, Hongge; Sosso, Davide; Li, Ting; Frommer, Wolf B.; Yang, Bing; White, Frank F.; Wang, Nian; Jones, Jeffrey B.

2014-01-01

Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccAw, induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations. PMID:24474801

Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease.

PubMed

Hu, Yang; Zhang, Junli; Jia, Hongge; Sosso, Davide; Li, Ting; Frommer, Wolf B; Yang, Bing; White, Frank F; Wang, Nian; Jones, Jeffrey B

2014-01-28

Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccA(w), induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations.

Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

PubMed Central

Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.

2012-01-01

Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment. PMID:22247133

Information transmission in microbial and fungal communication: from classical to quantum.

PubMed

Majumdar, Sarangam; Pal, Sukla

2018-06-01

Microbes have their own communication systems. Secretion and reception of chemical signaling molecules and ion-channels mediated electrical signaling mechanism are yet observed two special ways of information transmission in microbial community. In this article, we address the aspects of various crucial machineries which set the backbone of microbial cell-to-cell communication process such as quorum sensing mechanism (bacterial and fungal), quorum sensing regulated biofilm formation, gene expression, virulence, swarming, quorum quenching, role of noise in quorum sensing, mathematical models (therapy model, evolutionary model, molecular mechanism model and many more), synthetic bacterial communication, bacterial ion-channels, bacterial nanowires and electrical communication. In particular, we highlight bacterial collective behavior with classical and quantum mechanical approaches (including quantum information). Moreover, we shed a new light to introduce the concept of quantum synthetic biology and possible cellular quantum Turing test.

Lipopolysaccharide and Lipoteichoic Acid Virulence Deactivation by Stannous Fluoride.

PubMed

Haught, Chris; Xie, Sancai; Circello, Ben; Tansky, Cheryl S; Khambe, Deepa; Klukowska, Malgorzata; Huggins, Tom; White, Donald J

2016-09-01

Oral bacterial pathogens promote gingivitis and periodontal disease. Bacterial endotoxins, also known as lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs), are known to enhance bacterial pathogenicity through binding with Toll-like receptors (TLRs), a group of pattern recognition receptors critical to the activation of innate immunity, that are expressed on host cells. Both LPS and LTA contain lipophilic domains and anionic charges that may be susceptible to reactivity with stannous fluoride, a commonly used ingredient clinically proven for the treatment and prevention of gingivitis. This study examined the effects of stannous fluoride on Toll-like receptor activation in response to bacterially derived LPS and LTA in select cell lines and secretion of inflammatory cytokines from human primary peripheral monocytes likewise exposed to LPS. TLR4 and TLR2 transfected HEK293 cells and THP1-Dual™ cells were exposed to bacterial LPS and LTA in the presence of increasing concentrations of stannous fluoride. Gene expression was assessed by measurement of secreted embryonic alkaline phosphatase (SEAP) reporter gene for HEK293 cells and SEAP and luciferase for THP-1 cells. Cell viability was confirmed using PrestoBlue. Human primary monocytes were treated with LPS with various concentrations of supplemented stannous fluoride, and cytokine expression was directly measured. Stannous fluoride inhibited gene expression response of TLR4 and TLR2 in HEK293 cells and THP1-Dual™ cells in a dose response fashion, producing complete inhibition at micromolar concentrations. The addition of stannous fluoride suppressed production of TNF-a, IFN-g, IL12p70, IL10, IL-1b, IL2, and IL-6, and also increased secretion of Il-8 in dose response fashion. Viability assays confirmed no effects of LPS or stannous fluoride on viability of HEK293, THP-1, and primary human monocytes. These results support the potential for stannous fluoride to provide clinical gingivitis benefits by directly decreasing the pathogenicity of plaque biofilms by blocking reactivity of LPS and LTA ligands with tissue receptors associated with inflammation. These learnings may influence recommendations for patients at risk for plaque-related diseases.

A Rice CPYC-Type Glutaredoxin OsGRX20 in Protection against Bacterial Blight, Methyl Viologen and Salt Stresses.

PubMed

Ning, Xi; Sun, Yao; Wang, Changchun; Zhang, Weilin; Sun, Meihao; Hu, Haitao; Liu, Jianzhong; Yang, Ling

2018-01-01

Glutaredoxins (GRXs) belong to the antioxidants involved in the cellular stress responses. In spite of the identification 48 GRX genes in rice genomes, the biological functions of most of them remain unknown. Especially, the biological roles of members of GRX family in disease resistance are still lacking. Our proteomic analysis found that OsGRX20 increased by 2.7-fold after infection by bacterial blight. In this study, we isolated and characterized the full-length nucleotide sequences of the rice OsGRX20 gene, which encodes a GRX family protein with CPFC active site of CPYC-type class. OsGRX20 protein was localized in nucleus and cytosol, and its transcripts were expressed predominantly in leaves. Several stress- and hormone-related motifs putatively acting as regulatory elements were found in the OsGRX20 promoter. Real-time quantitative PCR analysis indicated that OsGRX20 was expressed at a significantly higher level in leaves of a resistant or tolerant rice genotype, Yongjing 50A, than in a sensitive genotype, Xiushui 11, exposed to bacterial blight, methyl viologen, heat, and cold. Its expression could be induced by salt, PEG-6000, 2,4-D, salicylic acid, jasmonic acid, and abscisic acid treatments in Yongjing 50A. Overexpression of OsGRX20 in rice Xiushui 11 significantly enhanced its resistance to bacterial blight attack, and tolerance to methyl viologen and salt stresses. In contrast, interference of OsGRX20 in Yongjing 50A led to increased susceptibility to bacterial blight, methyl viologen and salt stresses. OsGRX20 restrained accumulation of superoxide radicals in aerial tissue during methyl viologen treatment. Consistently, alterations in OsGRX20 expression affect the ascorbate/dehydroascorbate ratio and the abundance of transcripts encoding four reactive oxygen species scavenging enzymes after methyl viologen-induced stress. Our results demonstrate that OsGRX20 functioned as a positive regulator in rice tolerance to multiple stresses, which may be of significant use in the genetic improvement of rice resistance.

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 The Society for Applied Microbiology.

Intestinal alkaline phosphatase deficiency leads to dysbiosis and bacterial translocation in the newborn intestine.

PubMed

Fawley, Jason; Koehler, Shannon; Cabrera, Susan; Lam, Vy; Fredrich, Katherine; Hessner, Martin; Salzman, Nita; Gourlay, David

2017-10-01

Intestinal alkaline phosphatase (IAP) has been shown to help maintain intestinal homeostasis. Decreased expression of IAP has been linked with pediatric intestinal diseases associated with bacterial overgrowth and subsequent inflammation. We hypothesize that the absence of IAP leads to dysbiosis, with increased inflammation and permeability of the newborn intestine. Sprague-Dawley heterozygote IAP cross-matches were bred. Pups were dam fed ad lib and euthanized at weaning. The microbiotas of terminal ileum (TI) and colon was determined by quantitative real-time polymerase chain reaction (qRT-PCR) of subphylum-specific bacterial 16S ribosomal RNA. RT-PCR was performed on TI for inflammatory cytokines. Intestinal permeability was quantified by fluorescein isothiocyanate-dextran permeability and bacterial translocation by qRT-PCR for bacterial 16S ribosomal RNA in mesenteric lymph nodes. Statistical analysis was done by chi-square analysis. All three genotypes had similar concentrations of bacteria in the TI and colon. However, IAP knockout (IAP-KO) had significantly decreased diversity of bacterial species in their colonic stool compared with heterozygous and wild-type (WT). IAP-KO pups had a nonstatistically significant 3.9-fold increased inducible nitric oxide synthase messenger RNA expression compared with WT (IAP-KO, 3.92 ± 1.36; WT, 1.0 ± 0.27; P = 0.03). IAP-KO also had significantly increased bacterial translocation to mesenteric lymph nodes occurred in IAP-KO (IAP-KO, 7625 RFU/g ± 3469; WT, 4957 RFU/g ± 1552; P = 0.04). Furthermore, IAP-KO had increased permeability (IAP-KO, 0.297 mg/mL ± 0.2; WT, 0.189 mg/mL ± 0.15 P = 0.07), but was not statistically significant. Deficiency of IAP in the newborn intestine is associated with dysbiosis and increased inflammation, permeability, and bacterial translocation. Copyright © 2017 Elsevier Inc. All rights reserved.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

PubMed

Liu, Cunbao; Yang, Xu; Yao, Yufeng; Huang, Weiwei; Sun, Wenjia; Ma, Yanbing

2014-05-01

Two versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.