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Sample records for bacterial structured rnas

  1. Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis.

    PubMed

    Weinberg, Zasha; Perreault, Jonathan; Meyer, Michelle M; Breaker, Ronald R

    2009-12-03

    Estimates of the total number of bacterial species indicate that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins and RNAs. Bioinformatics searches of genomic DNA from bacteria commonly identify new noncoding RNAs (ncRNAs) such as riboswitches. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, indicating that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.

  2. Bacterial transfer RNAs

    PubMed Central

    Shepherd, Jennifer; Ibba, Michael

    2015-01-01

    Transfer RNA is an essential adapter molecule that is found across all three domains of life. The primary role of transfer RNA resides in its critical involvement in the accurate translation of messenger RNA codons during protein synthesis and, therefore, ultimately in the determination of cellular gene expression. This review aims to bring together the results of intensive investigations into the synthesis, maturation, modification, aminoacylation, editing and recycling of bacterial transfer RNAs. Codon recognition at the ribosome as well as the ever-increasing number of alternative roles for transfer RNA outside of translation will be discussed in the specific context of bacterial cells. PMID:25796611

  3. Modulation of Host miRNAs by Intracellular Bacterial Pathogens

    PubMed Central

    Das, Kishore; Garnica, Omar; Dhandayuthapani, Subramanian

    2016-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein coding genes of viruses and eukaryotes at the post-transcriptional level. The eukaryotic genes regulated by miRNAs include those whose products are critical for biological processes such as cell proliferation, metabolic pathways, immune response, and development. It is now increasingly recognized that modulation of miRNAs associated with biological processes is one of the strategies adopted by bacterial pathogens to survive inside host cells. In this review, we present an overview of the recent findings on alterations of miRNAs in the host cells by facultative intracellular bacterial pathogens. In addition, we discuss how the altered miRNAs help in the survival of these pathogens in the intracellular environment. PMID:27536558

  4. 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

  5. Comparative genomics boosts target prediction for bacterial small RNAs.

    PubMed

    Wright, Patrick R; Richter, Andreas S; Papenfort, Kai; Mann, Martin; Vogel, Jörg; Hess, Wolfgang R; Backofen, Rolf; Georg, Jens

    2013-09-10

    Small RNAs (sRNAs) constitute a large and heterogeneous class of bacterial gene expression regulators. Much like eukaryotic microRNAs, these sRNAs typically target multiple mRNAs through short seed pairing, thereby acting as global posttranscriptional regulators. In some bacteria, evidence for hundreds to possibly more than 1,000 different sRNAs has been obtained by transcriptome sequencing. However, the experimental identification of possible targets and, therefore, their confirmation as functional regulators of gene expression has remained laborious. Here, we present a strategy that integrates phylogenetic information to predict sRNA targets at the genomic scale and reconstructs regulatory networks upon functional enrichment and network analysis (CopraRNA, for Comparative Prediction Algorithm for sRNA Targets). Furthermore, CopraRNA precisely predicts the sRNA domains for target recognition and interaction. When applied to several model sRNAs, CopraRNA revealed additional targets and functions for the sRNAs CyaR, FnrS, RybB, RyhB, SgrS, and Spot42. Moreover, the mRNAs gdhA, lrp, marA, nagZ, ptsI, sdhA, and yobF-cspC were suggested as regulatory hubs targeted by up to seven different sRNAs. The verification of many previously undetected targets by CopraRNA, even for extensively investigated sRNAs, demonstrates its advantages and shows that CopraRNA-based analyses can compete with experimental target prediction approaches. A Web interface allows high-confidence target prediction and efficient classification of bacterial sRNAs.

  6. Comparative genomics boosts target prediction for bacterial small RNAs

    PubMed Central

    Wright, Patrick R.; Richter, Andreas S.; Papenfort, Kai; Mann, Martin; Vogel, Jörg; Hess, Wolfgang R.; Backofen, Rolf; Georg, Jens

    2013-01-01

    Small RNAs (sRNAs) constitute a large and heterogeneous class of bacterial gene expression regulators. Much like eukaryotic microRNAs, these sRNAs typically target multiple mRNAs through short seed pairing, thereby acting as global posttranscriptional regulators. In some bacteria, evidence for hundreds to possibly more than 1,000 different sRNAs has been obtained by transcriptome sequencing. However, the experimental identification of possible targets and, therefore, their confirmation as functional regulators of gene expression has remained laborious. Here, we present a strategy that integrates phylogenetic information to predict sRNA targets at the genomic scale and reconstructs regulatory networks upon functional enrichment and network analysis (CopraRNA, for Comparative Prediction Algorithm for sRNA Targets). Furthermore, CopraRNA precisely predicts the sRNA domains for target recognition and interaction. When applied to several model sRNAs, CopraRNA revealed additional targets and functions for the sRNAs CyaR, FnrS, RybB, RyhB, SgrS, and Spot42. Moreover, the mRNAs gdhA, lrp, marA, nagZ, ptsI, sdhA, and yobF-cspC were suggested as regulatory hubs targeted by up to seven different sRNAs. The verification of many previously undetected targets by CopraRNA, even for extensively investigated sRNAs, demonstrates its advantages and shows that CopraRNA-based analyses can compete with experimental target prediction approaches. A Web interface allows high-confidence target prediction and efficient classification of bacterial sRNAs. PMID:23980183

  7. Transfer RNAs with novel cloverleaf structures

    DOE PAGES

    Mukai, Takahito; Vargas-Rodriguez, Oscar; Englert, Markus; ...

    2016-10-05

    We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter Tstem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function asmore » missense and nonsense suppressor tRNAs and/or regulatory noncod ing RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species inEscherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.« less

  8. Transfer RNAs with novel cloverleaf structures

    SciTech Connect

    Mukai, Takahito; Vargas-Rodriguez, Oscar; Englert, Markus; Tripp, H. James; Ivanova, Natalia N.; Rubin, Edward M.; Kyrpides, Nikos C.; Söll, Dieter

    2016-10-05

    We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter Tstem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncod ing RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species inEscherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

  9. Prediction of bacterial small RNAs in the RsmA (CsrA) and ToxT pathways: a machine learning approach.

    PubMed

    Fakhry, Carl Tony; Kulkarni, Prajna; Chen, Ping; Kulkarni, Rahul; Zarringhalam, Kourosh

    2017-08-22

    Small RNAs (sRNAs) constitute an important class of post-transcriptional regulators that control critical cellular processes in bacteria. Recent research using high-throughput transcriptomic approaches has led to a dramatic increase in the discovery of bacterial sRNAs. However, it is generally believed that the currently identified sRNAs constitute a limited subset of the bacterial sRNA repertoire. In several cases, sRNAs belonging to a specific class are already known and the challenge is to identify additional sRNAs belonging to the same class. In such cases, machine-learning approaches can be used to predict novel sRNAs in a given class. In this work, we develop novel bioinformatics approaches that integrate sequence and structure-based features to train machine-learning models for the discovery of bacterial sRNAs. We show that features derived from recurrent structural motifs in the ensemble of low energy secondary structures can distinguish the RNA classes with high accuracy. We apply this approach to predict new members in two broad classes of bacterial small RNAs: 1) sRNAs that bind to the RNA-binding protein RsmA/CsrA in diverse bacterial species and 2) sRNAs regulated by the master regulator of virulence, ToxT, in Vibrio cholerae. The involvement of sRNAs in bacterial adaptation to changing environments is an increasingly recurring theme in current research in microbiology. It is likely that future research, combining experimental and computational approaches, will discover many more examples of sRNAs as components of critical regulatory pathways in bacteria. We have developed a novel approach for prediction of small RNA regulators in important bacterial pathways. This approach can be applied to specific classes of sRNAs for which several members have been identified and the challenge is to identify additional sRNAs.

  10. 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

  11. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    PubMed

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  12. Structure and Gene-Silencing Mechanisms of Small Noncoding RNAs

    NASA Astrophysics Data System (ADS)

    Chu, Chia-Ying; Rana, Tariq M.

    Small (19-31-nucleotides) noncoding RNAs were identified in the past 10 years for their distinct function in gene silencing. The best known gene-silencing phenomenon, RNA interference (RNAi), is triggered in a sequence-specific manner by endogenously produced or exogenously introduced small doubled-stranded RNAs. As knowledge of the structure and function of the RNAi machinery has expanded, this phenomenon has become a powerful tool for biochemical research; it has enormous potential for therapeutics. This chapter summarizes significant aspects of three major classes of small noncoding, regulatory RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Here, we focus on the biogenesis of these small RNAs, their structural features and coupled effectors as well as the mechanisms of each small regulatory RNA pathway which reveal fascinating ways by which gene silencing is controlled and fine-tuned at an epigenetic level.

  13. A Long Journey Ahead: Long Non-coding RNAs in Bacterial Infections

    PubMed Central

    zur Bruegge, Jennifer; Einspanier, Ralf; Sharbati, Soroush

    2017-01-01

    Bacterial pathogens have coevolved with their hosts and acquired strategies to circumvent defense mechanisms of host cells. It was shown that bacteria interfere with the expression of mammalian microRNAs to modify immune signaling, autophagy, or the apoptotic machinery. Recently, a new class of regulatory RNAs, long non-coding RNAs (lncRNAs), was reported to have a pivotal role in the regulation of eukaryotic gene expression. A growing body of literature reports on specific involvement of lncRNAs in the host cell response toward bacterial infections. This mini review summarizes recent data that focuses on lncRNA function in host cells during bacterial infection and provides a perspective where future research in this regard may be going. PMID:28401065

  14. Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaea, and their metagenomes

    PubMed Central

    2010-01-01

    Background Structured noncoding RNAs perform many functions that are essential for protein synthesis, RNA processing, and gene regulation. Structured RNAs can be detected by comparative genomics, in which homologous sequences are identified and inspected for mutations that conserve RNA secondary structure. Results By applying a comparative genomics-based approach to genome and metagenome sequences from bacteria and archaea, we identified 104 candidate structured RNAs and inferred putative functions for many of these. Twelve candidate metabolite-binding RNAs were identified, three of which were validated, including one reported herein that binds the coenzyme S-adenosylmethionine. Newly identified cis-regulatory RNAs are implicated in photosynthesis or nitrogen regulation in cyanobacteria, purine and one-carbon metabolism, stomach infection by Helicobacter, and many other physiological processes. A candidate riboswitch termed crcB is represented in both bacteria and archaea. Another RNA motif may control gene expression from 3'-untranslated regions of mRNAs, which is unusual for bacteria. Many noncoding RNAs that likely act in trans are also revealed, and several of the noncoding RNA candidates are found mostly or exclusively in metagenome DNA sequences. Conclusions This work greatly expands the variety of highly structured noncoding RNAs known to exist in bacteria and archaea and provides a starting point for biochemical and genetic studies needed to validate their biologic functions. Given the sustained rate of RNA discovery over several similar projects, we expect that far more structured RNAs remain to be discovered from bacterial and archaeal organisms. PMID:20230605

  15. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs.

    PubMed

    Cahová, Hana; Winz, Marie-Luise; Höfer, Katharina; Nübel, Gabriele; Jäschke, Andres

    2015-03-19

    A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

  16. Structure Prediction: New Insights into Decrypting Long Noncoding RNAs

    PubMed Central

    Yan, Kun; Arfat, Yasir; Li, Dijie; Zhao, Fan; Chen, Zhihao; Yin, Chong; Sun, Yulong; Hu, Lifang; Yang, Tuanmin; Qian, Airong

    2016-01-01

    Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological functions such as dosage compensation, chromatin regulation, genomic imprinting, and nuclear organization. However, understanding the wide range of functions of lncRNAs related to various processes of cellular networks remains a great experimental challenge. Structural versatility is critical for RNAs to perform various functions and provides new insights into probing the functions of lncRNAs. In recent years, the computational method of RNA structure prediction has been developed to analyze the structure of lncRNAs. This novel methodology has provided basic but indispensable information for the rapid, large-scale and in-depth research of lncRNAs. This review focuses on mainstream RNA structure prediction methods at the secondary and tertiary levels to offer an additional approach to investigating the functions of lncRNAs. PMID:26805815

  17. Enumerating secondary structures and structural moieties for circular RNAs.

    PubMed

    Cuesta, Jose A; Manrubia, Susanna

    2017-04-21

    A quantitative characterization of the relationship between molecular sequence and structure is essential to improve our understanding of how function emerges. This particular genotype-phenotype map has been often studied in the context of RNA sequences, with the folded configurations standing as a proxy for the phenotype. Here, we count the secondary structures of circular RNAs of length n and calculate the asymptotic distributions of different structural moieties, such as stems or hairpin loops, by means of symbolic combinatorics. Circular RNAs differ in essential ways from their linear counterparts. From the mathematical viewpoint, the enumeration of the corresponding secondary structures demands the use of combinatorial techniques additional to those used for linear RNAs. The asymptotic number of secondary structures for circular RNAs grows as a(n)n(-5/2), with a depending on particular constraints applied to the secondary structure. As it occurs with linear RNA, the abundance of any structural moiety is normally distributed in the limit n→∞, with a mean and a variance that increase linearly with n. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. microRNAs Databases: Developmental Methodologies, Structural and Functional Annotations.

    PubMed

    Singh, Nagendra Kumar

    2017-09-01

    microRNA (miRNA) is an endogenous and evolutionary conserved non-coding RNA, involved in post-transcriptional process as gene repressor and mRNA cleavage through RNA-induced silencing complex (RISC) formation. In RISC, miRNA binds in complementary base pair with targeted mRNA along with Argonaut proteins complex, causes gene repression or endonucleolytic cleavage of mRNAs and results in many diseases and syndromes. After the discovery of miRNA lin-4 and let-7, subsequently large numbers of miRNAs were discovered by low-throughput and high-throughput experimental techniques along with computational process in various biological and metabolic processes. The miRNAs are important non-coding RNA for understanding the complex biological phenomena of organism because it controls the gene regulation. This paper reviews miRNA databases with structural and functional annotations developed by various researchers. These databases contain structural and functional information of animal, plant and virus miRNAs including miRNAs-associated diseases, stress resistance in plant, miRNAs take part in various biological processes, effect of miRNAs interaction on drugs and environment, effect of variance on miRNAs, miRNAs gene expression analysis, sequence of miRNAs, structure of miRNAs. This review focuses on the developmental methodology of miRNA databases such as computational tools and methods used for extraction of miRNAs annotation from different resources or through experiment. This study also discusses the efficiency of user interface design of every database along with current entry and annotations of miRNA (pathways, gene ontology, disease ontology, etc.). Here, an integrated schematic diagram of construction process for databases is also drawn along with tabular and graphical comparison of various types of entries in different databases. Aim of this paper is to present the importance of miRNAs-related resources at a single place.

  19. Structured RNAs that evade or confound exonucleases: function follows form

    PubMed Central

    Akiyama, Benjamin M.; Eiler, Daniel; Kieft, Jeffrey S.

    2016-01-01

    Cells contain powerful RNA decay machinery to eliminate unneeded RNA from the cell, and this process is an important and regulated part of controlling gene expression. However, certain structured RNAs have been found that can robustly resist degradation and extend the lifetime of an RNA. In this review, we present three RNA structures that use a specific three-dimensional fold to provide protection from RNA degradation, and discuss how the recently-solved structures of these RNAs explain their function. Specifically, we describe the Xrn1-resistant RNAs from arthropod-borne flaviviruses, exosome-resistant long non-coding RNAs associated with lung cancer metastasis and found in Kaposi’s Sarcoma-associated herpesvirus, and tRNA-like sequences occurring in certain plant viruses. These three structures reveal three different mechanisms to protect RNAs from decay and suggest RNA structure-based nuclease resistance may be a widespread mechanism of regulation. PMID:26797676

  20. Functional characterization of bacterial sRNAs using a network biology approach.

    PubMed

    Modi, Sheetal R; Camacho, Diogo M; Kohanski, Michael A; Walker, Graham C; Collins, James J

    2011-09-13

    Small RNAs (sRNAs) are important components of posttranscriptional regulation. These molecules are prevalent in bacterial and eukaryotic organisms, and involved in a variety of responses to environmental stresses. The functional characterization of sRNAs is challenging and requires highly focused and extensive experimental procedures. Here, using a network biology approach and a compendium of gene expression profiles, we predict functional roles and regulatory interactions for sRNAs in Escherichia coli. We experimentally validate predictions for three sRNAs in our inferred network: IsrA, GlmZ, and GcvB. Specifically, we validate a predicted role for IsrA and GlmZ in the SOS response, and we expand on current knowledge of the GcvB sRNA, demonstrating its broad role in the regulation of amino acid metabolism and transport. We also show, using the inferred network coupled with experiments, that GcvB and Lrp, a transcription factor, repress each other in a mutually inhibitory network. This work shows that a network-based approach can be used to identify the cellular function of sRNAs and characterize the relationship between sRNAs and transcription factors.

  1. Structure based approaches for targeting non-coding RNAs with small molecules

    PubMed Central

    Shortridge, Matthew D.; Varani, Gabriele

    2015-01-01

    The increasing appreciation of the central role of non-coding RNAs (miRNAs and long non coding RNAs) in chronic and degenerative human disease makes them attractive therapeutic targets. This would not be unprecedented: the bacterial ribosomal RNA is a mainstay for antibacterial treatment, while the conservation and functional importance of viral RNA regulatory elements has long suggested they would constitute attractive targets for new antivirals. Oligonucleotide-based chemistry has obvious appeals but also considerable pharmacological limitations that are yet to be addressed satisfactorily. Recent studies identifying small molecules targeting non-coding RNAs may provide an alternative approach to oligonucleotide methods. Here we review recent work investigating new structural and chemical principles for targeting RNA with small molecules. PMID:25687935

  2. Temperature-dependent structural variability of RNAs: spliced leader RNAs and their evolutionary history.

    PubMed

    Marz, Manja; Vanzo, Nathalie; Stadler, Peter F

    2010-02-01

    The structures attained by RNA molecules depend not only on their sequence but also on environmental parameters such as their temperature. So far, this effect has been largely neglected in bioinformatics studies. Here, we show that structural comparisons can be facilitated and more coherent structural models can be obtained when differences in environmental parameters are taken into account. We re-evaluate the secondary structures of the spliced leader (SL) RNAs from the seven eukaryotic phyla in which SL RNA trans-splicing has been described. Adjusting structure prediction to the natural growth temperatures and considering energetically similar secondary structures, we observe striking similarities among Euglenida, Kinetoplastida, Dinophyceae, Cnidaria, Rotifera, Nematoda, Platyhelminthes, and Tunicata that cannot be explained easily by the independent innovation of SL RNAs in each of these phyla. Supplementary Table is available at http://www.worldscinet.com/jbcb/.

  3. Riboswitches: discovery of drugs that target bacterial gene-regulatory RNAs.

    PubMed

    Deigan, Katherine E; Ferré-D'Amaré, Adrian R

    2011-12-20

    Riboswitches are messenger RNA (mRNA) domains that regulate gene function in response to the intracellular concentration of a variety of metabolites and second messengers. They control essential genes in many pathogenic bacteria, thus representing an inviting new class of biomolecular target for the development of antibiotics and chemical-biological tools. In this Account, we briefly review the discovery of riboswitches in the first years of the 21st century and their ensuing characterization over the past decade. We then discuss the progress achieved so far in using riboswitches as a focus for drug discovery, considering both the value of past serendipity and the particular challenges that confront current researchers. Five mechanisms of gene regulation have been determined for riboswitches. Most bacterial riboswitches modulate either transcription termination or translation initiation in response to ligand binding. All known examples of eukaryotic riboswitches, and some bacterial riboswitches, control gene expression by alternative splicing. The glmS riboswitch, which is widespread in Gram-positive bacteria, is a catalytic RNA activated by ligand binding: its self-cleavage destabilizes the mRNA of which it is part. Finally, one example of a trans-acting riboswitch is known. Three-dimensional structures have been determined for representatives of 13 structurally distinct riboswitch classes, providing atomic-level insight into their mechanisms of ligand recognition. While cellular and viral RNAs have attracted widespread interest as potential drug targets, riboswitches show special promise due to the diversity of small-molecule recognition strategies that are on display in their ligand-binding pockets. Moreover, riboswitches have evolved to recognize small-molecule ligands, which is unique among known structured RNA domains. Structural and biochemical advances in the study of riboswitches provide an impetus for the development of methods for the discovery of novel

  4. Identification of Circular RNAs in Kiwifruit and Their Species-Specific Response to Bacterial Canker Pathogen Invasion

    PubMed Central

    Wang, Zupeng; Liu, Yifei; Li, Dawei; Li, Li; Zhang, Qiong; Wang, Shuaibin; Huang, Hongwen

    2017-01-01

    Research studies have recently focused on circle RNAs (circRNAs) in relation to their regulatory functions in animals. However, the systematic identification of circRNAs in plants, especially non-model plants, is limited. In addition, raw report on the prediction of the potential role of circRNAs in plant response to pathogen invasion is currently available. We conducted the systematic identification of circRNAs from four materials originating from three species belonging to genus Actinidia under different situations using ribosomal RNA (rRNA) depleted RNA-Seq data. A total of 3,582 circRNAs were identified in Actinidia, of which 64.01, 21.44, and 14.55% were intergenic circRNAs, exonic circRNAs, and intronic circRNAs, respectively. Tissue-specific expression of circRNAs was observed in kiwifruit, and a species-specific response was detected when infected with Pseudomonas syringae pv. actinidiae (Psa), which is the causative agent of kiwifruit bacterial canker disease. Furthermore, we found that both exonic and intronic circRNAs were significantly positively correlated to parent protein-coding genes, and intronic circRNAs are a class of highly remarkable regulators the parent genes comparing to that of exonic circRNAs. Expression and weighted gene co-expression network analysis (WGCNA) identified a set of circRNAs that were closely associated with plant defense response. The findings of the presents study suggest that circRNAs exhibit tissue- and species-specific expression, as well as play an important role in plant immune response. PMID:28396678

  5. Prediction of common folding structures of homologous RNAs.

    PubMed Central

    Han, K; Kim, H J

    1993-01-01

    We have developed an algorithm and a computer program for simultaneously folding homologous RNA sequences. Given an alignment of M homologous sequences of length N, the program performs phylogenetic comparative analysis and predicts a common secondary structure conserved in the sequences. When the structure is not uniquely determined, it infers multiple structures which appear most plausible. This method is superior to energy minimization methods in the sense that it is not sensitive to point mutation of a sequence. It is also superior to usual phylogenetic comparative methods in that it does not require manual scrutiny for covariation or secondary structures. The most plausible 1-5 structures are produced in O(MN2 + N3) time and O(N2) space, which are the same requirements as those of widely used dynamic programs based on energy minimization for folding a single sequence. This is the first algorithm probably practical both in terms of time and space for finding secondary structures of homologous RNA sequences. The algorithm has been implemented in C on a Sun SparcStation, and has been verified by testing on tRNAs, 5S rRNAs, 16S rRNAs, TAR RNAs of human immunodeficiency virus type 1 (HIV-1), and RRE RNAs of HIV-1. We have also applied the program to cis-acting packaging sequences of HIV-1, for which no generally accepted structures yet exist, and propose potentially stable structures. Simulation of the program with random sequences with the same base composition and the same degree of similarity as the above sequences shows that structures common to homologous sequences are very unlikely to occur by chance in random sequences. PMID:7681944

  6. Basis for Structural Diversity in Homologous RNAs

    SciTech Connect

    Krasilnikov, Andrey S.; Xiao, Yinghua; Pan, Tao; Mondragon, Alfonso

    2010-03-08

    Large RNA molecules, such as ribozymes, fold with well-defined tertiary structures that are important for their activity. There are many instances of ribozymes with identical function but differences in their secondary structures, suggesting alternative tertiary folds. Here, we report a crystal structure of the 161-nucleotide specificity domain of an A-type ribonuclease P that differs in secondary and tertiary structure from the specificity domain of a B-type molecule. Despite the differences, the cores of the domains have similar three-dimensional structure. Remarkably, the similar geometry of the cores is stabilized by a different set of interactions involving distinct auxiliary elements.

  7. S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization

    PubMed Central

    Sierant, Malgorzata; Leszczynska, Grazyna; Sadowska, Klaudia; Dziergowska, Agnieszka; Rozanski, Michal; Sochacka, Elzbieta; Nawrot, Barbara

    2016-01-01

    Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies. PMID:27566149

  8. S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization.

    PubMed

    Sierant, Malgorzata; Leszczynska, Grazyna; Sadowska, Klaudia; Dziergowska, Agnieszka; Rozanski, Michal; Sochacka, Elzbieta; Nawrot, Barbara

    2016-12-15

    Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNA(Lys3) sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

  9. Probing-directed identification of novel structured RNAs

    PubMed Central

    Vinogradova, Svetlana V.; Sutormin, Roman A.; Mironov, Andrey A.; Soldatov, Ruslan A.

    2016-01-01

    ABSTRACT Transcripts often harbor RNA elements, which regulate cell processes co- or post-transcriptionally. The functions of many regulatory RNA elements depend on their structure, thus it is important to determine the structure as well as to scan genomes for structured elements. State of the art ab initio approaches to predict structured RNAs rely on DNA sequence analysis. They use 2 major types of information inferred from a sequence: thermodynamic stability of an RNA structure and evolutionary footprints of base-pair interactions. In recent years, chemical probing of RNA has arisen as an alternative source of structural information. RNA probing experiments detect positions accessible to specific types of chemicals or enzymes indicating their propensity to be in a paired or unpaired state. There exist several strategies to integrate probing data into RNA secondary structure prediction algorithms that substantially improve the prediction quality. However, whether and how probing data could contribute to detection of structured RNAs remains an open question. We previously developed the energy-based approach RNASurface to detect locally optimal structured RNA elements. Here, we integrate probing data into the RNASurface energy model using a general framework. We show that the use of experimental data allows for better discrimination of ncRNAs from other transcripts. Application of RNASurface to genome-wide analysis of the human transcriptome with PARS data identifies previously undetectable segments, with evidence of functionality for some of them. PMID:26732206

  10. Small Non-Coding RNAs: New Insights in Modulation of Host Immune Response by Intracellular Bacterial Pathogens

    PubMed Central

    Ahmed, Waqas; Zheng, Ke; Liu, Zheng-Fei

    2016-01-01

    Pathogenic bacteria possess intricate regulatory networks that temporally control the production of virulence factors and enable the bacteria to survive and proliferate within host cell. Small non-coding RNAs (sRNAs) have been identified as important regulators of gene expression in diverse biological contexts. Recent research has shown bacterial sRNAs involved in growth and development, cell proliferation, differentiation, metabolism, cell signaling, and immune response through regulating protein–protein interactions or via their ability to base pair with RNA and DNA. In this review, we provide a brief overview of mechanism of action employed by immune-related sRNAs, their known functions in immunity, and how they can be integrated into regulatory circuits that govern virulence, which will facilitate our understanding of pathogenesis and the development of novel, more effective therapeutic approaches to treat infections caused by intracellular bacterial pathogens. PMID:27803700

  11. Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

    SciTech Connect

    Zhu, Jianyu; Korostelev, Andrei; Costantino, David A.; Donohue, John P.; Noller, Harry F.; Kieft, Jeffrey S.

    2011-08-24

    Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA-mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.

  12. An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

    PubMed Central

    Pichon, Christophe; du Merle, Laurence; Caliot, Marie Elise; Trieu-Cuot, Patrick; Le Bouguénec, Chantal

    2012-01-01

    Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli. PMID:22139924

  13. Structures of ribonuclease P RNAs of Vibrio core species.

    PubMed

    Maeda, T; Furushita, M; Hamamura, K; Shiba, T

    2001-05-01

    The structures of an RNA component of ribonuclease P (RNase P RNA) were examined for Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio natriegens, Vibrio campbellii, Vibrio proteolyticus, Vibrio pelagius and Vibrio harveyi to clearly determine their genetic differences. The RNase P RNAs ranged from 382 to 454 nucleotides (nt) in size, and were remarkably different from each other in the structure of two helices, P3 and P12. The P3 helices were comprised of tandem repeats of a palindromic sequence (24 nt), resulting in the longitudinal repetition of a stem structure. The number of repetitions ranged from four in V. harveyi, to one in both V. alginolyticus and V. proteolyticus. The genes for the RNase P RNAs of all species were located between two open reading frames, the amino acid sequences of which were similar to the hypothetical proteins located at 70.92 and 1.94 min in the Escherichia coli chromosome.

  14. Automated 3D structure composition for large RNAs.

    PubMed

    Popenda, Mariusz; Szachniuk, Marta; Antczak, Maciej; Purzycka, Katarzyna J; Lukasiak, Piotr; Bartol, Natalia; Blazewicz, Jacek; Adamiak, Ryszard W

    2012-08-01

    Understanding the numerous functions that RNAs play in living cells depends critically on knowledge of their three-dimensional structure. Due to the difficulties in experimentally assessing structures of large RNAs, there is currently great demand for new high-resolution structure prediction methods. We present the novel method for the fully automated prediction of RNA 3D structures from a user-defined secondary structure. The concept is founded on the machine translation system. The translation engine operates on the RNA FRABASE database tailored to the dictionary relating the RNA secondary structure and tertiary structure elements. The translation algorithm is very fast. Initial 3D structure is composed in a range of seconds on a single processor. The method assures the prediction of large RNA 3D structures of high quality. Our approach needs neither structural templates nor RNA sequence alignment, required for comparative methods. This enables the building of unresolved yet native and artificial RNA structures. The method is implemented in a publicly available, user-friendly server RNAComposer. It works in an interactive mode and a batch mode. The batch mode is designed for large-scale modelling and accepts atomic distance restraints. Presently, the server is set to build RNA structures of up to 500 residues.

  15. Automated 3D structure composition for large RNAs

    PubMed Central

    Popenda, Mariusz; Szachniuk, Marta; Antczak, Maciej; Purzycka, Katarzyna J.; Lukasiak, Piotr; Bartol, Natalia; Blazewicz, Jacek; Adamiak, Ryszard W.

    2012-01-01

    Understanding the numerous functions that RNAs play in living cells depends critically on knowledge of their three-dimensional structure. Due to the difficulties in experimentally assessing structures of large RNAs, there is currently great demand for new high-resolution structure prediction methods. We present the novel method for the fully automated prediction of RNA 3D structures from a user-defined secondary structure. The concept is founded on the machine translation system. The translation engine operates on the RNA FRABASE database tailored to the dictionary relating the RNA secondary structure and tertiary structure elements. The translation algorithm is very fast. Initial 3D structure is composed in a range of seconds on a single processor. The method assures the prediction of large RNA 3D structures of high quality. Our approach needs neither structural templates nor RNA sequence alignment, required for comparative methods. This enables the building of unresolved yet native and artificial RNA structures. The method is implemented in a publicly available, user-friendly server RNAComposer. It works in an interactive mode and a batch mode. The batch mode is designed for large-scale modelling and accepts atomic distance restraints. Presently, the server is set to build RNA structures of up to 500 residues. PMID:22539264

  16. Operon mRNAs are organized into ORF-centric structures that predict translation efficiency

    PubMed Central

    Burkhardt, David H; Rouskin, Silvi; Zhang, Yan; Li, Gene-Wei; Weissman, Jonathan S; Gross, Carol A

    2017-01-01

    Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF. DOI: http://dx.doi.org/10.7554/eLife.22037.001 PMID:28139975

  17. Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

    PubMed Central

    Robertson, Kelly L.

    2012-01-01

    We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population. PMID:22057868

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-02-01

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

  20. Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

    PubMed Central

    Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

    2013-01-01

    Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

  1. Sequence and Structural Analyses for Functional Non-coding RNAs

    NASA Astrophysics Data System (ADS)

    Sakakibara, Yasubumi; Sato, Kengo

    Analysis and detection of functional RNAs are currently important topics in both molecular biology and bioinformatics research. Several computational methods based on stochastic context-free grammars (SCFGs) have been developed for modeling and analysing functional RNA sequences. These grammatical methods have succeeded in modeling typical secondary structures of RNAs and are used for structural alignments of RNA sequences. Such stochastic models, however, are not sufficient to discriminate member sequences of an RNA family from non-members, and hence to detect non-coding RNA regions from genome sequences. Recently, the support vector machine (SVM) and kernel function techniques have been actively studied and proposed as a solution to various problems in bioinformatics. SVMs are trained from positive and negative samples and have strong, accurate discrimination abilities, and hence are more appropriate for the discrimination tasks. A few kernel functions that extend the string kernel to measure the similarity of two RNA sequences from the viewpoint of secondary structures have been proposed. In this article, we give an overview of recent progress in SCFG-based methods for RNA sequence analysis and novel kernel functions tailored to measure the similarity of two RNA sequences and developed for use with support vector machines (SVM) in discriminating members of an RNA family from non-members.

  2. DMS Footprinting of Structured RNAs and RNA-Protein Complexes

    PubMed Central

    Tijerina, Pilar; Mohr, Sabine; Russell, Rick

    2008-01-01

    We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nucleotides). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as ‘control’ conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis (PAGE), and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a ‘footprint’), it is possible to detect local changes in RNA secondary and tertiary structure, as well as the formation of RNA-protein contacts. This protocol takes 1.5–3 days to complete, depending on the type of analysis used. PMID:17948004

  3. Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

    PubMed Central

    Wroblewska, Zuzanna; Olejniczak, Mikolaj

    2016-01-01

    The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs. PMID:27154968

  4. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477-42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  5. Structural Biology of Bacterial RNA Polymerase

    PubMed Central

    Murakami, Katsuhiko S.

    2015-01-01

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477–42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription. PMID:25970587

  6. Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

    PubMed

    Hoernes, Thomas Philipp; Clementi, Nina; Faserl, Klaus; Glasner, Heidelinde; Breuker, Kathrin; Lindner, Herbert; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-29

    Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced m(5)C, m(6)A, Ψ or 2'-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m(5)C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Satellite RNAs of plant viruses: structures and biological effects.

    PubMed Central

    Roossinck, M J; Sleat, D; Palukaitis, P

    1992-01-01

    Plant viruses often contain parasites of their own, referred to as satellites. Satellite RNAs are dependent on their associated (helper) virus for both replication and encapsidation. Satellite RNAs vary from 194 to approximately 1,500 nucleotides (nt). The larger satellites (900 to 1,500 nt) contain open reading frames and express proteins in vitro and in vivo, whereas the smaller satellites (194 to 700 nt) do not appear to produce functional proteins. The smaller satellites contain a high degree of secondary structure involving 49 to 73% of their sequences, with the circular satellites containing more base pairing than the linear satellites. Many of the smaller satellites produce multimeric forms during replication. There are various models to account for their formation and role in satellite replication. Some of these smaller satellites encode ribozymes and are able to undergo autocatalytic cleavage. The enzymology of satellite replication is poorly understood, as is the replication of their helper viruses. In many cases the coreplication of satellites suppresses the replication of the helper virus genome. This is usually paralleled by a reduction in the disease induced by the helper virus; however, there are notable exceptions in which the satellite exacerbates the pathogenicity of the helper virus, albeit on only a limited number of hosts. The ameliorative satellites are being assessed as biocontrol agents of virus-induced disease. In greenhouse studies, satellites have been known to "spontaneously" appear in virus cultures. The possible origin of satellites will be briefly considered. PMID:1620065

  8. Non-coding sRNAs regulate virulence in the bacterial pathogen Vibrio cholerae

    PubMed Central

    Bardill, J. Patrick; Hammer, Brian

    2012-01-01

    Vibrio cholerae is the waterborne bacterium responsible for worldwide outbreaks of the acute, potentially fatal cholera diarrhea. The primary factors this human pathogen uses to cause the disease are controlled by a complex regulatory program linking extracellular signaling inputs to changes in expression of several critical virulence genes. Recently it has been uncovered that many non-coding regulatory sRNAs are important components of the V. cholerae virulence regulon. Most of these sRNAs appear to require the RNA-binding protein, Hfq, to interact with and alter the expression of target genes, while a few sRNAs appear to function by an Hfq-independent mechanism. Direct base-pairing between the sRNAs and putative target mRNAs has been shown in a few cases but the extent of each sRNAs regulon is not fully known. Genetic and biochemical methods, coupled with computational and genomics approaches, are being used to validate known sRNAs and also to identify many additional putative sRNAs that may play a role in the pathogenic lifestyle of V. cholerae. PMID:22546941

  9. Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

    PubMed

    Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

    2014-01-01

    Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

  10. Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

    PubMed Central

    Villet, Régis; Fonvielle, Matthieu; Busca, Patricia; Chemama, Maryline; Maillard, Antoine P.; Hugonnet, Jean-Emmanuel; Dubost, Lionel; Marie, Arul; Josseaume, Nathalie; Mesnage, Stéphane; Mayer, Claudine; Valéry, Jean-Marc; Ethève-Quelquejeu, Mélanie; Arthur, Michel

    2007-01-01

    The FemXWv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNAAla to UDP-MurNAc-pentadepsipeptide. FemXWv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemXWv is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemXWv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1–C72 and G2–C71 base pairs of the acceptor stem as critical for FemXWv activity in agreement with modeling of tRNAAla in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNAAla harboring nucleotide substitutions in the G3–U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemXWv. The different modes of recognition of the acceptor stem indicate that specific inhibition of FemXWv could be achieved by targeting the distal portion of tRNAAla for the design of substrate analogues. PMID:17932062

  11. Identification of ta-siRNAs and Cis-nat-siRNAs in Cassava and Their Roles in Response to Cassava Bacterial Blight

    PubMed Central

    Quintero, Andrés; Pérez-Quintero, Alvaro L.; López, Camilo

    2013-01-01

    Trans-acting small interfering RNAs (ta-siRNAs) and natural cis-antisense siRNAs (cis-nat-siRNAs) are recently discovered small RNAs (sRNAs) involved in post-transcriptional gene silencing. ta-siRNAs are transcribed from genomic loci and require processing by microRNAs (miRNAs). cis-nat-siRNAs are derived from antisense RNAs produced by the simultaneous transcription of overlapping antisense genes. Their roles in many plant processes, including pathogen response, are mostly unknown. In this work, we employed a bioinformatic approach to identify ta-siRNAs and cis-nat-siRNAs in cassava from two sRNA libraries, one constructed from healthy cassava plants and one from plants inoculated with the bacterium Xanthomonas axonopodis pv. manihotis (Xam). A total of 54 possible ta-siRNA loci were identified in cassava, including a homolog of TAS3, the best studied plant ta-siRNA. Fifteen of these loci were induced, while 39 were repressed in response to Xam infection. In addition, 15 possible cis-natural antisense transcript (cis-NAT) loci producing siRNAs were identified from overlapping antisense regions in the genome, and were found to be differentially expressed upon Xam infection. Roles of sRNAs were predicted by sequence complementarity and our results showed that many sRNAs identified in this work might be directed against various transcription factors. This work represents a significant step toward understanding the roles of sRNAs in the immune response of cassava. PMID:23665476

  12. RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs.

    PubMed

    Strobel, Eric J; Watters, Kyle E; Loughrey, David; Lucks, Julius B

    2016-06-01

    RNAs assume sophisticated structures that are active in myriad cellular processes. In this review, we highlight newly identified ribozymes, riboswitches, and small RNAs, some of which control the function of cellular metabolic and gene expression networks. We then examine recent developments in genome-wide RNA structure probing technologies that are yielding new insights into the structural landscape of the transcriptome. Finally, we discuss how these RNA 'structomic' methods can address emerging questions in RNA systems biology, from the mechanisms behind long non-coding RNAs to new bases for human diseases.

  13. From structure prediction to genomic screens for novel non-coding RNAs.

    PubMed

    Gorodkin, Jan; Hofacker, Ivo L

    2011-08-01

    Non-coding RNAs (ncRNAs) are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs). A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction of RNA structure with the aim of assisting in functional analysis. With the discovery of more and more ncRNAs, it has become clear that a large fraction of these are highly structured. Interestingly, a large part of the structure is comprised of regular Watson-Crick and GU wobble base pairs. This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other.

  14. Small RNAs in plant defense responses during viral and bacterial interactions: similarities and differences

    PubMed Central

    Peláez, Pablo; Sanchez, Federico

    2013-01-01

    Small non-coding RNAs constitute an important class of gene expression regulators that control different biological processes in most eukaryotes. In plants, several small RNA (sRNA) silencing pathways have evolved to produce a wide range of small RNAs with specialized functions. Evidence for the diverse mode of action of the small RNA pathways has been highlighted during plant–microbe interactions. Host sRNAs and small RNA silencing pathways have been recognized as essential components of plant immunity. One way plants respond and defend against pathogen infections is through the small RNA silencing immune system. To deal with plant defense responses, pathogens have evolved sophisticated mechanisms to avoid and counterattack this defense strategy. The relevance of the small RNA-mediated plant defense responses during viral infections has been well-established. Recent evidence points out its importance also during plant–bacteria interactions. Herein, this review discusses recent findings, similarities and differences about the small RNA-mediated arms race between plants and these two groups of microbes, including the small RNA silencing pathway components that contribute to plant immune responses, the pathogen-responsive endogenous sRNAs and the pathogen-delivered effector proteins. PMID:24046772

  15. Small RNAs in plant defense responses during viral and bacterial interactions: similarities and differences.

    PubMed

    Peláez, Pablo; Sanchez, Federico

    2013-09-05

    Small non-coding RNAs constitute an important class of gene expression regulators that control different biological processes in most eukaryotes. In plants, several small RNA (sRNA) silencing pathways have evolved to produce a wide range of small RNAs with specialized functions. Evidence for the diverse mode of action of the small RNA pathways has been highlighted during plant-microbe interactions. Host sRNAs and small RNA silencing pathways have been recognized as essential components of plant immunity. One way plants respond and defend against pathogen infections is through the small RNA silencing immune system. To deal with plant defense responses, pathogens have evolved sophisticated mechanisms to avoid and counterattack this defense strategy. The relevance of the small RNA-mediated plant defense responses during viral infections has been well-established. Recent evidence points out its importance also during plant-bacteria interactions. Herein, this review discusses recent findings, similarities and differences about the small RNA-mediated arms race between plants and these two groups of microbes, including the small RNA silencing pathway components that contribute to plant immune responses, the pathogen-responsive endogenous sRNAs and the pathogen-delivered effector proteins.

  16. The Sequence and Structure Determine the Function of Mature Human miRNAs

    PubMed Central

    Wawrzyniak, Dariusz; Jeleniewicz, Jaroslaw; Barciszewska, Miroslawa Z.; Barciszewski, Jan

    2016-01-01

    Micro RNAs (miRNAs) (19–25 nucleotides in length) belong to the group of non-coding RNAs are the most abundant group of posttranscriptional regulators in multicellular organisms. They affect a gene expression by binding of fully or partially complementary sequences to the 3’-UTR of target mRNA. Furthermore, miRNAs present a mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated at the post-transcriptional level. However, little is known about the specific pathways through which miRNAs with specific sequence or structural motifs regulate the cellular processes. In this paper we showed the broad and deep characteristics of mature miRNAs according to their sequence and structural motifs. We investigated a distinct group of miRNAs characterized by the presence of specific sequence motifs, such as UGUGU, GU-repeats and purine/pyrimidine contents. Using computational function and pathway analysis of their targeted genes, we were able to observe the relevance of sequence and the type of targeted mRNAs. As the consequence of the sequence analysis we finally provide the comprehensive description of pathways, biological processes and proteins associated with the distinct group of characterized miRNAs. Here, we found that the specific group of miRNAs with UGUGU can activate the targets associated to the interferon induction pathway or pathways prominently observed during carcinogenesis. GU-rich miRNAs are prone to regulate mostly processes in neurogenesis, whereas purine/pyrimidine rich miRNAs could be involved rather in transport and/or degradation of RNAs. Additionally, we have also analyzed the simple sequence repeats (SSRs). Their variation within mature miRNAs might be critical for normal miRNA regular activity. Expansion or contraction of SSRs in mature miRNA might directly affect its mRNA interaction or even change the function of that distinct miRNA. Our results prove that due to the specific sequence features, these

  17. Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

    PubMed Central

    Zhu, Jianyu; Korostelev, Andrei; Costantino, David A.; Donohue, John P.; Noller, Harry F.; Kieft, Jeffrey S.

    2011-01-01

    Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA•mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines. PMID:21245352

  18. Structural signatures of thermal adaptation of bacterial ribosomal RNA, transfer RNA, and messenger RNA.

    PubMed

    Jegousse, Clara; Yang, Yuedong; Zhan, Jian; Wang, Jihua; Zhou, Yaoqi

    2017-01-01

    Temperature adaptation of bacterial RNAs is a subject of both fundamental and practical interest because it will allow a better understanding of molecular mechanism of RNA folding with potential industrial application of functional thermophilic or psychrophilic RNAs. Here, we performed a comprehensive study of rRNA, tRNA, and mRNA of more than 200 bacterial species with optimal growth temperatures (OGT) ranging from 4°C to 95°C. We investigated temperature adaptation at primary, secondary and tertiary structure levels. We showed that unlike mRNA, tRNA and rRNA were optimized for their structures at compositional levels with significant tertiary structural features even for their corresponding randomly permutated sequences. tRNA and rRNA are more exposed to solvent but remain structured for hyperthermophiles with nearly OGT-independent fluctuation of solvent accessible surface area within a single RNA chain. mRNA in hyperthermophiles is essentially the same as random sequences without tertiary structures although many mRNA in mesophiles and psychrophiles have well-defined tertiary structures based on their low overall solvent exposure with clear separation of deeply buried from partly exposed bases as in tRNA and rRNA. These results provide new insight into temperature adaptation of different RNAs.

  19. Prediction of plant pre-microRNAs and their microRNAs in genome-scale sequences using structure-sequence features and support vector machine.

    PubMed

    Meng, Jun; Liu, Dong; Sun, Chao; Luan, Yushi

    2014-12-30

    MicroRNAs (miRNAs) are a family of non-coding RNAs approximately 21 nucleotides in length that play pivotal roles at the post-transcriptional level in animals, plants and viruses. These molecules silence their target genes by degrading transcription or suppressing translation. Studies have shown that miRNAs are involved in biological responses to a variety of biotic and abiotic stresses. Identification of these molecules and their targets can aid the understanding of regulatory processes. Recently, prediction methods based on machine learning have been widely used for miRNA prediction. However, most of these methods were designed for mammalian miRNA prediction, and few are available for predicting miRNAs in the pre-miRNAs of specific plant species. Although the complete Solanum lycopersicum genome has been published, only 77 Solanum lycopersicum miRNAs have been identified, far less than the estimated number. Therefore, it is essential to develop a prediction method based on machine learning to identify new plant miRNAs. A novel classification model based on a support vector machine (SVM) was trained to identify real and pseudo plant pre-miRNAs together with their miRNAs. An initial set of 152 novel features related to sequential structures was used to train the model. By applying feature selection, we obtained the best subset of 47 features for use with the Back Support Vector Machine-Recursive Feature Elimination (B-SVM-RFE) method for the classification of plant pre-miRNAs. Using this method, 63 features were obtained for plant miRNA classification. We then developed an integrated classification model, miPlantPreMat, which comprises MiPlantPre and MiPlantMat, to identify plant pre-miRNAs and their miRNAs. This model achieved approximately 90% accuracy using plant datasets from nine plant species, including Arabidopsis thaliana, Glycine max, Oryza sativa, Physcomitrella patens, Medicago truncatula, Sorghum bicolor, Arabidopsis lyrata, Zea mays and Solanum

  20. Expression profiling and structural characterization of microRNAs in adipose tissues of hibernating ground squirrels.

    PubMed

    Wu, Cheng-Wei; Biggar, Kyle K; Storey, Kenneth B

    2014-12-01

    MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P<0.05), which was 16%-54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32-2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%-70% of control), while only expression of miR-138 was significantly upregulated (2.91±0.8-fold of the control, P<0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked to distinct

  1. Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

    PubMed Central

    Wu, Cheng-Wei; Biggar, Kyle K.; Storey, Kenneth B.

    2014-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 miRNAs in brown (BAT) and white adipose tissue (WAT) during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six miRNAs including let-7a, let-7b, miR-107, miR-150, miR-222 and miR-31 was significantly downregulated in WAT (P < 0.05), which was 16%–54% of euthermic non-torpid control squirrels, whereas expression of three miRNAs including miR-143, miR-200a and miR-519d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more miRNAs was downregulated in BAT during torpor. We detected reduced expression of 6 miRNAs including miR-103a, miR-107, miR-125b, miR-21, miR-221 and miR-31 (48%–70% of control), while only expression of miR-138 was significantly upregulated (2.91 ± 0.8-fold of the control, P < 0.05). Interestingly, miRNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas miRNAs with altered expression in BAT during torpor were linked to mitochondrial β-oxidation. miRPath target prediction analysis showed that miRNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase (MAPK) signaling, while the miRNAs upregulated in WAT were linked to transforming growth factor β (TGFβ) signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-miRNAs for the miRNAs used in this study, suggesting no structure-influenced changes in pre-miRNA processing efficiency in the squirrel. As well, the expression of miRNA processing enzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of miRNA expression in adipose tissues may be linked

  2. Bioinformatics of prokaryotic RNAs.

    PubMed

    Backofen, Rolf; Amman, Fabian; Costa, Fabrizio; Findeiß, Sven; Richter, Andreas S; Stadler, Peter F

    2014-01-01

    The genome of most prokaryotes gives rise to surprisingly complex transcriptomes, comprising not only protein-coding mRNAs, often organized as operons, but also harbors dozens or even hundreds of highly structured small regulatory RNAs and unexpectedly large levels of anti-sense transcripts. Comprehensive surveys of prokaryotic transcriptomes and the need to characterize also their non-coding components is heavily dependent on computational methods and workflows, many of which have been developed or at least adapted specifically for the use with bacterial and archaeal data. This review provides an overview on the state-of-the-art of RNA bioinformatics focusing on applications to prokaryotes.

  3. Bioinformatics of prokaryotic RNAs

    PubMed Central

    Backofen, Rolf; Amman, Fabian; Costa, Fabrizio; Findeiß, Sven; Richter, Andreas S; Stadler, Peter F

    2014-01-01

    The genome of most prokaryotes gives rise to surprisingly complex transcriptomes, comprising not only protein-coding mRNAs, often organized as operons, but also harbors dozens or even hundreds of highly structured small regulatory RNAs and unexpectedly large levels of anti-sense transcripts. Comprehensive surveys of prokaryotic transcriptomes and the need to characterize also their non-coding components is heavily dependent on computational methods and workflows, many of which have been developed or at least adapted specifically for the use with bacterial and archaeal data. This review provides an overview on the state-of-the-art of RNA bioinformatics focusing on applications to prokaryotes. PMID:24755880

  4. Dealing with stable structures at ribosome binding sites: bacterial translation and ribosome standby.

    PubMed

    Unoson, Cecilia; Wagner, E Gerhart H

    2007-11-01

    Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.

  5. Structural correlations in bacterial metabolic networks

    PubMed Central

    2011-01-01

    Background Evolution of metabolism occurs through the acquisition and loss of genes whose products acts as enzymes in metabolic reactions, and from a presumably simple primordial metabolism the organisms living today have evolved complex and highly variable metabolisms. We have studied this phenomenon by comparing the metabolic networks of 134 bacterial species with known phylogenetic relationships, and by studying a neutral model of metabolic network evolution. Results We consider the 'union-network' of 134 bacterial metabolisms, and also the union of two smaller subsets of closely related species. Each reaction-node is tagged with the number of organisms it belongs to, which we denote organism degree (OD), a key concept in our study. Network analysis shows that common reactions are found at the centre of the network and that the average OD decreases as we move to the periphery. Nodes of the same OD are also more likely to be connected to each other compared to a random OD relabelling based on their occurrence in the real data. This trend persists up to a distance of around five reactions. A simple growth model of metabolic networks is used to investigate the biochemical constraints put on metabolic-network evolution. Despite this seemingly drastic simplification, a 'union-network' of a collection of unrelated model networks, free of any selective pressure, still exhibit similar structural features as their bacterial counterpart. Conclusions The OD distribution quantifies topological properties of the evolutionary history of bacterial metabolic networks, and lends additional support to the importance of horizontal gene transfer during bacterial metabolic evolution where new reactions are attached at the periphery of the network. The neutral model of metabolic network growth can reproduce the main features of real networks, but we observe that the real networks contain a smaller common core, while they are more similar at the periphery of the network. This suggests

  6. High resolution structure of bacterial cell sacculi

    NASA Astrophysics Data System (ADS)

    Dutcher, John; Touhami, Ahmed; Matias, Valerio; Clarke, Anthony; Jericho, Manfred; Beveridge, Terry

    2008-03-01

    The major structural component of bacterial cell walls is the peptidoglycan sacculus, which is one of nature's strongest and largest macromolecules that allows the cell to maintain a large internal pressure while allowing the transport of molecules into and out of the cell and cell growth. The three-dimensional structure of this unique biopolymer is controversial, and two models have been proposed: the planar model, in which the glycan strands lie in the plane of the cell surface, and the scaffold model, in which the glycan strands lie perpendicular to the cell surface. In this study we have used atomic force microscopy (AFM) to investigate the high resolution structure of isolated, intact sacculi of both Gram-positive and Gram-negative bacterial cells. We have observed a sponge-like structure for both types of sacculi with pore diameters between 5 to 15 nm. Our data for Gram-positive sacculi provide evidence for the validity of the scaffold model, whereas our data for Gram-negative sacculi indicate an orientation along the short axis of the cell which is consistent with the planar model. To further elucidate the structure, we have exposed sacculi to the tAmiB enzyme which cleaves peptide-peptide bonds.

  7. Structure and operation of bacterial tripartite pumps.

    PubMed

    Hinchliffe, Philip; Symmons, Martyn F; Hughes, Colin; Koronakis, Vassilis

    2013-01-01

    In bacteria such as Pseudomonas aeruginosa and Escherichia coli, tripartite membrane machineries, or pumps, determine the efflux of small noxious molecules, such as detergents, heavy metals, and antibiotics, and the export of large proteins including toxins. They are therefore influential in bacterial survival, particularly during infections caused by multidrug-resistant pathogens. In these tripartite pumps an inner membrane transporter, typically an ATPase or proton antiporter, binds and translocates export or efflux substrates. In cooperation with a periplasmic adaptor protein it recruits and opens a TolC family cell exit duct, which is anchored in the outer membrane and projects across the periplasmic space between inner and outer membranes. Assembled tripartite pumps thus span the entire bacterial cell envelope. We review the atomic structures of each of the three pump components and discuss how these have allowed high-resolution views of tripartite pump assembly, operation, and possible inhibition.

  8. Structural diversity of bacterial flagellar motors

    PubMed Central

    Chen, Songye; Beeby, Morgan; Murphy, Gavin E; Leadbetter, Jared R; Hendrixson, David R; Briegel, Ariane; Li, Zhuo; Shi, Jian; Tocheva, Elitza I; Müller, Axel; Dobro, Megan J; Jensen, Grant J

    2011-01-01

    The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species. PMID:21673657

  9. Structural and metabolic characterization of RNAs from rats with experimental Guerin tumor - I. Nucleotide composition of RNAs from the liver and tumor tissues of rats.

    PubMed

    Ratkiewicz, A; Galasinski, W

    1976-01-01

    The characteristics of the ribonucleic acids of Guerin tumor was the subject of this work. The effect of tumor development on the structure of the ribonucleic acids in the liver of tumor bearing rats was studied. Some differences of nucleotide compositions in RNAs isolated from subcellular fractions of liver of control and tumor bearing rats and of cancer tissue were observed. The nucleotide compositions of cancer nuclear RNA is distinctly different from liver RNA. The changes in primary structure of liver RNAs due by development of tumor in rats may be result of metabolic peculiarities of these RNAs.

  10. Role of long non-coding RNAs in bacterial cold water disease pathogenesis in rainbow trout

    USDA-ARS?s Scientific Manuscript database

    Bacterial cold water disease (BCWD) caused by Flavobacterium psychrophilum is one of the major causes of mortality in salmonids. Three genetic lines of rainbow trout designated as ARS-Fp-R (resistant), ARS-Fp-C (control) and ARS-Fp-S (susceptible) have significant differences in survival rate follow...

  11. Bacterial phylogeny structures soil resistomes across habitats

    NASA Astrophysics Data System (ADS)

    Forsberg, Kevin J.; Patel, Sanket; Gibson, Molly K.; Lauber, Christian L.; Knight, Rob; Fierer, Noah; Dantas, Gautam

    2014-05-01

    Ancient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil, including genes identical to those in human pathogens. Despite the apparent overlap between soil and clinical resistomes, factors influencing ARG composition in soil and their movement between genomes and habitats remain largely unknown. General metagenome functions often correlate with the underlying structure of bacterial communities. However, ARGs are proposed to be highly mobile, prompting speculation that resistomes may not correlate with phylogenetic signatures or ecological divisions. To investigate these relationships, we performed functional metagenomic selections for resistance to 18 antibiotics from 18 agricultural and grassland soils. The 2,895 ARGs we discovered were mostly new, and represent all major resistance mechanisms. We demonstrate that distinct soil types harbour distinct resistomes, and that the addition of nitrogen fertilizer strongly influenced soil ARG content. Resistome composition also correlated with microbial phylogenetic and taxonomic structure, both across and within soil types. Consistent with this strong correlation, mobility elements (genes responsible for horizontal gene transfer between bacteria such as transposases and integrases) syntenic with ARGs were rare in soil by comparison with sequenced pathogens, suggesting that ARGs may not transfer between soil bacteria as readily as is observed between human pathogens. Together, our results indicate that bacterial community composition is the primary determinant of soil ARG content, challenging previous hypotheses that horizontal gene transfer effectively decouples resistomes from phylogeny.

  12. Bacterial phylogeny structures soil resistomes across habitats.

    PubMed

    Forsberg, Kevin J; Patel, Sanket; Gibson, Molly K; Lauber, Christian L; Knight, Rob; Fierer, Noah; Dantas, Gautam

    2014-05-29

    Ancient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil, including genes identical to those in human pathogens. Despite the apparent overlap between soil and clinical resistomes, factors influencing ARG composition in soil and their movement between genomes and habitats remain largely unknown. General metagenome functions often correlate with the underlying structure of bacterial communities. However, ARGs are proposed to be highly mobile, prompting speculation that resistomes may not correlate with phylogenetic signatures or ecological divisions. To investigate these relationships, we performed functional metagenomic selections for resistance to 18 antibiotics from 18 agricultural and grassland soils. The 2,895 ARGs we discovered were mostly new, and represent all major resistance mechanisms. We demonstrate that distinct soil types harbour distinct resistomes, and that the addition of nitrogen fertilizer strongly influenced soil ARG content. Resistome composition also correlated with microbial phylogenetic and taxonomic structure, both across and within soil types. Consistent with this strong correlation, mobility elements (genes responsible for horizontal gene transfer between bacteria such as transposases and integrases) syntenic with ARGs were rare in soil by comparison with sequenced pathogens, suggesting that ARGs may not transfer between soil bacteria as readily as is observed between human pathogens. Together, our results indicate that bacterial community composition is the primary determinant of soil ARG content, challenging previous hypotheses that horizontal gene transfer effectively decouples resistomes from phylogeny.

  13. Self Containment, a Property of Modular RNA Structures, Distinguishes microRNAs

    PubMed Central

    Lee, Miler T.; Kim, Junhyong

    2008-01-01

    RNA molecules will tend to adopt a folded conformation through the pairing of bases on a single strand; the resulting so-called secondary structure is critical to the function of many types of RNA. The secondary structure of a particular substring of functional RNA may depend on its surrounding sequence. Yet, some RNAs such as microRNAs retain their specific structures during biogenesis, which involves extraction of the substructure from a larger structural context, while other functional RNAs may be composed of a fusion of independent substructures. Such observations raise the question of whether particular functional RNA substructures may be selected for invariance of secondary structure to their surrounding nucleotide context. We define the property of self containment to be the tendency for an RNA sequence to robustly adopt the same optimal secondary structure regardless of whether it exists in isolation or is a substring of a longer sequence of arbitrary nucleotide content. We measured degree of self containment using a scoring method we call the self-containment index and found that miRNA stem loops exhibit high self containment, consistent with the requirement for structural invariance imposed by the miRNA biogenesis pathway, while most other structured RNAs do not. Further analysis revealed a trend toward higher self containment among clustered and conserved miRNAs, suggesting that high self containment may be a characteristic of novel miRNAs acquiring new genomic contexts. We found that miRNAs display significantly enhanced self containment compared to other functional RNAs, but we also found a trend toward natural selection for self containment in most functional RNA classes. We suggest that self containment arises out of selection for robustness against perturbations, invariance during biogenesis, and modular composition of structural function. Analysis of self containment will be important for both annotation and design of functional RNAs. A Python

  14. Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human CellsD⃞

    PubMed Central

    Valgardsdottir, Rut; Chiodi, Ilaria; Giordano, Manuela; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2005-01-01

    Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs. PMID:15788562

  15. Structural model of an mRNA in complex with the bacterial chaperone Hfq

    SciTech Connect

    Peng, Yi; Curtis, Joseph E.; Fang, Xianyang; Woodson, Sarah A.

    2014-11-17

    The Sm-like protein Hfq (host factor Q-beta phage) facilitates regulation by bacterial small noncoding RNAs (sRNAs) in response to stress and other environmental signals. In this paper, we present a low-resolution model of Escherichia coli Hfq bound to the rpoS mRNA, a bacterial stress response gene that is targeted by three different sRNAs. Selective 2'-hydroxyl acylation and primer extension, small-angle X-ray scattering, and Monte Carlo molecular dynamics simulations show that the distal face and lateral rim of Hfq interact with three sites in the rpoS leader, folding the RNA into a compact tertiary structure. These interactions are needed for sRNA regulation of rpoS translation and position the sRNA target adjacent to an sRNA binding region on the proximal face of Hfq. Finally, our results show how Hfq specifically distorts the structure of the rpoS mRNA to enable sRNA base pairing and translational control.

  16. Structural model of an mRNA in complex with the bacterial chaperone Hfq

    DOE PAGES

    Peng, Yi; Curtis, Joseph E.; Fang, Xianyang; ...

    2014-11-17

    The Sm-like protein Hfq (host factor Q-beta phage) facilitates regulation by bacterial small noncoding RNAs (sRNAs) in response to stress and other environmental signals. In this paper, we present a low-resolution model of Escherichia coli Hfq bound to the rpoS mRNA, a bacterial stress response gene that is targeted by three different sRNAs. Selective 2'-hydroxyl acylation and primer extension, small-angle X-ray scattering, and Monte Carlo molecular dynamics simulations show that the distal face and lateral rim of Hfq interact with three sites in the rpoS leader, folding the RNA into a compact tertiary structure. These interactions are needed for sRNAmore » regulation of rpoS translation and position the sRNA target adjacent to an sRNA binding region on the proximal face of Hfq. Finally, our results show how Hfq specifically distorts the structure of the rpoS mRNA to enable sRNA base pairing and translational control.« less

  17. Structural model of an mRNA in complex with the bacterial chaperone Hfq.

    PubMed

    Peng, Yi; Curtis, Joseph E; Fang, Xianyang; Woodson, Sarah A

    2014-12-02

    The Sm-like protein Hfq (host factor Q-beta phage) facilitates regulation by bacterial small noncoding RNAs (sRNAs) in response to stress and other environmental signals. Here, we present a low-resolution model of Escherichia coli Hfq bound to the rpoS mRNA, a bacterial stress response gene that is targeted by three different sRNAs. Selective 2'-hydroxyl acylation and primer extension, small-angle X-ray scattering, and Monte Carlo molecular dynamics simulations show that the distal face and lateral rim of Hfq interact with three sites in the rpoS leader, folding the RNA into a compact tertiary structure. These interactions are needed for sRNA regulation of rpoS translation and position the sRNA target adjacent to an sRNA binding region on the proximal face of Hfq. Our results show how Hfq specifically distorts the structure of the rpoS mRNA to enable sRNA base pairing and translational control.

  18. Biocomputational prediction of small non-coding RNAs in Streptomyces

    PubMed Central

    Pánek, Josef; Bobek, Jan; Mikulík, Karel; Basler, Marek; Vohradský, Jiří

    2008-01-01

    Background The first systematic study of small non-coding RNAs (sRNA, ncRNA) in Streptomyces is presented. Except for a few exceptions, the Streptomyces sRNAs, as well as the sRNAs in other genera of the Actinomyces group, have remained unstudied. This study was based on sequence conservation in intergenic regions of Streptomyces, localization of transcription termination factors, and genomic arrangement of genes flanking the predicted sRNAs. Results Thirty-two potential sRNAs in Streptomyces were predicted. Of these, expression of 20 was detected by microarrays and RT-PCR. The prediction was validated by a structure based computational approach. Two predicted sRNAs were found to be terminated by transcription termination factors different from the Rho-independent terminators. One predicted sRNA was identified computationally with high probability as a Streptomyces 6S RNA. Out of the 32 predicted sRNAs, 24 were found to be structurally dissimilar from known sRNAs. Conclusion Streptomyces is the largest genus of Actinomyces, whose sRNAs have not been studied. The Actinomyces is a group of bacterial species with unique genomes and phenotypes. Therefore, in Actinomyces, new unique bacterial sRNAs may be identified. The sequence and structural dissimilarity of the predicted Streptomyces sRNAs demonstrated by this study serve as the first evidence of the uniqueness of Actinomyces sRNAs. PMID:18477385

  19. Search for characteristic structural features of mammalian mitochondrial tRNAs.

    PubMed Central

    Helm, M; Brulé, H; Friede, D; Giegé, R; Pütz, D; Florentz, C

    2000-01-01

    A number of mitochondrial (mt) tRNAs have strong structural deviations from the classical tRNA cloverleaf secondary structure and from the conventional L-shaped tertiary structure. As a consequence, there is a general trend to consider all mitochondrial tRNAs as "bizarre" tRNAs. Here, a large sequence comparison of the 22 tRNA genes within 31 fully sequenced mammalian mt genomes has been performed to define the structural characteristics of this specific group of tRNAs. Vertical alignments define the degree of conservation/variability of primary sequences and secondary structures and search for potential tertiary interactions within each of the 22 families. Further horizontal alignments ascertain that, with the exception of serine-specific tRNAs, mammalian mt tRNAs do fold into cloverleaf structures with mostly classical features. However, deviations exist and concern large variations in size of the D- and T-loops. The predominant absence of the conserved nucleotides G18G19 and T54T55C56, respectively in these loops, suggests that classical tertiary interactions between both domains do not take place. Classification of the tRNA sequences according to their genomic origin (G-rich or G-poor DNA strand) highlight specific features such as richness/poorness in mismatches or G-T pairs in stems and extremely low G-content or C-content in the D- and T-loops. The resulting 22 "typical" mammalian mitochondrial sequences built up a phylogenetic basis for experimental structural and functional investigations. Moreover, they are expected to help in the evaluation of the possible impacts of those point mutations detected in human mitochondrial tRNA genes and correlated with pathologies. PMID:11073213

  20. Identification of candidate structured RNAs in the marine organism 'Candidatus Pelagibacter ubique'

    PubMed Central

    Meyer, Michelle M; Ames, Tyler D; Smith, Daniel P; Weinberg, Zasha; Schwalbach, Michael S; Giovannoni, Stephen J; Breaker, Ronald R

    2009-01-01

    Background Metagenomic sequence data are proving to be a vast resource for the discovery of biological components. Yet analysis of this data to identify functional RNAs lags behind efforts to characterize protein diversity. The genome of 'Candidatus Pelagibacter ubique' HTCC 1062 is the closest match for approximately 20% of marine metagenomic sequence reads. It is also small, contains little non-coding DNA, and has strikingly low GC content. Results To aid the discovery of RNA motifs within the marine metagenome we exploited the genomic properties of 'Cand. P. ubique' by targeting our search to long intergenic regions (IGRs) with relatively high GC content. Analysis of known RNAs (rRNA, tRNA, riboswitches etc.) shows that structured RNAs are significantly enriched in such IGRs. To identify additional candidate structured RNAs, we examined other IGRs with similar characteristics from 'Cand. P. ubique' using comparative genomics approaches in conjunction with marine metagenomic data. Employing this strategy, we discovered four candidate structured RNAs including a new riboswitch class as well as three additional likely cis-regulatory elements that precede genes encoding ribosomal proteins S2 and S12, and the cytoplasmic protein component of the signal recognition particle. We also describe four additional potential RNA motifs with few or no examples occurring outside the metagenomic data. Conclusion This work begins the process of identifying functional RNA motifs present in the metagenomic data and illustrates how existing completed genomes may be used to aid in this task. PMID:19531245

  1. Ab initio identification of human microRNAs based on structure motifs.

    PubMed

    Brameier, Markus; Wiuf, Carsten

    2007-12-18

    MicroRNAs (miRNAs) are short, non-coding RNA molecules that are directly involved in post-transcriptional regulation of gene expression. The mature miRNA sequence binds to more or less specific target sites on the mRNA. Both their small size and sequence specificity make the detection of completely new miRNAs a challenging task. This cannot be based on sequence information alone, but requires structure information about the miRNA precursor. Unlike comparative genomics approaches, ab initio approaches are able to discover species-specific miRNAs without known sequence homology. MiRPred is a novel method for ab initio prediction of miRNAs by genome scanning that only relies on (predicted) secondary structure to distinguish miRNA precursors from other similar-sized segments of the human genome. We apply a machine learning technique, called linear genetic programming, to develop special classifier programs which include multiple regular expressions (motifs) matched against the secondary structure sequence. Special attention is paid to scanning issues. The classifiers are trained on fixed-length sequences as these occur when shifting a window in regular steps over a genome region. Various statistical and empirical evidence is collected to validate the correctness of and increase confidence in the predicted structures. Among other things, we propose a new criterion to select miRNA candidates with a higher stability of folding that is based on the number of matching windows around their genome location. An ensemble of 16 motif-based classifiers achieves 99.9 percent specificity with sensitivity remaining on an acceptable high level when requiring all classifiers to agree on a positive decision. A low false positive rate is considered more important than a low false negative rate, when searching larger genome regions for unknown miRNAs. 117 new miRNAs have been predicted close to known miRNAs on human chromosome 19. All candidate structures match the free energy

  2. Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination.

    PubMed Central

    Le Guyader, F; Apaire-Marchais, V; Brillet, J; Billaudel, S

    1993-01-01

    Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts). Images PMID:8285700

  3. LocARNA-P: Accurate boundary prediction and improved detection of structural RNAs

    PubMed Central

    Will, Sebastian; Joshi, Tejal; Hofacker, Ivo L.; Stadler, Peter F.; Backofen, Rolf

    2012-01-01

    Current genomic screens for noncoding RNAs (ncRNAs) predict a large number of genomic regions containing potential structural ncRNAs. The analysis of these data requires highly accurate prediction of ncRNA boundaries and discrimination of promising candidate ncRNAs from weak predictions. Existing methods struggle with these goals because they rely on sequence-based multiple sequence alignments, which regularly misalign RNA structure and therefore do not support identification of structural similarities. To overcome this limitation, we compute columnwise and global reliabilities of alignments based on sequence and structure similarity; we refer to these structure-based alignment reliabilities as STARs. The columnwise STARs of alignments, or STAR profiles, provide a versatile tool for the manual and automatic analysis of ncRNAs. In particular, we improve the boundary prediction of the widely used ncRNA gene finder RNAz by a factor of 3 from a median deviation of 47 to 13 nt. Post-processing RNAz predictions, LocARNA-P's STAR score allows much stronger discrimination between true- and false-positive predictions than RNAz's own evaluation. The improved accuracy, in this scenario increased from AUC 0.71 to AUC 0.87, significantly reduces the cost of successive analysis steps. The ready-to-use software tool LocARNA-P produces structure-based multiple RNA alignments with associated columnwise STARs and predicts ncRNA boundaries. We provide additional results, a web server for LocARNA/LocARNA-P, and the software package, including documentation and a pipeline for refining screens for structural ncRNA, at http://www.bioinf.uni-freiburg.de/Supplements/LocARNA-P/. PMID:22450757

  4. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla.

    PubMed

    Rafels-Ybern, Àlbert; Torres, Adrian Gabriel; Grau-Bove, Xavier; Ruiz-Trillo, Iñaki; de Pouplana, Lluís Ribas

    2017-09-07

    The modification of adenosine to inosine at position 34 of tRNA anticodons has a profound impact upon codon-anticodon recognition. In bacteria, I34 is thought to exist only in tRNA(Arg), while in eukaryotes the modification is present in eight different tRNAs. In eukaryotes, the widespread use of I34 strongly influenced the evolution of genomes in terms of tRNA gene abundance and codon usage. In humans, codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Here we extend the analysis of distribution of codons that are recognized by I34 containing tRNAs to all phyla known to use this modification. We find that the preference for codons recognized by such tRNAs in genes with highly biased codon compositions is universal among eukaryotes, and we report that, unexpectedly, some bacterial phyla show a similar preference. We demonstrate that the genomes of these bacterial species contain previously undescribed tRNA genes that are potential substrates for deamination at position 34.

  5. Structure of bacterial respiratory complex I.

    PubMed

    Berrisford, John M; Baradaran, Rozbeh; Sazanov, Leonid A

    2016-07-01

    Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. It is the largest protein assembly of respiratory chains and one of the most elaborate redox membrane proteins known. Bacterial enzyme is about half the size of mitochondrial and thus provides its important "minimal" model. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The L-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. We have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and recently of the intact, entire complex I from T. thermophilus (536 kDa, 16 subunits, 9 iron-sulphur clusters, 64 transmembrane helices). The 95Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

  6. Cis-encoded non-coding antisense RNAs in streptococci and other low GC Gram (+) bacterial pathogens

    PubMed Central

    Cho, Kyu Hong; Kim, Jeong-Ho

    2015-01-01

    Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory non-coding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small non-coding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded non-coding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+) bacteria to provide a guide for future studies. PMID:25859258

  7. Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

    PubMed

    Bao, Weier; Greenwold, Matthew J; Sawyer, Roger H

    2016-10-15

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and mRNAs

  8. Comparative analysis of structured RNAs in S. cerevisiae indicates a multitude of different functions

    PubMed Central

    Steigele, Stephan; Huber, Wolfgang; Stocsits, Claudia; Stadler, Peter F; Nieselt, Kay

    2007-01-01

    Background Non-coding RNAs (ncRNAs) are an emerging focus for both computational analysis and experimental research, resulting in a growing number of novel, non-protein coding transcripts with often unknown functions. Whole genome screens in higher eukaryotes, for example, provided evidence for a surprisingly large number of ncRNAs. To supplement these searches, we performed a computational analysis of seven yeast species and searched for new ncRNAs and RNA motifs. Results A comparative analysis of the genomes of seven yeast species yielded roughly 2800 genomic loci that showed the hallmarks of evolutionary conserved RNA secondary structures. A total of 74% of these regions overlapped with annotated non-coding or coding genes in yeast. Coding sequences that carry predicted structured RNA elements belong to a limited number of groups with common functions, suggesting that these RNA elements are involved in post-transcriptional regulation and/or cellular localization. About 700 conserved RNA structures were found outside annotated coding sequences and known ncRNA genes. Many of these predicted elements overlapped with UTR regions of particular classes of protein coding genes. In addition, a number of RNA elements overlapped with previously characterized antisense transcripts. Transcription of about 120 predicted elements located in promoter regions and other, previously un-annotated, intergenic regions was supported by tiling array experiments, ESTs, or SAGE data. Conclusion Our computational predictions strongly suggest that yeasts harbor a substantial pool of several hundred novel ncRNAs. In addition, we describe a large number of RNA structures in coding sequences and also within antisense transcripts that were previously characterized using tiling arrays. PMID:17577407

  9. Higher order structural elements in ribosomal RNAs: pseudo-knots and the use of noncanonical pairs.

    PubMed

    Gutell, R R; Woese, C R

    1990-01-01

    The data base of prokaryotic small subunit ribosomal RNAs alone now numbers more than 400 sequences, while that for the large subunit rRNAs numbers more than 70 when eukaryotic, mitochondrial, and plastid sequences are also included. Comparisons among these rRNA sequences reveal a number of positions that covary in composition, suggestive of higher order structural elements; 5 such structures are reported for the small subunit rRNA and 15 for the large subunit rRNA. While some of these are properly (small) secondary structural elements, the majority would have to be classified as more complex "tertiary" interactions, which in some cases bring together diverse areas in the secondary structural diagram. A number of the covariances are not of the canonical type, indicating non-Watson-Crick interactions.

  10. Fitting the structurally diverse animal mitochondrial tRNAs(Ser) to common three-dimensional constraints.

    PubMed

    Steinberg, S; Gautheret, D; Cedergren, R

    1994-03-04

    We propose three-dimensional models for animal mitochondrial (amt) tRNAs lacking the D-domain based on consideration of universal constraints on tRNA to maintain functionality. The available tRNA sequences are classified into two groups, and distinct models are proposed for both classes derived from common structural features. The distance between the anticodon and the acceptor stem is comparable in the models and corresponds to that observed in conventional tRNAs. This fact averts the problem of how a shorter mitochondrial tRNA could function within the context of a protein synthesis machinery suited to full-sized tRNAs. In the models, the angle which defines the relationship between the helical domains composed of the acceptor/T-stem and the anticodon/D-stem is greater than in conventional tRNAs. These structures resemble more a "boomerang" than an "L". However, even in the boomerang model, the inner surface of tRNA would be sufficiently uncluttered to avoid steric clashes when two tRNA molecules cohabit the ribosome.

  11. Jellyfish Modulate Bacterial Dynamic and Community Structure

    PubMed Central

    Tinta, Tinkara; Kogovšek, Tjaša; Malej, Alenka; Turk, Valentina

    2012-01-01

    Jellyfish blooms have increased in coastal areas around the world and the outbreaks have become longer and more frequent over the past few decades. The Mediterranean Sea is among the heavily affected regions and the common bloom - forming taxa are scyphozoans Aurelia aurita s.l., Pelagia noctiluca, and Rhizostoma pulmo. Jellyfish have few natural predators, therefore their carcasses at the termination of a bloom represent an organic-rich substrate that supports rapid bacterial growth, and may have a large impact on the surrounding environment. The focus of this study was to explore whether jellyfish substrate have an impact on bacterial community phylotype selection. We conducted in situ jellyfish - enrichment experiment with three different jellyfish species. Bacterial dynamic together with nutrients were monitored to assess decaying jellyfish-bacteria dynamics. Our results show that jellyfish biomass is characterized by protein rich organic matter, which is highly bioavailable to ‘jellyfish - associated’ and ‘free - living’ bacteria, and triggers rapid shifts in bacterial population dynamics and composition. Based on 16S rRNA clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis, we observed a rapid shift in community composition from unculturable Alphaproteobacteria to culturable species of Gammaproteobacteria and Flavobacteria. The results of sequence analyses of bacterial isolates and of total bacterial community determined by culture independent genetic analysis showed the dominance of the Pseudoalteromonadaceae and the Vibrionaceae families. Elevated levels of dissolved proteins, dissolved organic and inorganic nutrient release, bacterial abundance and carbon production as well as ammonium concentrations characterized the degradation process. The biochemical composition of jellyfish species may influence changes in the amount of accumulated dissolved organic and inorganic nutrients. Our results can contribute insights into

  12. Structural Genomics of Bacterial Virulence Factors

    DTIC Science & Technology

    2006-05-01

    level of expression in E. coli. The highest expression was obtained for pX01-118 using B. megaterium; nevertheless, the expression level per gram of...within the B. anthracis Stern strain can be used as anti-bacterial agents for the treatment and prophylaxis of anthrax and other Gram positive bacterial...homology (68% identity). This minimum catalytic domains will be tested on other Gram positive bacteria strains in the near future, as soon as they become

  13. Jellyfish modulate bacterial dynamic and community structure.

    PubMed

    Tinta, Tinkara; Kogovšek, Tjaša; Malej, Alenka; Turk, Valentina

    2012-01-01

    Jellyfish blooms have increased in coastal areas around the world and the outbreaks have become longer and more frequent over the past few decades. The Mediterranean Sea is among the heavily affected regions and the common bloom-forming taxa are scyphozoans Aurelia aurita s.l., Pelagia noctiluca, and Rhizostoma pulmo. Jellyfish have few natural predators, therefore their carcasses at the termination of a bloom represent an organic-rich substrate that supports rapid bacterial growth, and may have a large impact on the surrounding environment. The focus of this study was to explore whether jellyfish substrate have an impact on bacterial community phylotype selection. We conducted in situ jellyfish-enrichment experiment with three different jellyfish species. Bacterial dynamic together with nutrients were monitored to assess decaying jellyfish-bacteria dynamics. Our results show that jellyfish biomass is characterized by protein rich organic matter, which is highly bioavailable to 'jellyfish-associated' and 'free-living' bacteria, and triggers rapid shifts in bacterial population dynamics and composition. Based on 16S rRNA clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis, we observed a rapid shift in community composition from unculturable Alphaproteobacteria to culturable species of Gammaproteobacteria and Flavobacteria. The results of sequence analyses of bacterial isolates and of total bacterial community determined by culture independent genetic analysis showed the dominance of the Pseudoalteromonadaceae and the Vibrionaceae families. Elevated levels of dissolved proteins, dissolved organic and inorganic nutrient release, bacterial abundance and carbon production as well as ammonium concentrations characterized the degradation process. The biochemical composition of jellyfish species may influence changes in the amount of accumulated dissolved organic and inorganic nutrients. Our results can contribute insights into possible changes in

  14. Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity

    SciTech Connect

    Gupta,Y.; Nair, D.; Wharton, R.; Aggarwal, A.

    2008-01-01

    Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycBreverse and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, in effect, acting as a 'spacer.' The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.

  15. Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs.

    PubMed

    Hickey, Scott F; Hammond, Ming C

    2014-03-20

    Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease.

    PubMed

    Lim, Youngbin; Bak, So Young; Sung, Keewon; Jeong, Euihwan; Lee, Seung Hwan; Kim, Jin-Soo; Bae, Sangsu; Kim, Seong Keun

    2016-11-02

    The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.

  17. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

    PubMed Central

    Lim, Youngbin; Bak, So Young; Sung, Keewon; Jeong, Euihwan; Lee, Seung Hwan; Kim, Jin-Soo; Bae, Sangsu; Kim, Seong Keun

    2016-01-01

    The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA–DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system. PMID:27804953

  18. Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline

    PubMed Central

    Weinberg, Zasha; Barrick, Jeffrey E.; Yao, Zizhen; Roth, Adam; Kim, Jane N.; Gore, Jeremy; Wang, Joy Xin; Lee, Elaine R.; Block, Kirsten F.; Sudarsan, Narasimhan; Neph, Shane; Tompa, Martin; Ruzzo, Walter L.

    2007-01-01

    We applied a computational pipeline based on comparative genomics to bacteria, and identified 22 novel candidate RNA motifs. We predicted six to be riboswitches, which are mRNA elements that regulate gene expression on binding a specific metabolite. In separate studies, we confirmed that two of these are novel riboswitches. Three other riboswitch candidates are upstream of either a putative transporter gene in the order Lactobacillales, citric acid cycle genes in Burkholderiales or molybdenum cofactor biosynthesis genes in several phyla. The remaining riboswitch candidate, the widespread Genes for the Environment, for Membranes and for Motility (GEMM) motif, is associated with genes important for natural competence in Vibrio cholerae and the use of metal ions as electron acceptors in Geobacter sulfurreducens. Among the other motifs, one has a genetic distribution similar to a previously published candidate riboswitch, ykkC/yxkD, but has a different structure. We identified possible non-coding RNAs in five phyla, and several additional cis-regulatory RNAs, including one in ε-proteobacteria (upstream of purD, involved in purine biosynthesis), and one in Cyanobacteria (within an ATP synthase operon). These candidate RNAs add to the growing list of RNA motifs involved in multiple cellular processes, and suggest that many additional RNAs remain to be discovered. PMID:17621584

  19. Non-coding Y RNAs as tethers and gates

    PubMed Central

    Wolin, Sandra L; Belair, Cedric; Boccitto, Marco; Chen, Xinguo; Sim, Soyeong; Taylor, David W; Wang, Hong-Wei

    2013-01-01

    Non-coding RNAs (ncRNAs) called Y RNAs are abundant components of both animal cells and a variety of bacteria. In all species examined, these ~100 nt RNAs are bound to the Ro 60 kDa (Ro60) autoantigen, a ring-shaped protein that also binds misfolded ncRNAs in some vertebrate nuclei. Although the function of Ro60 RNPs has been mysterious, we recently reported that a bacterial Y RNA tethers Ro60 to the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase) to form RYPER (Ro60/Y RNA/PNPase Exoribonuclease RNP), a new RNA degradation machine. PNPase is a homotrimeric ring that degrades single-stranded RNA, and Y RNA-mediated tethering of Ro60 increases the effectiveness of PNPase in degrading structured RNAs. Single particle electron microscopy of RYPER suggests that RNA threads through the Ro60 ring into the PNPase cavity. Further studies indicate that Y RNAs may also act as gates to regulate entry of RNA substrates into the Ro60 channel. These findings reveal novel functions for Y RNAs and raise questions about how the bacterial findings relate to the roles of these ncRNAs in animal cells. Here we review the literature on Y RNAs, highlighting their close relationship with Ro60 proteins and the hypothesis that these ncRNAs function generally to tether Ro60 rings to diverse RNA-binding proteins. PMID:24036917

  20. The structure of bacterial cell cycle and age structure of bacterial populations.

    PubMed

    Ivanov, V N; Svechnikova, T A; Stabnikova, E V; Gregirchak, N N

    1995-01-01

    Study of synchronous and asynchronous cultures of Bacillus megaterium, Bacillus thuringiensis and Bacillus licheniformis has shown that the duration of chromosomal DNA replication (period C) is proportional to the generation time, and time between two cycles of the DNA replication (known as period I). The duration of period C is nearly constant and makes up from 0.5 to 1.0 hour at the variations of the generation time from 1.5 to 2.75 hours. The duration of period B (the time between the termination of the cell division and initiation of DNA replication), and period D (the time between the termination of DNA replication and initiation of cell division) were experimentally revealed as stochastic parameters. The theoretical model of the bacterial cell cycle and the age structure of bacterial population was suggested. The main points of this theory are that periods C and I may be stochastically disposed in the division cycle of individual cells and a sum of duration of C- and I-periods is equal to generation time. The data calculated from the theoretical model were confirmed by the experimental data of flow cytofluorometric analysis of the age structure of synchronous and asynchronous cultures of the bacilli.

  1. Novel insights into the response of Atlantic salmon (Salmo salar) to Piscirickettsia salmonis: Interplay of coding genes and lncRNAs during bacterial infection.

    PubMed

    Valenzuela-Miranda, Diego; Gallardo-Escárate, Cristian

    2016-12-01

    Despite the high prevalence and impact to Chilean salmon aquaculture of the intracellular bacterium Piscirickettsia salmonis, the molecular underpinnings of host-pathogen interactions remain unclear. Herein, the interplay of coding and non-coding transcripts has been proposed as a key mechanism involved in immune response. Therefore, the aim of this study was to evidence how coding and non-coding transcripts are modulated during the infection process of Atlantic salmon with P. salmonis. For this, RNA-seq was conducted in brain, spleen, and head kidney samples, revealing different transcriptional profiles according to bacterial load. Additionally, while most of the regulated genes annotated for diverse biological processes during infection, a common response associated with clathrin-mediated endocytosis and iron homeostasis was present in all tissues. Interestingly, while endocytosis-promoting factors and clathrin inductions were upregulated, endocytic receptors were mainly downregulated. Furthermore, the regulation of genes related to iron homeostasis suggested an intracellular accumulation of iron, a process in which heme biosynthesis/degradation pathways might play an important role. Regarding the non-coding response, 918 putative long non-coding RNAs were identified, where 425 were newly characterized for S. salar. Finally, co-localization and co-expression analyses revealed a strong correlation between the modulations of long non-coding RNAs and genes associated with endocytosis and iron homeostasis. These results represent the first comprehensive study of putative interplaying mechanisms of coding and non-coding RNAs during bacterial infection in salmonids.

  2. A computational proposal for designing structured RNA pools for in vitro selection of RNAs

    PubMed Central

    Kim, Namhee; Gan, Hin Hark; Schlick, Tamar

    2007-01-01

    Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a “mixing matrix” approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems. PMID:17322501

  3. RNA structures are involved in the thermoregulation of bacterial virulence-associated traits.

    PubMed

    Grosso-Becera, María Victoria; Servín-González, Luis; Soberón-Chávez, Gloria

    2015-08-01

    Pathogenic bacteria are exposed to temperature changes during colonization of the human body and during exposure to environmental conditions. Virulence-associated traits are mainly expressed by pathogenic bacteria at 37°C. We review different cases of post-transcriptional regulation of virulence-associated proteins through RNA structures (called RNA thermometers or RNATs) that modulate the translation of mRNAs. The analysis of RNATs in pathogenic bacteria has started to produce a comprehensive picture of the structures involved, and of the genes regulated by this mechanism. However, we are still not able to predict the functionality of putative RNATs predicted by bioinformatics methods, and there is not a global approach to measure the effect of these RNA structures in gene regulation during bacterial infections. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Structural insights into bacterial modulation of the host cytoskeleton.

    PubMed

    Stebbins, C Erec

    2004-12-01

    Many bacterial pathogens manipulate the host cell cytoskeleton during infection. Such cytoskeletal modulation can occur at several points of contact between the pathogen and the host, and involves extracellular receptors, intracellular signal transduction and cytoskeletal proteins themselves. The field of bacterial pathogenesis has progressed dramatically over the past decade, such that structural knowledge is both timely and essential for a full appreciation of the biology at the pathogen-host interface. Several recent examples involving bacterial proteins that target actin, Rho family GTPases and extracellular receptors have contributed to a structural understanding of eukaryotic cytoskeletal modulation by pathogens.

  5. The 3D Structure of Some Diarrheal Causing Bacterial Toxins

    DTIC Science & Technology

    1991-07-01

    AD-A242 202 AD THE 3D STRUCTURE OF SOME DIARRHEAL CAUSING BACTERIAL TOXINS ANNUAL REPORT MARTIN SAX JULY 1, 1991 Supported by U.S. ARMY MEDICAL...21702-5012 162787A 162787A8714] AA tDA313380 11. TITLE (Include SeCUrit Clauftcatton) The 3D Structure of Some Diarrheal Causing Bacterial Toxins 12...speculative. 4 RBPRRlINCZB . 1. Marrack, P. T Kappler, J. Science 24.8, 705-711 (1990). 2. Spero et al. In Bacterial Toxins , VI 4, Ed. C Hardegree and A

  6. Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

    SciTech Connect

    Sonenberg, N.; Guertin, D.; Lee, K.A.W.

    1982-12-01

    Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

  7. Reconstitution and structure of a bacterial Pnkp1RnlHen1 RNA repair complex

    SciTech Connect

    Wang, Pei; Selvadurai, Kiruthika; Huang, Raven H.

    2016-01-22

    Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. We report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins—Pnkp1, Rnl and Hen1—that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1–Rnl–Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. Furthermore, the architecture and the locations of the active sites of the Pnkp1–Rnl–Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.

  8. Secondary structures of rRNAs from all three domains of life.

    PubMed

    Petrov, Anton S; Bernier, Chad R; Gulen, Burak; Waterbury, Chris C; Hershkovits, Eli; Hsiao, Chiaolong; Harvey, Stephen C; Hud, Nicholas V; Fox, George E; Wartell, Roger M; Williams, Loren Dean

    2014-01-01

    Accurate secondary structures are important for understanding ribosomes, which are extremely large and highly complex. Using 3D structures of ribosomes as input, we have revised and corrected traditional secondary (2°) structures of rRNAs. We identify helices by specific geometric and molecular interaction criteria, not by co-variation. The structural approach allows us to incorporate non-canonical base pairs on parity with Watson-Crick base pairs. The resulting rRNA 2° structures are up-to-date and consistent with three-dimensional structures, and are information-rich. These 2° structures are relatively simple to understand and are amenable to reproduction and modification by end-users. The 2° structures made available here broadly sample the phylogenetic tree and are mapped with a variety of data related to molecular interactions and geometry, phylogeny and evolution. We have generated 2° structures for both large subunit (LSU) 23S/28S and small subunit (SSU) 16S/18S rRNAs of Escherichia coli, Thermus thermophilus, Haloarcula marismortui (LSU rRNA only), Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. We provide high-resolution editable versions of the 2° structures in several file formats. For the SSU rRNA, the 2° structures use an intuitive representation of the central pseudoknot where base triples are presented as pairs of base pairs. Both LSU and SSU secondary maps are available (http://apollo.chemistry.gatech.edu/RibosomeGallery). Mapping of data onto 2° structures was performed on the RiboVision server (http://apollo.chemistry.gatech.edu/RiboVision).

  9. Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements

    NASA Astrophysics Data System (ADS)

    Fernandez-Chamorro, Javier; Lozano, Gloria; Garcia-Martin, Juan Antonio; Ramajo, Jorge; Dotu, Ivan; Clote, Peter; Martinez-Salas, Encarnacion

    2016-04-01

    The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

  10. Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS.

    PubMed

    Yin, Chaoran; Zhang, Ting; Li, Fang; Yang, Fan; Putatunda, Raj; Young, Won-Bin; Khalili, Kamel; Hu, Wenhui; Zhang, Yonggang

    2016-05-15

    There is an urgent need for the development of HIV-1 genome eradication strategies that lead to a permanent cure for HIV-1/AIDS. We previously reported that four guide RNAs (gRNAs) targeting HIV-1 long terminal repeats (LTR) effectively eradicated the entire HIV-1 genome. In this study, we sought to identify the best gRNAs targeting HIV-1 LTR and viral structural region and optimize gRNA pairing that can efficiently eradicate the HIV-1 genome. Highly specific gRNAs were designed using bioinformatics tools, and their capacity of guiding CRISPR-associated system 9 to cleave HIV-1 proviral DNA was evaluated using high-throughput HIV-1 luciferase reporter assay and rapid Direct-PCR genotyping. The target seed sequences for each gRNA were cloned into lentiviral vectors. HEK293T cells were cotransfected with a pEcoHIV-NL4-3-firefly-luciferase reporter vector (1 : 20) over lentiviral vectors carrying CRISPR-associated system 9 and single gRNA or various combinations of gRNAs. The EcoHIV DNA cleaving efficiency was evaluated by Direct-PCR genotyping, and the EcoHIV transcription/replication activity was examined by a luciferase reporter assay. Most of the designed gRNAs are effective to eliminate the predicted HIV-1 genome sequence between the selected two target sites. This is evidenced by the presence of PCR genotypic deletion/insertion and the decrease of luciferase reporter activity. In particular, a combination of viral structural gRNAs with LTR gRNAs provided a higher efficiency of genome eradication and an easier approach for PCR genotyping. Our screening strategy can specifically and effectively identify gRNAs targeting HIV-1 LTR and structural region to excise proviral HIV-1 from the host genome.

  11. Structural and biological studies on bacterial nitric oxide synthase inhibitors

    PubMed Central

    Holden, Jeffrey K.; Li, Huiying; Jing, Qing; Kang, Soosung; Richo, Jerry; Silverman, Richard B.; Poulos, Thomas L.

    2013-01-01

    Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria. PMID:24145412

  12. Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc.

    PubMed

    Voigt, Franka; Reuter, Michael; Kasaruho, Anisa; Schulz, Eike C; Pillai, Ramesh S; Barabas, Orsolya

    2012-12-01

    Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcripts has been long sought. A Phospholipase D-like protein, Zucchini, that is essential for piRNA processing has been proposed to be a nuclease acting in piRNA biogenesis. Here we describe the crystal structure of Zucchini from Drosophila melanogaster and show that it is very similar to the bacterial endonuclease, Nuc. The structure also reveals that homodimerization induces major conformational changes assembling the active site. The active site is situated on the dimer interface at the bottom of a narrow groove that can likely accommodate single-stranded nucleic acid substrates. Furthermore, biophysical analysis identifies protein segments essential for dimerization and provides insights into regulation of Zucchini's activity.

  13. tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter

    PubMed Central

    Keiler, Kenneth C.; Shapiro, Lucy; Williams, Kelly P.

    2000-01-01

    A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis. We have identified a circularly permuted version of the tmRNA gene in α-proteobacteria as well as in a lineage of cyanobacteria. The genes in these two groups seem to have arisen from two independent permutation events. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained. Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the α-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA. PMID:10884408

  14. Unique Gene-Silencing and Structural Properties of 2;#8242;-Fluoro-Modified siRNAs

    SciTech Connect

    Manoharan, Muthiah; Akinc, Akin; Pandey, Rajendra K.; Qin, June; Hadwiger, Philipp; John, Matthias; Mills, Kathy; Charisse, Klaus; Maier, Martin A.; Nechev, Lubomir; Greene, Emily M.; Pallan, Pradeep S.; Rozners, Eriks; Rajeev, Kallanthottathil G.; Egli, Martin

    2015-10-15

    With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.

  15. Spatial structuring of bacterial communities within individual Ginkgo biloba trees.

    PubMed

    Leff, Jonathan W; Del Tredici, Peter; Friedman, William E; Fierer, Noah

    2015-07-01

    Plant-associated microorganisms affect the health of their hosts in diverse ways, yet the distribution of these organisms within individual plants remains poorly understood. To address this knowledge gap, we assessed the spatial variability in bacterial community diversity and composition found on and in aboveground tissues of individual Ginkgo biloba trees. We sampled bacterial communities from > 100 locations per tree, including leaf, branch and trunk samples and used high-throughput sequencing of the 16S rRNA gene to determine the diversity and composition of these communities. Bacterial community structure differed strongly between bark and leaf samples, with bark samples harbouring much greater bacterial diversity and a community composition distinct from leaves. Within sample types, we observed clear spatial patterns in bacterial diversity and community composition that corresponded to the samples' proximity to the exterior of the tree. The composition of the bacterial communities found on trees is highly variable, but this variability is predictable and dependent on sampling location. Moreover, this work highlights the importance of carefully considering plant spatial structure when characterizing the microbial communities associated with plants and their impacts on plant hosts. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Structure, Mechanism, and Mutation of Bacterial Luciferase.

    PubMed

    Tinikul, Ruchanok; Chaiyen, Pimchai

    2016-01-01

    : Bacterial luciferase is a flavin-dependent monooxygenase found in bioluminescent bacteria. The enzyme catalyzes a light-emitting reaction by using reduced flavin, long chain aldehyde, and oxygen as substrates and yields oxidized flavin, carboxylic acid, and water as products with concomitant emission of blue-green light around 485-490 nm. The enzyme is a heterodimer consisting of two homologous subunits, designated as the α- and β-subunits. The reactive reaction center is located in the α-subunit, whereas the β-subunit is required for maintaining the active conformation of the α-subunit. The enzyme reaction occurs through the generation of a reactive C4a-oxygenflavin adduct, presumably C4a-peroxyflavin, before the light-emitting species is generated from the decomposition of an adduct between the C4a-peroxyflavin and the aldehyde. Because the luciferase reaction generates light, the enzyme has the potential to be used as a bioreporter for a wide variety of applications. With the recent invention of the fusion enzyme that can be expressed in mammalian cells, future possibilities for the development of additional bioreporter applications are promising.

  17. Bacterial membrane lipids: diversity in structures and pathways.

    PubMed

    Sohlenkamp, Christian; Geiger, Otto

    2016-01-01

    For many decades, Escherichia coli was the main model organism for the study of bacterial membrane lipids. The results obtained served as a blueprint for membrane lipid biochemistry, but it is clear now that there is no such thing as a typical bacterial membrane lipid composition. Different bacterial species display different membrane compositions and even the membrane composition of cells belonging to a single species is not constant, but depends on the environmental conditions to which the cells are exposed. Bacterial membranes present a large diversity of amphiphilic lipids, including the common phospholipids phosphatidylglycerol, phosphatidylethanolamine and cardiolipin, the less frequent phospholipids phosphatidylcholine, and phosphatidylinositol and a variety of other membrane lipids, such as for example ornithine lipids, glycolipids, sphingolipids or hopanoids among others. In this review, we give an overview about the membrane lipid structures known in bacteria, the different metabolic pathways involved in their formation, and the distribution of membrane lipids and metabolic pathways across taxonomical groups.

  18. Structural Genomics of Bacterial Virulence Factors

    DTIC Science & Technology

    2004-05-01

    drug design . In this first year of funding we have focused our attention on plasmid annotation, target selection, protein expression, purification and crystallization of proteins encoded by the Bacillus anthracis pXOl plasmid. We have cloned and expressed a total of 35 new proteins, and structural analysis of several of these is underway. Currently, 3 new crystal structures are essentially complete, and 6 crystal structures of anthrax Lethal Factor in complex with small molecule inhibitors provided by our collaborators have been determined, and lodged in the public data

  19. Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

    SciTech Connect

    Zhang, R.; Evans, G.; Rotella, F. J.; Westbrook, E. M.; Beno, D.; Huberman, E.; Joachimiak, A.; Collart, F. R.

    1999-01-01

    IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K{sub m} for NAD (1180 {mu}M) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 {angstrom} with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione {beta}-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

  20. DGCR8 recognizes primary transcripts of microRNAs through highly cooperative binding and formation of higher-order structures

    PubMed Central

    Faller, Michael; Toso, Daniel; Matsunaga, Michio; Atanasov, Ivo; Senturia, Rachel; Chen, Yanqiu; Zhou, Z. Hong; Guo, Feng

    2010-01-01

    DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8–pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8–pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha. PMID:20558544

  1. Bacterial Community Structures in Freshwater Polar Environments of Svalbard

    PubMed Central

    Ntougias, Spyridon; Polkowska, Żaneta; Nikolaki, Sofia; Dionyssopoulou, Eva; Stathopoulou, Panagiota; Doudoumis, Vangelis; Ruman, Marek; Kozak, Katarzyna; Namieśnik, Jacek; Tsiamis, George

    2016-01-01

    Two thirds of Svalbard archipelago islands in the High Arctic are permanently covered with glacial ice and snow. Polar bacterial communities in the southern part of Svalbard were characterized using an amplicon sequencing approach. A total of 52,928 pyrosequencing reads were analyzed in order to reveal bacterial community structures in stream and lake surface water samples from the Fuglebekken and Revvatnet basins of southern Svalbard. Depending on the samples examined, bacterial communities at a higher taxonomic level mainly consisted either of Bacteroidetes, Betaproteobacteria, and Microgenomates (OP11) or Planctomycetes, Betaproteobacteria, and Bacteroidetes members, whereas a population of Microgenomates was prominent in 2 samples. At the lower taxonomic level, bacterial communities mostly comprised Microgenomates, Comamonadaceae, Flavobacteriaceae, Legionellales, SM2F11, Parcubacteria (OD1), and TM7 members at different proportions in each sample. The abundance of OTUs shared in common among samples was greater than 70%, with the exception of samples in which the proliferation of Planctomycetaceae, Phycisphaeraceae, and Candidatus Methylacidiphilum spp. lowered their relative abundance. A multi-variable analysis indicated that As, Pb, and Sb were the main environmental factors influencing bacterial profiles. We concluded that the bacterial communities in the polar aquatic ecosystems examined mainly consisted of freshwater and marine microorganisms involved in detritus mineralization, with a high proportion of zooplankton-associated taxa also being identified. PMID:27725345

  2. Structure of bacterial communities in diverse freshwater habitats.

    PubMed

    Aizenberg-Gershtein, Yana; Vaizel-Ohayon, Dalit; Halpern, Malka

    2012-03-01

    The structures and dynamics of bacterial communities from raw source water, groundwater, and drinking water before and after filtration were studied in four seasons of a year, with culture-independent methods. Genomic DNA from water samples was analyzed by the polymerase chain reaction - denaturing gradient gel electrophoresis system and by cloning of the 16S rRNA gene. Water samples exhibited complex denaturing gradient gel electrophoresis genetic profiles composed of many bands, corresponding to a great variety of bacterial taxa. The bacterial communities of different seasons from the four sampling sites clustered into two major groups: (i) water before and after filtration, and (ii) source water and groundwater. Phylogenetic analyses of the clones from the autumn sampling revealed 13 phyla, 19 classes, and 155 operational taxonomic units. Of the clones, 66% showed less than 97% similarities to known bacterial species. Representatives of the phyla Proteobacteria, Bacteroidetes, and Actinobacteria were found at all four sampling sites. Species belonging to the phylum Firmicutes were an important component of the microbial community in filtered water. Representatives of Enterobacteriaceae were not detected, indicating the absence of fecal pollution in the drinking water. Differences were found in the bacterial populations that were sampled from the same sites in different seasons. Each water habitat had a unique bacterial profile. Drinking water harbors diverse and dynamic microbial communities, part of which may be active and resilient to chlorine disinfection. This study provides, for the first time, basic data for uncultivable drinking water bacteria in Israel.

  3. Structural basis of transcription by bacterial and eukaryotic RNA polymerases.

    PubMed

    Sekine, Shun-ichi; Tagami, Shunsuke; Yokoyama, Shigeyuki

    2012-02-01

    DNA-dependent RNA polymerase (RNAP) is responsible for cellular gene transcription. Although crystallographic studies on prokaryotic and eukaryotic RNAPs have elucidated the basic RNAP architectures, the structural details of many essential events during transcription initiation, elongation, and termination are still largely unknown. Recent crystallographic studies on a bacterial RNAP and yeast RNAP II have revealed different RNAP structural states from that of the normal transcribing complex, as well as the basis of transcription factor functions, advancing our understanding of transcription. These studies have highlighted unexpected similarities in many fundamental aspects of transcription mechanisms between the bacterial and eukaryotic transcription machineries. Remarkable differences also exist between the bacterial and eukaryotic transcription systems, suggesting directions for future studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Bacterial cell surface structures in Yersinia enterocolitica.

    PubMed

    Białas, Nataniel; Kasperkiewicz, Katarzyna; Radziejewska-Lebrecht, Joanna; Skurnik, Mikael

    2012-06-01

    Yersinia enterocolitica is a widespread member of the family of Enterobacteriaceae that contains both non-virulent and virulent isolates. Pathogenic Y. enterocolitica strains, especially belonging to serotypes O:3, O:5,27, O:8 and O:9 are etiologic agents of yersiniosis in animals and humans. Y. enterocolitica cell surface structures that play a significant role in virulence have been subject to many investigations. These include outer membrane (OM) glycolipids such as lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) and several cell surface adhesion proteins present only in virulent Y. enterocolitica, i.e., Inv, YadA and Ail. While the yadA gene is located on the Yersinia virulence plasmid the Ail, Inv, LPS and ECA are chromosomally encoded. These structures ensure the correct architecture of the OM, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms.

  5. Interconnected Cavernous Structure of Bacterial Fruiting Bodies

    DOE PAGES

    Harvey, Cameron W.; Du, Huijing; Xu, Zhiliang; ...

    2012-12-27

    The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicelular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to Imitations at different imaging methods. A new technique using Infrared Opticalmore » Coherence Tomography (OCT) revealed previously unknown details of the Internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative nigh and low spore density regions. Here, to make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The Integration of novel OCT experimental techniques with computational simulations can provide new insight Into the mechanisms that can give rise to the pattern formation seen In other biological systems such as dlctyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.« less

  6. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    SciTech Connect

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  7. Interconnected Cavernous Structure of Bacterial Fruiting Bodies

    PubMed Central

    Harvey, Cameron W.; Du, Huijing; Xu, Zhiliang; Kaiser, Dale; Aranson, Igor; Alber, Mark

    2012-01-01

    The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions. PMID:23300427

  8. Towards revealing the structure of bacterial inclusion bodies

    PubMed Central

    2009-01-01

    Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6–12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies. PMID:19806034

  9. Towards revealing the structure of bacterial inclusion bodies.

    PubMed

    Wang, Lei

    2009-01-01

    Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6-12 nm, they are comprised of residue-specific cross-beta structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.

  10. Use of the U1A protein to facilitate crystallization and structure determination of large RNAs

    PubMed Central

    Ferré-D’Amaré, Adrian R.

    2016-01-01

    Summary The preparation of well-ordered crystals of RNAs with complex three-dimensional architecture can be facilitated by engineering a binding site for the spliceosomal protein U1A into a functionally and structurally dispensable stem-loop of the RNA of interest. Once suitable crystals are obtained, the U1A protein, of known structure, can be employed to facilitate preparation of heavy atom or anomalously scattering atom derivatives, or as a source of partial model phases for the molecular replacement method. Here, we describe the methods for making U1A preparations suitable for cocrystallization with RNA. As an example, the cocrystallization of the tetracycline aptamer with U1A is also described. PMID:26227038

  11. Bacterial community structure in secondary wastewater treatment

    SciTech Connect

    Lin, K.T.J.

    1984-01-01

    Practically all process problems encountered in activated sludge treatment, such as bulking, sludge rising, septic sludge, foaming, dispersed growth, deflocculation, and pinpoint floc, are caused by poor separation of sludge in the secondary sedimentation tank. This occurs when the microorganisms fail to produce floc which settle well and results in an increase in effluent suspended solids and biochemical oxygen demand. In the past, attempts at control and prevention of such failures have been almost entirely empirical. In order to better understand these flocculation problems, a study of the community structure of the bacteria as in activated sludge has been conducted. In addition to activated sludge, samples from a trickling filter and a rotating biological contactor (RBC) also were examined for comparison.

  12. Myriad Triple-Helix-Forming Structures in the Transposable Element RNAs of Plants and Fungi.

    PubMed

    Tycowski, Kazimierz T; Shu, Mei-Di; Steitz, Joan A

    2016-05-10

    The ENE (element for nuclear expression) is a cis-acting RNA structure that protects viral or cellular noncoding RNAs (ncRNAs) from nuclear decay through triple-helix formation with the poly(A) tail or 3'-terminal A-rich tract. We expanded the roster of nine known ENEs by bioinformatic identification of ∼200 distinct ENEs that reside in transposable elements (TEs) of numerous non-metazoan and one fish species and in four Dicistrovirus genomes. Despite variation within the ENE core, none of the predicted triple-helical stacks exceeds five base triples. Increased accumulation of reporter transcripts in human cells demonstrated functionality for representative ENEs. Location close to the poly(A) tail argues that ENEs are active in TE transcripts. Their presence in intronless, but not intron-containing, hAT transposase genes supports the idea that TEs acquired ENEs to counteract the RNA-destabilizing effects of intron loss, a potential evolutionary consequence of TE horizontal transfer in organisms that couple RNA silencing to splicing deficits.

  13. Identification of conserved secondary structures and expansion segments in enod40 RNAs reveals new enod40 homologues in plants

    PubMed Central

    Gultyaev, Alexander P.; Roussis, Andreas

    2007-01-01

    enod40 is a plant gene that participates in the regulation of symbiotic interaction between leguminous plants and bacteria or fungi. Furthermore, it has been suggested to play a general role in non-symbiotic plant development. Although enod40 seems to have multiple functions, being present in many land plants, the molecular mechanisms of its activity are unclear; they may be determined though, by short peptides and/or RNA structures encoded in the enod40 genes. We utilized conserved RNA structures in enod40 sequences to search nucleotide sequence databases and identified a number of new enod40 homologues in plant species that belong to known, but also, to yet unknown enod40-containing plant families. RNA secondary structure predictions and comparative sequence analysis of enod40 RNAs allowed us to determine the most conserved structural features, present in all known enod40 genes. Remarkably, the topology and evolution of one of the conserved structural domains are similar to those of the expansion segments found in structural RNAs such as rRNAs, RNase P and SRP RNAs. Surprisingly, the enod40 RNA structural elements are much more stronger conserved than the encoded peptides. This finding suggests that some general functions of enod40 gene could be determined by the encoded RNA structure, whereas short peptides may be responsible for more diverse functions found only in certain plant families. PMID:17452360

  14. Experimental sulfate amendment alters peatland bacterial community structure.

    PubMed

    Strickman, R J S; Fulthorpe, R R; Coleman Wasik, J K; Engstrom, D R; Mitchell, C P J

    2016-10-01

    As part of a long-term, peatland-scale sulfate addition experiment, the impact of varying sulfate deposition on bacterial community responses was assessed using 16S tag encoded pyrosequencing. In three separate areas of the peatland, sulfate manipulations included an eight year quadrupling of atmospheric sulfate deposition (experimental), a 3-year recovery to background deposition following 5years of elevated deposition (recovery), and a control area. Peat concentrations of methylmercury (MeHg), a bioaccumulative neurotoxin, were measured, the production of which is attributable to a growing list of microorganisms, including many sulfate-reducing Deltaproteobacteria. The total bacterial and Deltaproteobacterial community structures in the experimental treatment differed significantly from those in the control and recovery treatments that were either indistinguishable or very similar to one another. Notably, the relatively rapid return (within three years) of bacterial community structure in the recovery treatment to a state similar to the control, demonstrates significant resilience of the peatland bacterial community to changes in atmospheric sulfate deposition. Changes in MeHg accumulation between sulfate treatments correlated with changes in the Deltaproteobacterial community, suggesting that sulfate may affect MeHg production through changes in the community structure of this group.

  15. A size-structured model of bacterial growth and reproduction.

    PubMed

    Ellermeyer, S F; Pilyugin, S S

    2012-01-01

    We consider a size-structured bacterial population model in which the rate of cell growth is both size- and time-dependent and the average per capita reproduction rate is specified as a model parameter. It is shown that the model admits classical solutions. The population-level and distribution-level behaviours of these solutions are then determined in terms of the model parameters. The distribution-level behaviour is found to be different from that found in similar models of bacterial population dynamics. Rather than convergence to a stable size distribution, we find that size distributions repeat in cycles. This phenomenon is observed in similar models only under special assumptions on the functional form of the size-dependent growth rate factor. Our main results are illustrated with examples, and we also provide an introductory study of the bacterial growth in a chemostat within the framework of our model.

  16. Changes in soil bacterial community structure with increasing disturbance frequency.

    PubMed

    Kim, Mincheol; Heo, Eunjung; Kang, Hojeong; Adams, Jonathan

    2013-07-01

    Little is known of the responsiveness of soil bacterial community structure to disturbance. In this study, we subjected a soil microcosm to physical disturbance, sterilizing 90 % of the soil volume each time, at a range of frequencies. We analysed the bacterial community structure using 454 pyrosequencing of the 16S rRNA gene. Bacterial diversity was found to decline with the increasing disturbance frequencies. Total bacterial abundance was, however, higher at intermediate and high disturbance frequencies, compared to low and no-disturbance treatments. Changing disturbance frequency also led to changes in community composition, with changes in overall species composition and some groups becoming abundant at the expense of others. Some phylogenetic groups were found to be relatively more disturbance-sensitive or tolerant than others. With increasing disturbance frequency, phylogenetic species variability (an index of community composition) itself became more variable from one sample to another, suggesting a greater role of chance in community composition. Compared to the tightly clustered community of the original undisturbed soil, in all the aged disturbed soils the lists of most abundant operational taxonomic units (OTUs) in each replicate were very different, suggesting a possible role of stochasticity in resource colonization and exploitation in the aged and disturbed soils. For example, colonization may be affected by whichever localized concentrations of bacterial populations happen to survive the last disturbance and be reincorporated in abundance into each pot. Overall, it appears that the soil bacterial community is very sensitive to physical disturbance, losing diversity, and that certain groups have identifiable 'high disturbance' vs. 'low disturbance' niches.

  17. Structural RNAs of known and unknown function identified in malaria parasites by comparative genomics and RNA analysis

    PubMed Central

    Chakrabarti, Kausik; Pearson, Michael; Grate, Leslie; Sterne-Weiler, Timothy; Deans, Jonathan; Donohue, John Paul; Ares, Manuel

    2007-01-01

    As the genomes of more eukaryotic pathogens are sequenced, understanding how molecular differences between parasite and host might be exploited to provide new therapies has become a major focus. Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. In this article we identify by comparative genomics and validate by RNA analysis numerous previously unknown structural RNAs encoded by the Plasmodium falciparum genome, including the telomerase RNA, U3, 31 snoRNAs, as well as previously predicted spliceosomal snRNAs, SRP RNA, MRP RNA, and RNAse P RNA. Furthermore, we identify six new RNA coding genes of unknown function. To investigate the relationships of the RNA coding genes to other genomic features in related parasites, we developed a genome browser for P. falciparum (http://areslab.ucsc.edu/cgi-bin/hgGateway). Additional experiments provide evidence supporting the prediction that snoRNAs guide methylation of a specific position on U4 snRNA, as well as predicting an snRNA promoter element particular to Plasmodium sp. These findings should allow detailed structural comparisons between the RNA components of the gene expression machinery of the parasite and its vertebrate hosts. PMID:17901154

  18. Actin: Structure, Function, Dynamics, and Interactions with Bacterial Toxins.

    PubMed

    Kühn, Sonja; Mannherz, Hans Georg

    Actin is one of the most abundant proteins in any eukaryotic cell and an indispensable component of the cytoskeleton. In mammalian organisms, six highly conserved actin isoforms can be distinguished, which differ by only a few amino acids. In non-muscle cells, actin polymerizes into actin filaments that form actin structures essential for cell shape stabilization, and participates in a number of motile activities like intracellular vesicle transport, cytokinesis, and also cell locomotion. Here, we describe the structure of monomeric and polymeric actin, the polymerization kinetics, and its regulation by actin-binding proteins. Probably due to its conserved nature and abundance, actin and its regulating factors have emerged as prefered targets of bacterial toxins and effectors, which subvert the host actin cytoskeleton to serve bacterial needs.

  19. Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures.

    PubMed Central

    Hernández, C; Flores, R

    1992-01-01

    Peach latent mosaic viroid (PLMVd), the causal agent of peach latent mosaic disease, has been sequenced and found to be a circular RNA molecule of 337 nucleotide residues, which adopts a branched conformation when it is folded in the model of lowest free energy. PLMVd exhibits limited homologies with other viroids and some satellite RNAs, but it does not have any of the central conserved sequences characteristic of the subgroups of typical viroids. However, a segment of approximately one-third of the PLMVd sequence has the elements required to form in the RNAs of both polarities the hammerhead structures proposed to act in the in vitro self-cleavage of avocado sunblotch viroid (ASBVd) and some satellite RNAs. Plus and minus partial- and full-length RNA transcripts of PLMVd containing the hammerhead structures displayed self-cleavage during transcription and after purification as predicted by these structures. These data are consistent with the high stability of the PLMVd hammerhead structures, more similar to the corresponding structures of some satellite RNAs than to those of ASBVd, and indicate that the self-cleavage reactions of PLMVd are most probably mediated by single hammerhead structures. Our results support the inclusion of PLMVd in a viroid subgroup represented by ASBVd, whose members are characterized by their ability to self-cleave in vitro, and probably in vivo, through hammerhead structures. A consensus phylogenetic tree has been obtained suggesting that PLMVd, together with ASBVd, may represent an evolutionary link between viroids and viroid-like satellite RNAs. Images PMID:1373888

  20. Identification and Characterization of Novel Small RNAs in Rickettsia prowazekii

    PubMed Central

    Schroeder, Casey L. C.; Narra, Hema P.; Sahni, Abha; Rojas, Mark; Khanipov, Kamil; Patel, Jignesh; Shah, Riya; Fofanov, Yuriy; Sahni, Sanjeev K.

    2016-01-01

    Emerging evidence implicates a critically important role for bacterial small RNAs (sRNAs) as post-transcriptional regulators of physiology, metabolism, stress/adaptive responses, and virulence, but the roles of sRNAs in pathogenic Rickettsia species remain poorly understood. Here, we report on the identification of both novel and well-known bacterial sRNAs in Rickettsia prowazekii, known to cause epidemic typhus in humans. RNA sequencing of human microvascular endothelial cells (HMECs), the preferred targets during human rickettsioses, infected with R. prowazekii revealed the presence of 35 trans-acting and 23 cis-acting sRNAs, respectively. Of these, expression of two trans-acting (Rp_sR17 and Rp_sR60) and one cis-acting (Rp_sR47) novel sRNAs and four well-characterized bacterial sRNAs (RNaseP_bact_a, α-tmRNA, 4.5S RNA, 6S RNA) was further confirmed by Northern blot or RT-PCR analyses. The transcriptional start sites of five novel rickettsial sRNAs and 6S RNA were next determined using 5′ RLM-RACE yielding evidence for their independent biogenesis in R. prowazekii. Finally, computational approaches were employed to determine the secondary structures and potential mRNA targets of novel sRNAs. Together, these results establish the presence and expression of sRNAs in R. prowazekii during host cell infection and suggest potential functional roles for these important post-transcriptional regulators in rickettsial biology and pathogenesis. PMID:27375581

  1. Bacterial, plant, and fungal carbohydrate structure databases: daily usage.

    PubMed

    Toukach, Philip V; Egorova, Ksenia S

    2015-01-01

    Natural carbohydrates play important roles in living systems and therefore are used as diagnostic and therapeutic targets. The main goal of glycomics is systematization of carbohydrates and elucidation of their role in human health and disease. The amount of information on natural carbohydrates accumulates rapidly, but scientists still lack databases and computer-assisted tools needed for orientation in the glycomic information space. Therefore, freely available, regularly updated, and cross-linked databases are demanded. Bacterial Carbohydrate Structure Database (Bacterial CSDB) was developed for provision of structural, bibliographic, taxonomic, NMR spectroscopic, and other related information on bacterial and archaeal carbohydrate structures. Its main features are (1) coverage above 90%, (2) high data consistence (above 90% of error-free records), and (3) presence of manually verified bibliographic, NMR spectroscopic, and taxonomic annotations. Recently, CSDB has been expanded to cover carbohydrates of plant and fungal origin. The achievement of full coverage in the plant and fungal domains is expected in the future. CSDB is freely available on the Internet as a web service at http://csdb.glycoscience.ru. This chapter aims at showing how to use CSDB in your daily scientific practice.

  2. Experimental warming effects on the bacterial community structure and diversity

    NASA Astrophysics Data System (ADS)

    Kim, W.; Han, S.; Adams, J.; Son, Y.

    2014-12-01

    The objective of this study is to investigate the responses of soil bacterial community to future temperature increase by conducting open-field warming experiment. We conducted an open-field experimental warming system using infra-red heater in 2011 and regulated the temperature of warmed plots by 3oC higher than that of control plots constantly. The seeds of Pinus densiflora, Abies holophylla, Abies koreana, Betula costata, Quercus variabilis, Fraxinus rhynchophylla, and Zelkova serrata were planted in each 1 m × 1 m plot (n=3) in April, 2012. We collected soil samples from the rhizosphere of 7 tree species. DNA was extracted and PCR-amplified for the bacterial 16S gene targeting V1-V3 region. The paired-end sequencing was performed at Beijing Genome Institute (BGI, Hong Kong, China) using 2× 100 bp Hiseq2000 (Illumina). This study aimed to answer the following prediction/hypothesis: 1) Experimental warming will change the structure of soil bacterial community, 2) There will be distinct 'indicator group' which response to warming treatment relatively more sensitive than other groups. 3) Warming treatment will enhance the microbial activity in terms of soil respiration. 4) The rhizoplane bacterial communities for each of 7 tree species will show different response pattern to warming treatment. Since the sequence data does not arrive before the submission deadline, therefore, we would like to present the results and discussions on December 2014, AGU Fall Meeting.

  3. The primary structure of oocyte and somatic 5S rRNAs from the loach Misgurnus fossilis.

    PubMed Central

    Mashkova, T D; Serenkova, T I; Mazo, A M; Avdonina, T A; Timofeyeva MYa; Kisselev, L L

    1981-01-01

    Somatic and oocyte 5S rRNAs from the liver and unfertilized eggs of the loach (Misgurnus fossilis have been sequenced and found to differ in six nucleotides. All the substitutions are confined to the 5'-half of the molecules; 4 of them are pyrimidine-pyrimidine substitutions, and 2 are purine-pyrimidine ones. Considerable differences, both in the position and the character of substitutions, have been established when these 5S rRNAs were compared with somatic and oocyte 5S rRNAs from Xenopus borealis and Xenopus laevis. Among the known primary structures, somatic 5S rRNA of M. fossilis is most similar to trout 5S rRNA. Images PMID:7197777

  4. Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea

    PubMed Central

    Toffano-Nioche, Claire; Gautheret, Daniel; Leclerc, Fabrice

    2015-01-01

    A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea. PMID:26240384

  5. Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression.

    PubMed

    Engreitz, Jesse M; Ollikainen, Noah; Guttman, Mitchell

    2016-12-01

    Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples - including Xist, which orchestrates X chromosome inactivation - has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus.

  6. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome

    PubMed Central

    Zhang, Jingji; Ieong, Ka-Weng; Johansson, Magnus; Ehrenberg, Måns

    2015-01-01

    We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200–80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNAHis and, as also seen in vivo, Glu-tRNAGlu. We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here. PMID:26195797

  7. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome.

    PubMed

    Zhang, Jingji; Ieong, Ka-Weng; Johansson, Magnus; Ehrenberg, Måns

    2015-08-04

    We used a cell-free system with pure Escherichia coli components to study initial codon selection of aminoacyl-tRNAs in ternary complex with elongation factor Tu and GTP on messenger RNA-programmed ribosomes. We took advantage of the universal rate-accuracy trade-off for all enzymatic selections to determine how the efficiency of initial codon readings decreased linearly toward zero as the accuracy of discrimination against near-cognate and wobble codon readings increased toward the maximal asymptote, the d value. We report data on the rate-accuracy variation for 7 cognate, 7 wobble, and 56 near-cognate codon readings comprising about 15% of the genetic code. Their d values varied about 400-fold in the 200-80,000 range depending on type of mismatch, mismatch position in the codon, and tRNA isoacceptor type. We identified error hot spots (d = 200) for U:G misreading in second and U:U or G:A misreading in third codon position by His-tRNA(His) and, as also seen in vivo, Glu-tRNA(Glu). We suggest that the proofreading mechanism has evolved to attenuate error hot spots in initial selection such as those found here.

  8. Interactions in Bacterial Biofilm Development: A Structural Perspective

    PubMed Central

    Garnett, James A; Matthews, Steve

    2012-01-01

    A community-based life style is the normal mode of growth and survival for many bacterial species. These cellular accretions or biofilms are initiated upon recognition of solid phases by cell surface exposed adhesive moieties. Further cell-cell interactions, cell signalling and bacterial replication leads to the establishment of dense populations encapsulated in a mainly self-produced extracellular matrix; this comprises a complex mixture of macromolecules. These fascinating architectures protect the inhabitants from radiation damage, dehydration, pH fluctuations and antimicrobial compounds. As such they can cause bacterial persistence in disease and problems in industrial applications. In this review we discuss the current understandings of these initial biofilm-forming processes based on structural data. We also briefly describe latter biofilm maturation and dispersal events, which although lack high-resolution insights, are the present focus for many structural biologists working in this field. Finally we give an overview of modern techniques aimed at preventing and disrupting problem biofilms. PMID:23305361

  9. From bacterial to human dihydrouridine synthase: automated structure determination

    SciTech Connect

    Whelan, Fiona Jenkins, Huw T.; Griffiths, Samuel C.; Byrne, Robert T.; Dodson, Eleanor J.; Antson, Alfred A.

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  10. Isolation of translating ribosomes containing peptidyl-tRNAs for functional and structural analyses.

    PubMed

    Shirole, Nitin; Balasubramanian, Sreeram; Yanofsky, Charles; Cruz-Vera, Luis

    2011-02-25

    Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism. Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled m

  11. Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs

    DOE PAGES

    Michalska, Karolina; Gucinski, Grant C.; Garza-Sanchez, Fernando; ...

    2017-08-11

    Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxinmore » specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Altogether, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.« less

  12. A neutral pH thermal hydrolysis method for quantification of structured RNAs

    PubMed Central

    Wilson, Stephen C.; Cohen, Daniel T.; Wang, Xin C.; Hammond, Ming C.

    2014-01-01

    Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative 31P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays. PMID:24860014

  13. Synthesis, Oxidation Behavior, Crystallization and Structure of 2'-Methylseleno Guanosine Containing RNAs

    SciTech Connect

    Moroder,H.; Kreutz, C.; Lang, K.; Serganov, A.; Micura, R.

    2006-01-01

    We have recently introduced a basic concept for the combined chemical and enzymatic preparation of site-specifically modified 2'-methylseleno RNAs which represent useful derivatives for phasing of X-ray crystallographic data during their three-dimensional structure determination. Here, we introduce the first synthesis of an appropriate guanosine phosphoramidite, which complements the thus far established set of 2'-methylseleno-modified uridine, cytidine, and adenosine building blocks for solid-phase synthesis. The novel building block was readily incorporated into RNA. Importantly, it was the 2'-methylseleno-guanosine-labeled RNA that allowed us to reveal the reversible oxidation/reduction behavior of the Se moiety and thus it represents a valuable contribution to the understanding of the action of threo-1,4-dimercapto-2,3-butanediol (DTT) required during solid-phase synthesis, deprotection, and crystallization of selenium-containing RNA. In addition, we investigated 2'-methylseleno RNA with respect to crystallization properties. Our studies revealed that the Se modification significantly increases the range of conditions leading to crystal growth. Moreover, we determined the crystal structures of model RNA helices and showed that the Se modification can affect crystal packing interactions, thus potentially expanding the possibilities for obtaining the best crystal form.

  14. Molecular medicine of microRNAs: structure, function and implications for diabetes.

    PubMed

    Hennessy, Erica; O'Driscoll, Lorraine

    2008-08-15

    MicroRNAs (miRNAs) are a family of endogenous small noncoding RNA molecules, of 19-28 nucleotides in length. In humans, up to 3% of all genes are estimated to encode these evolutionarily conserved sequences. miRNAs are thought to control expression of thousands of target mRNAs. Mammalian miRNAs generally negatively regulate gene expression by repressing translation, possibly through effects on mRNA stability and compartmentalisation, and/or the translation process itself. An extensive range of in silico and experimental techniques have been applied to our understanding of the occurrence and functional relevance of such sequences, and antisense technologies have been successfully used to control miRNA expression in vitro and in vivo. Interestingly, miRNAs have been identified in both normal and pathological conditions, including differentiation and development, metabolism, proliferation, cell death, viral infection and cancer. Of specific relevance and excitement to the area of diabetes research, miRNA regulation has been implicated in insulin secretion from pancreatic beta-cells, diabetic heart conditions and nephropathy. Further analyses of miRNAs in vitro and in vivo will, undoubtedly, enable us determine their potential to be exploited as therapeutic targets in diabetes.

  15. Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing

    PubMed Central

    Beisel, Chase L.; Chen, Yvonne Y.; Culler, Stephanie J.; Hoff, Kevin G.; Smolke, Christina D.

    2011-01-01

    MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine. PMID:21149259

  16. From bacterial to human dihydrouridine synthase: automated structure determination

    PubMed Central

    Whelan, Fiona; Jenkins, Huw T.; Griffiths, Samuel C.; Byrne, Robert T.; Dodson, Eleanor J.; Antson, Alfred A.

    2015-01-01

    The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr_rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer. PMID:26143927

  17. The idiosyncrasy of spatial structure in bacterial competition.

    PubMed

    Hol, Felix J H; Galajda, Peter; Woolthuis, Rutger G; Dekker, Cees; Keymer, Juan E

    2015-06-17

    The spatial structure of a habitat can have a strong impact on community dynamics. Different experimental approaches exist to explore the effect of spatial structure on bacterial communities. To investigate the effect of 'space', a single implementation of spatial structure is often contrasted to bacterial community dynamics in well-mixed cultures. While such comparisons are useful, it is likely that the observed dynamics will be particular to the specific experimental implementation of spatial structure. In order to address this question, we track the community dynamics of a two-strain Escherichia coli community in various spatial habitats and relate the observed dynamics to the structure of a habitat. By tracking the community dynamics of rpoS wild-type and mutant E. coli in radially expanding colonies on solid and semi-solid agar plates, we find that the mutant strain outcompetes the wild-type on semi-solid agar plates, whereas the two strains coexist on solid agar. We compare these results to previous studies in which the same two strains were shown to coexist in habitats spatially structured by microfabrication, while the mutant outcompeted the wild-type in well-mixed batch cultures. Together, these observations show that different implementations of space may result in qualitatively different community dynamics. Furthermore, we argue that the same competitive outcome (e.g. coexistence) may arise from distinct underlying dynamics in different experimental implementations of spatial structure. Our observations demonstrate that different experimental implementations of spatial structure may not only lead to quantitatively different communities (changes in the relative abundance of types) but can also lead to qualitatively different outcomes of long-term community dynamics (coexistence versus extinction and loss of biodiversity).

  18. A coarse-grained model with implicit salt for RNAs: Predicting 3D structure, stability and salt effect

    SciTech Connect

    Shi, Ya-Zhou; Wang, Feng-Hua; Wu, Yuan-Yan; Tan, Zhi-Jie

    2014-09-14

    To bridge the gap between the sequences and 3-dimensional (3D) structures of RNAs, some computational models have been proposed for predicting RNA 3D structures. However, the existed models seldom consider the conditions departing from the room/body temperature and high salt (1M NaCl), and thus generally hardly predict the thermodynamics and salt effect. In this study, we propose a coarse-grained model with implicit salt for RNAs to predict 3D structures, stability, and salt effect. Combined with Monte Carlo simulated annealing algorithm and a coarse-grained force field, the model folds 46 tested RNAs (≤45 nt) including pseudoknots into their native-like structures from their sequences, with an overall mean RMSD of 3.5 Å and an overall minimum RMSD of 1.9 Å from the experimental structures. For 30 RNA hairpins, the present model also gives the reliable predictions for the stability and salt effect with the mean deviation ∼ 1.0 °C of melting temperatures, as compared with the extensive experimental data. In addition, the model could provide the ensemble of possible 3D structures for a short RNA at a given temperature/salt condition.

  19. Three separate classes of bacterial ice nucleation structures.

    PubMed

    Turner, M A; Arellano, F; Kozloff, L M

    1990-05-01

    Studies of the properties of the ice nucleation structure exposed on the surfaces of various bacteria such as Pseudomonas syringae, Erwinia herbicola, or various strains of Ice+ recombinant Escherichia coli have shown that there are clearly three major related but chemically distinct types of structures on these cells. First, the ability of Ice+ cells to nucleate super-cooled D2O has been examined, and it has been found that this ability (relative to the ability of the same cells to nucleate super-cooled H2O) exhibited three characteristic nucleating patterns. The rarest structure, called class A, is found on only a small fraction of cells in a culture, nucleates H2O at temperatures above -4.4 degrees C, and is an effective nucleator of super-cooled D2O. A second class of structure, called class B, is found on a larger portion of the cells, nucleates H2O between -4.8 and -5.7 degrees C, and is a relatively poor nucleator of super-cooled D2O. The class C structure is found on almost all cells and nucleates at -7.6 degrees C or colder. These three classes of structures were also differentiated by their sensitivities to low concentrations of water-miscible organic solvents such as dioxane or dimethyl sulfoxide. Depending on the specific bacterial strain, the addition of these solvents to bacterial suspensions lowered the nucleation activity of the class A structure by 1,000-fold or more. The nucleation activities of class B structures in the same culture were highly resistant to these compounds and were lowered only by 20 to 40%. The class C structures were more sensitive than Class B structures were, and the nucleation activities decreased 70 to 90%. Finally, the pH sensitivity of these three classes of structures was examined. The class A structure was destroyed in buffers at pH 4.5 lower but was stable in buffers at higher pHs. The class B structure was less sensitive to acidic buffers but was destroyed at pH 5.5 or lower and was stable at higher pHs. However, the

  20. Changes in the bacterial community structure in stored wormbed leachate.

    PubMed

    Romero-Tepal, Elda M; Contreras-Blancas, Eduardo; Navarro-Noya, Yendi E; Ruíz-Valdiviezo, Víctor M; Luna-Guido, Marco; Gutiérrez-Miceli, Federico A; Dendooven, Luc

    2014-01-01

    Organic wastes, such as cow manure, are often composted with earthworms (vermicomposting) while excess water is drained and collected. This wormbed leachate is nutrient-rich and it has been extensively used to fertilize plants. However, it is derived partially from a not yet finished compost process and could exhibit phytotoxicity or contain potentially hazardous microorganisms. The bacterial community in wormbed leachate derived from vermicomposting of cow manure was studied by pyrosequencing the 16S rRNA gene. The fresh wormbed leachate was rich in Mollicutes, particularly the genus Acholeplasma which contain phytopathogen species. The abundance of the Mollicutes decreased when the leachate was stored, while that of the Rhizobiales and the genus Pseudomonas increased. The bacterial communities changed rapidly in the leachate during storage. The changes in ammonium, nitrate and inorganic carbon content of the wormbed leachate when stored were correlated to changes in the bacterial community structure. It was found that storage of the wormbed leachate might be required before it can be applied to crops as large proportions of potentially plant pathogens were found in the fresh leachate.

  1. A structural comparison of bacterial microfossils versus nanobacteria and nanofossils

    NASA Astrophysics Data System (ADS)

    Southam, G.; Donald, R.

    1999-12-01

    The formation of bacterial microfossils results from the cell surface immobilization of soluble heavy metals (biomineralization) via passive ionic interactions or by the formation and release of chemical reactive metabolic by-products. These metal-encrusted cell surfaces are resistant to re-mobilization and are typically the only component of the cell that is preserved, for possibly as long as several billion years. The size and shape of microfossils are determined by bacterial morphology, which includes spherical, rod, filamentous, vibriod, helical and stalked structures. The examination of ultra-thin sections using transmission electron microscopy (TEM) reveals that mineralized bacterial cells have the basic shape of the original cell from which they formed and appear hollow. Even in rare cases when the cell envelope and the cytoplasm are mineralized, the cell envelope can be differentiated from the cytoplasm preserving the original cell morphology. Scanning electron microscopy (SEM) cannot differentiate between geochemical and geomicrobiological mineral precipitation. The term `nanobacteria' has been used to describe spherical or rod-shaped minerals (tens of nanometers in diameter) observed using SEM. While these minerals may represent mineralized portions of bacteria, e.g., membrane vesicles, stalks or flagella, they are too small to be bacteria. Conversely, `nanobacteria' may simply represent solid, inorganic precipitates.

  2. Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

    PubMed

    Matsuura, Yoshiyuki

    2016-05-22

    Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza.

  3. cis-Acting 5' hammerhead ribozyme optimization for in vitro transcription of highly structured RNAs.

    PubMed

    Meyer, Mélanie; Masquida, Benoît

    2014-01-01

    RNA-mediated biological processes usually require precise definition of 5' and 3' ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5' hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.

  4. Myriad Triple-Helix-Forming Structures in the Transposable Element RNAs of Plants and Fungi

    PubMed Central

    Tycowski, Kazimierz T.; Shu, Mei-Di; Steitz, Joan A.

    2016-01-01

    SUMMARY The ENE (element for nuclear expression) is a cis-acting RNA structure that protects viral or cellular noncoding (nc)RNAs from nuclear decay through triple-helix formation with the poly(A) tail or 3′-terminal A-rich tract. We expanded the roster of 9 known ENEs by bioinformatic identification of ~200 distinct ENEs that reside in transposable elements (TEs) of numerous non-metazoan and one fish species, and in four Dicistrovirus genomes. Despite variation within the ENE core, none of the predicted triple-helical stacks exceeds five base triples. Increased accumulation of reporter transcripts in human cells demonstrated functionality for representative ENEs. Location close to the poly(A) tail argues that ENEs are active in TE transcripts. Their presence in intronless but not intron-containing hAT transposase genes supports the idea that TEs acquired ENEs to counteract the RNA-destabilizing effects of intron loss, a potential evolutionary consequence of TE horizontal transfer in organisms that couple RNA silencing to splicing deficits. PMID:27134163

  5. Molecular, Cellular, and Structural Mechanisms of Cocaine Addiction: A Key Role for MicroRNAs

    PubMed Central

    Jonkman, Sietse; Kenny, Paul J

    2013-01-01

    The rewarding properties of cocaine play a key role in establishing and maintaining the drug-taking habit. However, as exposure to cocaine increases, drug use can transition from controlled to compulsive. Importantly, very little is known about the neurobiological mechanisms that control this switch in drug use that defines addiction. MicroRNAs (miRNAs) are small non-protein coding RNA transcripts that can regulate the expression of messenger RNAs that code for proteins. Because of their highly pleiotropic nature, each miRNA has the potential to regulate hundreds or even thousands of protein-coding RNA transcripts. This property of miRNAs has generated considerable interest in their potential involvement in complex psychiatric disorders such as addiction, as each miRNA could potentially influence the many different molecular and cellular adaptations that arise in response to drug use that are hypothesized to drive the emergence of addiction. Here, we review recent evidence supporting a key role for miRNAs in the ventral striatum in regulating the rewarding and reinforcing properties of cocaine in animals with limited exposure to the drug. Moreover, we discuss evidence suggesting that miRNAs in the dorsal striatum control the escalation of drug intake in rats with extended cocaine access. These findings highlight the central role for miRNAs in drug-induced neuroplasticity in brain reward systems that drive the emergence of compulsive-like drug use in animals, and suggest that a better understanding of how miRNAs control drug intake will provide new insights into the neurobiology of drug addiction. PMID:22968819

  6. 75 FR 52755 - Draft Guidance for Industry on Acute Bacterial Skin and Skin Structure Infections: Developing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-27

    ... HUMAN SERVICES Food and Drug Administration Draft Guidance for Industry on Acute Bacterial Skin and Skin... guidance for industry entitled ``Acute Bacterial Skin and Skin Structure Infections: Developing Drugs for... the development of antimicrobial drugs for the treatment of acute bacterial skin and skin structure...

  7. Genomewide analysis of Drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation

    PubMed Central

    Westholm, Jakub O.; Miura, Pedro; Olson, Sara; Shenker, Sol; Joseph, Brian; Sanfilippo, Piero; Celniker, Susan E.; Graveley, Brenton R.; Lai, Eric C.

    2014-01-01

    Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues and cultured cells, to rigorously annotate >2500 fruitfly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs, and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase dramatically relative to linear isoforms during CNS aging, and constitute a novel aging biomarker. PMID:25544350

  8. Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation

    DOE PAGES

    Westholm, Jakub  O.; Miura, Pedro; Olson, Sara; ...

    2014-11-26

    Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and codingmore » conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker.« less

  9. Bacterial Community Structure Response to Petroleum Concentration in Groundwater

    NASA Astrophysics Data System (ADS)

    Kitts, C. L.; Wrighton, K. C.; Phillips, W. A.; Cano, R. J.; Lundegard, P. D.

    2004-12-01

    This study characterized the bacterial community present in groundwater samples from the Guadalupe Dunes Restoration Project on the central California coast. The purpose of the study was to determine the changes in bacterial community structure and function in response to variations in the concentration of dissolved phase total petroleum hydrocarbons (TPH) in groundwater plumes at the site. For the purpose of this study groundwater samples were collected at varying distance from TPH source zones in 10 different plumes. All samples were analyzed for ammonia, phosphate, TPH, methane, oxygen, carbon dioxide, nitrate, sulfate, and dissolved iron levels. Chemical analysis revealed that the groundwater chemistry varied between plumes and on a well-to-well basis within a plume. Principle component analyses (PCA) demonstrated that TPH degradation related parameters explained 28% of the variation in the groundwater chemistry. In addition to the physical and chemical analyses, four liters of each groundwater sample were filtered and bacterial DNA was isolated to determine the relationship between groundwater chemistry and bacterial community structure and function. Specific Polymerase Chain Reaction (PCR) primers were used to characterize populations of Eubacteria, and Archaea, as well as function genes for sulfate reducing, methanotrophic, and methanogenic bacteria. Terminal Restriction Fragment (TRF) Length Polymorphisms (or T-RFLP) were used to analyze community structure. Eubacterial and Archaeal groundwater communities were separated into distinct clusters which did not clearly reflect changes in groundwater chemical parameters unless individual plumes were analyzed separately. However, specific Eubacterial and Archaeal TRF peaks did correspond to known petroleum degrading organisms and methanogenic bacteria, respectively. Only one sample produced a positive result for the sulfite reductase gene (dsrAB), indicating that sulfate reduction may not be a dominant process at

  10. Unraveling the Molecular Mechanisms Underlying the Nasopharyngeal Bacterial Community Structure

    PubMed Central

    de Steenhuijsen Piters, Wouter A. A.

    2016-01-01

    ABSTRACT The upper respiratory tract is colonized by a diverse array of commensal bacteria that harbor potential pathogens, such as Streptococcus pneumoniae. As long as the local microbial ecosystem—also called “microbiome”—is in balance, these potentially pathogenic bacterial residents cause no harm to the host. However, similar to macrobiological ecosystems, when the bacterial community structure gets perturbed, potential pathogens can overtake the niche and cause mild to severe infections. Recent studies using next-generation sequencing show that S. pneumoniae, as well as other potential pathogens, might be kept at bay by certain commensal bacteria, including Corynebacterium and Dolosigranulum spp. Bomar and colleagues are the first to explore a specific biological mechanism contributing to the antagonistic interaction between Corynebacterium accolens and S. pneumoniae in vitro [L. Bomar, S. D. Brugger, B. H. Yost, S. S. Davies, K. P. Lemon, mBio 7(1):e01725-15, 2016, doi:10.1128/mBio.01725-15]. The authors comprehensively show that C. accolens is capable of hydrolyzing host triacylglycerols into free fatty acids, which display antipneumococcal properties, suggesting that these bacteria might contribute to the containment of pneumococcus. This work exemplifies how molecular epidemiological findings can lay the foundation for mechanistic studies to elucidate the host-microbe and microbial interspecies interactions underlying the bacterial community structure. Next, translation of these results to an in vivo setting seems necessary to unveil the magnitude and importance of the observed effect in its natural, polymicrobial setting. PMID:26838716

  11. Mechanism and structure of the bacterial type IV secretion systems.

    PubMed

    Christie, Peter J; Whitaker, Neal; González-Rivera, Christian

    2014-08-01

    The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the 'archetypal' A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Structure-based design of bacterial nitric oxide synthase inhibitors.

    PubMed

    Holden, Jeffrey K; Kang, Soosung; Hollingsworth, Scott A; Li, Huiying; Lim, Nathan; Chen, Steven; Huang, He; Xue, Fengtian; Tang, Wei; Silverman, Richard B; Poulos, Thomas L

    2015-01-22

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial ( Holden , , Proc. Natl. Acad. Sci. U.S.A. 2013 , 110 , 18127 ). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

  13. Structure-Based Design of Bacterial Nitric Oxide Synthase Inhibitors

    PubMed Central

    2015-01-01

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial (Holden, , Proc. Natl. Acad. Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors. PMID:25522110

  14. Towards an automated analysis of bacterial peptidoglycan structure.

    PubMed

    Bern, Marshall; Beniston, Richard; Mesnage, Stéphane

    2017-01-01

    Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3-3 and 4-3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3-3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported. Graphical Abstract The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry.

  15. Structure-based design of bacterial nitric oxide synthase inhibitors

    DOE PAGES

    Holden, Jeffrey K.; Kang, Soosung; Hollingsworth, Scott A.; ...

    2014-12-18

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial. Here wemore » present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Lastly, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.« less

  16. Structure-based design of bacterial nitric oxide synthase inhibitors

    SciTech Connect

    Holden, Jeffrey K.; Kang, Soosung; Hollingsworth, Scott A.; Li, Huiying; Lim, Nathan; Chen, Steven; Huang, He; Xue, Fengtian; Tang, Wei; Silverman, Richard B.; Poulos, Thomas L.

    2014-12-18

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial. Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Lastly, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

  17. Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently

    PubMed Central

    Yano, Takehisa; Miyahara, Yoshiko; Morii, Noriyuki; Okano, Tetsuya

    2015-01-01

    The genus Methylobacterium tolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we found that the combination of BAC with particular alcohols at nonlethal concentrations in terms of their solitary uses significantly reduced bacterial viability after only 5 min of exposure. Among the alcohols, Raman spectroscopic analyses showed that pentanol (pentyl alcohol [PeA]) and benzyl alcohol (BzA) accelerated the cellular accumulation of BAC. Fluorescence spectroscopic assays and morphological assays with giant vesicles indicated that PeA rarely attacked membrane structures, while BzA increased the membrane fluidity and destabilized the structures. Other fluorescent spectroscopic assays indicated that PeA and BzA inactivate bacterial membrane proteins, including an efflux pump for BAC transportation. These findings suggested that the inactivation of membrane proteins by PeA and BzA led to the cellular accumulation but that only BzA also enhanced BAC penetration by membrane fluidization at nonlethal concentrations. PMID:26519389

  18. Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently.

    PubMed

    Yano, Takehisa; Miyahara, Yoshiko; Morii, Noriyuki; Okano, Tetsuya; Kubota, Hiromi

    2015-10-30

    The genus Methylobacterium tolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we found that the combination of BAC with particular alcohols at nonlethal concentrations in terms of their solitary uses significantly reduced bacterial viability after only 5 min of exposure. Among the alcohols, Raman spectroscopic analyses showed that pentanol (pentyl alcohol [PeA]) and benzyl alcohol (BzA) accelerated the cellular accumulation of BAC. Fluorescence spectroscopic assays and morphological assays with giant vesicles indicated that PeA rarely attacked membrane structures, while BzA increased the membrane fluidity and destabilized the structures. Other fluorescent spectroscopic assays indicated that PeA and BzA inactivate bacterial membrane proteins, including an efflux pump for BAC transportation. These findings suggested that the inactivation of membrane proteins by PeA and BzA led to the cellular accumulation but that only BzA also enhanced BAC penetration by membrane fluidization at nonlethal concentrations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Structure of a Bacterial Cell Surface Decaheme Electron Conduit

    SciTech Connect

    Clarke, Thomas A.; Edwards, Marcus; Gates, Andrew J.; Hall, Andrea; White, Gaye; Bradley, Justin; Reardon, Catherine L.; Shi, Liang; Beliaev, Alex S.; Marshall, Matthew J.; Wang, Zheming; Watmough, Nicholas; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.

    2011-05-23

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves deca-heme cytochromes that are located on the bacterial cell surface at the termini of trans-outermembrane (OM) electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular inter-cytochrome electron exchange along ‘nanowire’ appendages. We present a 3.2 Å crystal structure of one of these deca-heme cytochromes, MtrF, that allows the spatial organization of the ten hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65 Å octa-heme chain transects the length of the protein and is bisected by a planar 45 Å tetra-heme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g. minerals), soluble substrates (e.g. flavins) and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

  20. Structure of a bacterial cell surface decaheme electron conduit

    PubMed Central

    Clarke, Thomas A.; Edwards, Marcus J.; Gates, Andrew J.; Hall, Andrea; White, Gaye F.; Bradley, Justin; Reardon, Catherine L.; Shi, Liang; Beliaev, Alexander S.; Marshall, Matthew J.; Wang, Zheming; Watmough, Nicholas J.; Fredrickson, James K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.

    2011-01-01

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along “nanowire” appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface. PMID:21606337

  1. Structure of a bacterial cell surface decaheme electron conduit.

    PubMed

    Clarke, Thomas A; Edwards, Marcus J; Gates, Andrew J; Hall, Andrea; White, Gaye F; Bradley, Justin; Reardon, Catherine L; Shi, Liang; Beliaev, Alexander S; Marshall, Matthew J; Wang, Zheming; Watmough, Nicholas J; Fredrickson, James K; Zachara, John M; Butt, Julea N; Richardson, David J

    2011-06-07

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

  2. Structure of a bacterial homologue of vitamin K epoxide reductase

    SciTech Connect

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  3. Structural and metabolic characterization of RNAs from rats with experimental Guerin tumor - II. metabolic peculiarities of RNAs from the liver and tumor tissues of rats.

    PubMed

    Ratkiewicz, A; Galasinski, W

    1976-01-01

    Metabolic peculiarities of RNAs in the liver of the tumor bearing and in the tumor tissue were found. The synthesis of nuclear RNA in liver of tumor bearing rats is distinctly disordered in comparison to that of control rats. The level of 14C-orotic acid incorporation into RNA of cancer tissue is manifold lower than that into the liver RNA. The studies on turnover rate showed the metabolic heterogeneity of the nuclear RNAs. The part of them showed a short turnover, the other RNAs were degraded much slower.

  4. Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs

    PubMed Central

    Szarzynska, Bogna; Sobkowiak, Lukasz; Pant, Bikram Datt; Balazadeh, Salma; Scheible, Wolf-Rüdiger; Mueller-Roeber, Bernd; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia

    2009-01-01

    Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3′ and 5′ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5′ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs. PMID:19304749

  5. Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs.

    PubMed

    Szarzynska, Bogna; Sobkowiak, Lukasz; Pant, Bikram Datt; Balazadeh, Salma; Scheible, Wolf-Rüdiger; Mueller-Roeber, Bernd; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia

    2009-05-01

    Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3' and 5' RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5' splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

  6. Elucidation of operon structures across closely related bacterial genomes.

    PubMed

    Zhou, Chuan; Ma, Qin; Li, Guojun

    2014-01-01

    About half of the protein-coding genes in prokaryotic genomes are organized into operons to facilitate co-regulation during transcription. With the evolution of genomes, operon structures are undergoing changes which could coordinate diverse gene expression patterns in response to various stimuli during the life cycle of a bacterial cell. Here we developed a graph-based model to elucidate the diversity of operon structures across a set of closely related bacterial genomes. In the constructed graph, each node represents one orthologous gene group (OGG) and a pair of nodes will be connected if any two genes, from the corresponding two OGGs respectively, are located in the same operon as immediate neighbors in any of the considered genomes. Through identifying the connected components in the above graph, we found that genes in a connected component are likely to be functionally related and these identified components tend to form treelike topology, such as paths and stars, corresponding to different biological mechanisms in transcriptional regulation as follows. Specifically, (i) a path-structure component integrates genes encoding a protein complex, such as ribosome; and (ii) a star-structure component not only groups related genes together, but also reflects the key functional roles of the central node of this component, such as the ABC transporter with a transporter permease and substrate-binding proteins surrounding it. Most interestingly, the genes from organisms with highly diverse living environments, i.e., biomass degraders and animal pathogens of clostridia in our study, can be clearly classified into different topological groups on some connected components.

  7. Elucidation of Operon Structures across Closely Related Bacterial Genomes

    PubMed Central

    Li, Guojun

    2014-01-01

    About half of the protein-coding genes in prokaryotic genomes are organized into operons to facilitate co-regulation during transcription. With the evolution of genomes, operon structures are undergoing changes which could coordinate diverse gene expression patterns in response to various stimuli during the life cycle of a bacterial cell. Here we developed a graph-based model to elucidate the diversity of operon structures across a set of closely related bacterial genomes. In the constructed graph, each node represents one orthologous gene group (OGG) and a pair of nodes will be connected if any two genes, from the corresponding two OGGs respectively, are located in the same operon as immediate neighbors in any of the considered genomes. Through identifying the connected components in the above graph, we found that genes in a connected component are likely to be functionally related and these identified components tend to form treelike topology, such as paths and stars, corresponding to different biological mechanisms in transcriptional regulation as follows. Specifically, (i) a path-structure component integrates genes encoding a protein complex, such as ribosome; and (ii) a star-structure component not only groups related genes together, but also reflects the key functional roles of the central node of this component, such as the ABC transporter with a transporter permease and substrate-binding proteins surrounding it. Most interestingly, the genes from organisms with highly diverse living environments, i.e., biomass degraders and animal pathogens of clostridia in our study, can be clearly classified into different topological groups on some connected components. PMID:24959722

  8. Satellite RNA of cucumber mosaic virus forms a secondary structure with partial 3'-terminal homology to genomal RNAs.

    PubMed Central

    Gordon, K H; Symons, R H

    1983-01-01

    Sat-RNA is one of several replicating satellite RNAs which have been isolated from RNA encapsidated in cucumber mosaic virus (CMV) and which are totally dependent on CMV for replication. The 336 residue sequence of Sat-RNA obtained using the dideoxynucleotide chain termination and partial enzymic digestion procedures shows only a few short stretches (up to 11 residues) of sequence homology with one of the three CMV genomal RNAs so far sequenced. Sat-RNA has 88% sequence homology with another, previously sequenced, satellite RNA of CMV, CARNA 5. Analysis of partial digests of 5'- or 3' -32P-Sat-RNA with nuclease S1 or RNase T1 under non-denaturing conditions showed that only about 10% of the residues in Sat-RNA were cleaved. Further data on base-paired segments of Sat-RNA were obtained using digestion with RNase T1 followed by electrophoretic fractionation of the resulting fragments under both non-denaturing and denaturing conditions. On the basis of this data, a complete secondary structure model is proposed for Sat-RNA with 52% of its residues involved in base pairs. A prominent hairpin at the 3'-terminus of Sat-RNA shows considerable sequence and structural homology with parts of the 3'-terminal tRNA-like structure of the CMV genomal RNAs. Images PMID:6186989

  9. Structural insights into bacterial flagellar hooks similarities and specificities

    PubMed Central

    Yoon, Young-Ho; Barker, Clive S.; Bulieris, Paula V.; Matsunami, Hideyuki; Samatey, Fadel A.

    2016-01-01

    Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments. PMID:27759043

  10. An improved method for identification of small non-coding RNAs in bacteria using support vector machine.

    PubMed

    Barman, Ranjan Kumar; Mukhopadhyay, Anirban; Das, Santasabuj

    2017-04-06

    Bacterial small non-coding RNAs (sRNAs) are not translated into proteins, but act as functional RNAs. They are involved in diverse biological processes like virulence, stress response and quorum sensing. Several high-throughput techniques have enabled identification of sRNAs in bacteria, but experimental detection remains a challenge and grossly incomplete for most species. Thus, there is a need to develop computational tools to predict bacterial sRNAs. Here, we propose a computational method to identify sRNAs in bacteria using support vector machine (SVM) classifier. The primary sequence and secondary structure features of experimentally-validated sRNAs of Salmonella Typhimurium LT2 (SLT2) was used to build the optimal SVM model. We found that a tri-nucleotide composition feature of sRNAs achieved an accuracy of 88.35% for SLT2. We validated the SVM model also on the experimentally-detected sRNAs of E. coli and Salmonella Typhi. The proposed model had robustly attained an accuracy of 81.25% and 88.82% for E. coli K-12 and S. Typhi Ty2, respectively. We confirmed that this method significantly improved the identification of sRNAs in bacteria. Furthermore, we used a sliding window-based method and identified sRNAs from complete genomes of SLT2, S. Typhi Ty2 and E. coli K-12 with sensitivities of 89.09%, 83.33% and 67.39%, respectively.

  11. An improved method for identification of small non-coding RNAs in bacteria using support vector machine

    PubMed Central

    Barman, Ranjan Kumar; Mukhopadhyay, Anirban; Das, Santasabuj

    2017-01-01

    Bacterial small non-coding RNAs (sRNAs) are not translated into proteins, but act as functional RNAs. They are involved in diverse biological processes like virulence, stress response and quorum sensing. Several high-throughput techniques have enabled identification of sRNAs in bacteria, but experimental detection remains a challenge and grossly incomplete for most species. Thus, there is a need to develop computational tools to predict bacterial sRNAs. Here, we propose a computational method to identify sRNAs in bacteria using support vector machine (SVM) classifier. The primary sequence and secondary structure features of experimentally-validated sRNAs of Salmonella Typhimurium LT2 (SLT2) was used to build the optimal SVM model. We found that a tri-nucleotide composition feature of sRNAs achieved an accuracy of 88.35% for SLT2. We validated the SVM model also on the experimentally-detected sRNAs of E. coli and Salmonella Typhi. The proposed model had robustly attained an accuracy of 81.25% and 88.82% for E. coli K-12 and S. Typhi Ty2, respectively. We confirmed that this method significantly improved the identification of sRNAs in bacteria. Furthermore, we used a sliding window-based method and identified sRNAs from complete genomes of SLT2, S. Typhi Ty2 and E. coli K-12 with sensitivities of 89.09%, 83.33% and 67.39%, respectively. PMID:28383059

  12. An improved method for identification of small non-coding RNAs in bacteria using support vector machine

    NASA Astrophysics Data System (ADS)

    Barman, Ranjan Kumar; Mukhopadhyay, Anirban; Das, Santasabuj

    2017-04-01

    Bacterial small non-coding RNAs (sRNAs) are not translated into proteins, but act as functional RNAs. They are involved in diverse biological processes like virulence, stress response and quorum sensing. Several high-throughput techniques have enabled identification of sRNAs in bacteria, but experimental detection remains a challenge and grossly incomplete for most species. Thus, there is a need to develop computational tools to predict bacterial sRNAs. Here, we propose a computational method to identify sRNAs in bacteria using support vector machine (SVM) classifier. The primary sequence and secondary structure features of experimentally-validated sRNAs of Salmonella Typhimurium LT2 (SLT2) was used to build the optimal SVM model. We found that a tri-nucleotide composition feature of sRNAs achieved an accuracy of 88.35% for SLT2. We validated the SVM model also on the experimentally-detected sRNAs of E. coli and Salmonella Typhi. The proposed model had robustly attained an accuracy of 81.25% and 88.82% for E. coli K-12 and S. Typhi Ty2, respectively. We confirmed that this method significantly improved the identification of sRNAs in bacteria. Furthermore, we used a sliding window-based method and identified sRNAs from complete genomes of SLT2, S. Typhi Ty2 and E. coli K-12 with sensitivities of 89.09%, 83.33% and 67.39%, respectively.

  13. Massive Effect on LncRNAs in Human Monocytes During Fungal and Bacterial Infections and in Response to Vitamins A and D

    PubMed Central

    Riege, Konstantin; Hölzer, Martin; Klassert, Tilman E.; Barth, Emanuel; Bräuer, Julia; Collatz, Maximilian; Hufsky, Franziska; Mostajo, Nelly; Stock, Magdalena; Vogel, Bertram; Slevogt, Hortense; Marz, Manja

    2017-01-01

    Mycoses induced by C.albicans or A.fumigatus can cause important host damage either by deficient or exaggerated immune response. Regulation of chemokine and cytokine signaling plays a crucial role for an adequate inflammation, which can be modulated by vitamins A and D. Non-coding RNAs (ncRNAs) as transcription factors or cis-acting antisense RNAs are known to be involved in gene regulation. However, the processes during fungal infections and treatment with vitamins in terms of therapeutic impact are unknown. We show that in monocytes both vitamins regulate ncRNAs involved in amino acid metabolism and immune system processes using comprehensive RNA-Seq analyses. Compared to protein-coding genes, fungi and bacteria induced an expression change in relatively few ncRNAs, but with massive fold changes of up to 4000. We defined the landscape of long-ncRNAs (lncRNAs) in response to pathogens and observed variation in the isoforms composition for several lncRNA following infection and vitamin treatment. Most of the involved antisense RNAs are regulated and positively correlated with their sense protein-coding genes. We investigated lncRNAs with stimulus specific immunomodulatory activity as potential marker genes: LINC00595, SBF2-AS1 (A.fumigatus) and RP11-588G21.2, RP11-394l13.1 (C.albicans) might be detectable in the early phase of infection and serve as therapeutic targets in the future. PMID:28094339

  14. A structural basis for electron transfer in bacterial photosynthesis

    SciTech Connect

    Norris, J.R.; DiMagno, T.J.; Angerhofer, A.; Chang, C.H.; El-Kabbani, O.; Schiffer, M.

    1989-01-01

    Triplet data for the primary donor in single crystals of bacterial reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis are interpreted in terms of the corresponding x-ray structures. The analysis of electron paramagnetic resonance data from single crystals (triplet zero field splitting and cation and triplet linewidth of the primary special pair donor of bacterial reaction centers) is extended to systems of a non-crystalline nature. A unified interpretation based on frontier molecular orbitals concludes that the special pair behaves like a supermolecule in all wild-type bacteria investigated here. However, in heterodimers of Rb. capsulatus (His/sup M200/ changed to Leu or Phe with the result that the M-half of the special pair is converted to bacteriopheophytin) the special pair possesses the EPR properties more appropriately described in terms of a monomer. In all cases the triplet state and cation EPR properties appear to be dominated by the highest occupied molecular orbitals. These conclusions derived from EPR experiments are supplemented by data from Stark spectroscopy of reaction centers from Rb. capsulatus. 41 refs., 3 tabs.

  15. Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme*

    PubMed Central

    Basu, Ritwika S.; Warner, Brittany A.; Molodtsov, Vadim; Pupov, Danil; Esyunina, Daria; Fernández-Tornero, Carlos; Kulbachinskiy, Andrey; Murakami, Katsuhiko S.

    2014-01-01

    The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5′-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs. PMID:24973216

  16. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme.

    PubMed

    Basu, Ritwika S; Warner, Brittany A; Molodtsov, Vadim; Pupov, Danil; Esyunina, Daria; Fernández-Tornero, Carlos; Kulbachinskiy, Andrey; Murakami, Katsuhiko S

    2014-08-29

    The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Fisher: a program for the detection of H/ACA snoRNAs using MFE secondary structure prediction and comparative genomics – assessment and update

    PubMed Central

    Freyhult, Eva; Edvardsson, Sverker; Tamas, Ivica; Moulton, Vincent; Poole, Anthony M

    2008-01-01

    Background The H/ACA family of small nucleolar RNAs (snoRNAs) plays a central role in guiding the pseudouridylation of ribosomal RNA (rRNA). In an effort to systematically identify the complete set of rRNA-modifying H/ACA snoRNAs from the genome sequence of the budding yeast, Saccharomyces cerevisiae, we developed a program – Fisher – and previously presented several candidate snoRNAs based on our analysis [1]. Findings In this report, we provide a brief update of this work, which was aborted after the publication of experimentally-identified snoRNAs [2] identical to candidates we had identified bioinformatically using Fisher. Our motivation for revisiting this work is to report on the status of the candidate snoRNAs described in [1], and secondly, to report that a modified version of Fisher together with the available multiple yeast genome sequences was able to correctly identify several H/ACA snoRNAs for modification sites not identified by the snoGPS program [3]. While we are no longer developing Fisher, we briefly consider the merits of the Fisher algorithm relative to snoGPS, which may be of use for workers considering pursuing a similar search strategy for the identification of small RNAs. The modified source code for Fisher is made available as supplementary material. Conclusion Our results confirm the validity of using minimum free energy (MFE) secondary structure prediction to guide comparative genomic screening for RNA families with few sequence constraints. PMID:18710502

  18. Sequences of three molluscan 5 S ribosomal RNAs confirm the validity of a dynamic secondary structure model.

    PubMed Central

    Fang, B L; De Baere, R; Vandenberghe, A; De Wachter, R

    1982-01-01

    The collection of known 5 S rRNA primary structures is enriched with the sequences from three mollusca, the snails Helix pomatia and Arion rufus, and the mussel Mytilus edulis. The three sequences can be fitted in a five-helix secondary structure model previously shown (De Wachter et al. (1982) Biochimie 64, 311-329) to apply to all 5 S RNAs regardless of their origin. One of the helices in this model can undergo a bulge-internal loop transition. Within the metazoan kingdom, the dimensions of each helix and loop are rigidly conserved, except for one helix which can comprise either 6 or 7 base pairs. PMID:7133995

  19. Mechanical and structural property analysis of bacterial cellulose composites.

    PubMed

    Dayal, Manmeet Singh; Catchmark, Jeffrey M

    2016-06-25

    Bacterial cellulose (BC) exhibits unique properties including high mechanical strength and high crystallinity. Improvement in the mechanical properties of BC is sought for many applications ranging from food to structural composites to biomedical materials. In this study, different additives including carboxymethyl cellulose (CMC), pectin, gelatin, cornstarch, and corn steep liquor were included in the fermentation media to alter the BC produced. Three different concentrations (1%, 3% and 5%) were chosen for each of the additives, with no additive (0%) as the control. The produced BC was then analyzed to determine tensile and compression modulus. Amongst the tested additives, BC produced in media containing 3% (w/v) pectin had the maximum compressive modulus (142kPa), and BC produced in media containing 1% (w/v) gelatin exhibited the maximum tensile modulus (21MPa). Structural characteristics of BC and BC-additive composites were compared using X-Ray diffraction (XRD). The crystal size and crystallinity of BC was reduced when grown in the presence of CMC and gelatin while pectin only decreased the crystallite size. This suggested that CMC and gelatin may be incorporated into the BC fibril structure. The field emission scanning electron microscopy (FESEM) images showed the increased micro-fibril aggregation in BC pellicles grown in the presence of additives to the culture media. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Bacterial Inclusion Bodies Contain Amyloid-Like Structure

    PubMed Central

    Wang, Lei; Maji, Samir K; Sawaya, Michael R; Eisenberg, David; Riek, Roland

    2008-01-01

    Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized β-sheet–rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-β structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation. PMID:18684013

  1. Structural photoactivation of a full-length bacterial phytochrome

    PubMed Central

    Björling, Alexander; Berntsson, Oskar; Lehtivuori, Heli; Takala, Heikki; Hughes, Ashley J.; Panman, Matthijs; Hoernke, Maria; Niebling, Stephan; Henry, Léocadie; Henning, Robert; Kosheleva, Irina; Chukharev, Vladimir; Tkachenko, Nikolai V.; Menzel, Andreas; Newby, Gemma; Khakhulin, Dmitry; Wulff, Michael; Ihalainen, Janne A.; Westenhoff, Sebastian

    2016-01-01

    Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes. PMID:27536728

  2. RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core

    PubMed Central

    Anokhina, Maria; Bessonov, Sergey; Miao, Zhichao; Westhof, Eric; Hartmuth, Klaus; Lührmann, Reinhard

    2013-01-01

    Although U snRNAs play essential roles in splicing, little is known about the 3D arrangement of U2, U6, and U5 snRNAs and the pre-mRNA in active spliceosomes. To elucidate their relative spatial organization and dynamic rearrangement, we examined the RNA structure of affinity-purified, human spliceosomes before and after catalytic step 1 by chemical RNA structure probing. We found a stable 3-way junction of the U2/U6 snRNA duplex in active spliceosomes that persists minimally through step 1. Moreover, the formation of alternating, mutually exclusive, U2 snRNA conformations, as observed in yeast, was not detected in different assembly stages of human spliceosomal complexes (that is, B, Bact, or C complexes). Psoralen crosslinking revealed an interaction during/after step 1 between internal loop 1 of the U5 snRNA, and intron nucleotides immediately downstream of the branchpoint. Using the experimentally derived structural constraints, we generated a model of the RNA network of the step 1 spliceosome, based on the crystal structure of a group II intron through homology modelling. The model is topologically consistent with current genetic, biochemical, and structural data. PMID:24002212

  3. Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea.

    PubMed

    Toffano-Nioche, Claire; Gautheret, Daniel; Leclerc, Fabrice

    2015-09-18

    A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Molecular structure and dynamics in bacterial mercury resistance

    SciTech Connect

    Johs, Alexander; Shi, Liang; Miller, Susan M; Summers, Anne O; Liang, Liyuan

    2008-01-01

    Bacteria participate significantly in mercury transformation in natural and industrial environments. Previous studies have shown that bacterial mercury resistance is mediated by the mer operon, typically located on transposons or plasmids. It encodes specific genes that facilitate uptake of mercury species, cleavage of organomercurials, and reduction of Hg(II) to Hg(0). Expression of mer operon genes is regulated by MerR, a metal-responsive regulator protein on the level of transcription. In vitro studies have shown that MerR forms a non-transcribing pre-initiation complex with RNA polymerase and the promoter DNA. Binding of Hg(II) induces conformational changes in MerR and other components of the complex resulting in the transcription of mer operon genes. As part of ongoing investigations on allosteric conformational changes induced by Hg(II) in dimeric MerR, and the implications on the binding of RNA polymerase to the promoter of the mer operon, we applied small angle scattering to study the regulatory mechanism of MerR in the presence and absence of Hg(II). Our results show that in the presence of Hg(II) the MerR dimer undergoes a significant reorientation from a compact state to a conformation revealing two distinct domains. Bacterial reduction of Hg(II) can also occur at concentrations too low to induce mer operon functions. Dissimilatory metal reducing bacteria, such as Shewanella and Geobacter are able to reduce Hg(II) in the presence of mineral oxides. This process has been linked to the activity of outer membrane multiheme cytochromes. We isolated and purified a decaheme outer membrane cytochrome OmcA from Shewanella oneidensis MR-1 and characterized its envelope shape in solution by small angle x-ray scattering. Structural features were identified and compared to homology models. These results show that OmcA is an elongated macromolecule consisting of separate modules, which may be connected by flexible linkers.

  5. Bacterial community structure in aquifers corresponds to stratigraphy

    NASA Astrophysics Data System (ADS)

    Beyer, Andrea; Möller, Silke; Neumann, Stefan; Burow, Katja; Gutmann, Falko; Lindner, Julia; Müsse, Steffen; Kothe, Erika; Büchel, Georg

    2014-05-01

    So far, groundwater microbiology with respect to different host rocks has not been well described in the literature. However, factors influencing the communities would be of interest to provide a tool for mapping groundwater paths. The Thuringian Basin (Germany) studied here, contains formations of the Permian (Zechstein) and also Triassic period of Buntsandstein, Muschelkalk and Keuper, all of which can be found to crop out at the surface in different regions. We analyzed the bacterial community of nine natural springs and sixteen groundwater wells of the respective rock formations as well as core material from the Zechstein salts. For that we sampled in a mine 3 differnet salt rock samples (carnallitite, halite and sylvinitite). To validate the different approaches, similar rock formations were compared and a consistent microbial community for Buntsandstein could be verified. Similary, for Zechstein, the presence of halophiles was seen with cultivation, isolation directly from the rock material and also in groundwater with DNA-dependent approaches. A higher overlap between sandstone- and limestone-derived communities was visible as if compared to the salt formations. Principal component analysis confirmed formation specific patterns for Muschelkalk, Buntsandstein and Zechstein for the bacterial taxa present, with some overlaps. Bacilli and Gammaproteobacteria were the major groups, with the genera Pseudomonas, Marinomonas, Bacillus, Marinobacter and Pseudoalteromonas representing the communities. The bacteria are well adapted to their respective environment with survival strategies including a wide range of salinity which makes them suitable as tracers for fluid movement below the ground. The results indicate the usefulness and robustness of the approach taken here to investigate aquifer community structures in dependence of the stratigraphy of the groundwater reservoir.

  6. CentroidAlign: fast and accurate aligner for structured RNAs by maximizing expected sum-of-pairs score.

    PubMed

    Hamada, Michiaki; Sato, Kengo; Kiryu, Hisanori; Mituyama, Toutai; Asai, Kiyoshi

    2009-12-15

    The importance of accurate and fast predictions of multiple alignments for RNA sequences has increased due to recent findings about functional non-coding RNAs. Recent studies suggest that maximizing the expected accuracy of predictions will be useful for many problems in bioinformatics. We designed a novel estimator for multiple alignments of structured RNAs, based on maximizing the expected accuracy of predictions. First, we define the maximum expected accuracy (MEA) estimator for pairwise alignment of RNA sequences. This maximizes the expected sum-of-pairs score (SPS) of a predicted alignment under a probability distribution of alignments given by marginalizing the Sankoff model. Then, by approximating the MEA estimator, we obtain an estimator whose time complexity is O(L(3)+c(2)dL(2)) where L is the length of input sequences and both c and d are constants independent of L. The proposed estimator can handle uncertainty of secondary structures and alignments that are obstacles in Bioinformatics because it considers all the secondary structures and all the pairwise alignments as input sequences. Moreover, we integrate the probabilistic consistency transformation (PCT) on alignments into the proposed estimator. Computational experiments using six benchmark datasets indicate that the proposed method achieved a favorable SPS and was the fastest of many state-of-the-art tools for multiple alignments of structured RNAs. The software called CentroidAlign, which is an implementation of the algorithm in this article, is freely available on our website: http://www.ncrna.org/software/centroidalign/. hamada-michiaki@aist.go.jp Supplementary data are available at Bioinformatics online.

  7. Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases.

    PubMed

    Zhang, Yu-Zhong; Ran, Li-Yuan; Li, Chun-Yang; Chen, Xiu-Lan

    2015-09-01

    Bacterial collagenolytic proteases are important because of their essential role in global collagen degradation and because of their virulence in some human bacterial infections. Bacterial collagenolytic proteases include some metalloproteases of the M9 family from Clostridium or Vibrio strains, some serine proteases distributed in the S1, S8, and S53 families, and members of the U32 family. In recent years, there has been remarkable progress in discovering new bacterial collagenolytic proteases and in investigating the collagen-degrading mechanisms of bacterial collagenolytic proteases. This review provides comprehensive insight into bacterial collagenolytic proteases, especially focusing on the structures and collagen-degrading mechanisms of representative bacterial collagenolytic proteases in each family. The roles of bacterial collagenolytic proteases in human diseases and global nitrogen cycling, together with the biotechnological and medical applications for these proteases, are also briefly discussed.

  8. Diversity, Structures, and Collagen-Degrading Mechanisms of Bacterial Collagenolytic Proteases

    PubMed Central

    Zhang, Yu-Zhong; Ran, Li-Yuan; Li, Chun-Yang

    2015-01-01

    Bacterial collagenolytic proteases are important because of their essential role in global collagen degradation and because of their virulence in some human bacterial infections. Bacterial collagenolytic proteases include some metalloproteases of the M9 family from Clostridium or Vibrio strains, some serine proteases distributed in the S1, S8, and S53 families, and members of the U32 family. In recent years, there has been remarkable progress in discovering new bacterial collagenolytic proteases and in investigating the collagen-degrading mechanisms of bacterial collagenolytic proteases. This review provides comprehensive insight into bacterial collagenolytic proteases, especially focusing on the structures and collagen-degrading mechanisms of representative bacterial collagenolytic proteases in each family. The roles of bacterial collagenolytic proteases in human diseases and global nitrogen cycling, together with the biotechnological and medical applications for these proteases, are also briefly discussed. PMID:26150451

  9. X-ray structure of a bacterial oligosaccharyltransferase.

    PubMed

    Lizak, Christian; Gerber, Sabina; Numao, Shin; Aebi, Markus; Locher, Kaspar P

    2011-06-15

    Asparagine-linked glycosylation is a post-translational modification of proteins containing the conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host-pathogen interactions. The reaction is catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologues in bacteria and archaea. Here we report the X-ray structure of a bacterial OST, the PglB protein of Campylobacter lari, in complex with an acceptor peptide. The structure defines the fold of STT3 proteins and provides insight into glycosylation sequon recognition and amide nitrogen activation, both of which are prerequisites for the formation of the N-glycosidic linkage. We also identified and validated catalytically important, acidic amino acid residues. Our results provide the molecular basis for understanding the mechanism of N-linked glycosylation.

  10. Crystal structure of a chimaeric bacterial glutamate dehydrogenase

    SciTech Connect

    Oliveira, Tânia; Sharkey, Michael A.; Engel, Paul C.; Khan, Amir R.

    2016-05-23

    Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

  11. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  12. Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

    PubMed Central

    Niederer, Rachel O.

    2015-01-01

    Telomerase catalyzes the addition of nucleotides to the ends of chromosomes to complete genomic DNA replication in eukaryotes and is implicated in multiple diseases, including most cancers. The core enzyme is composed of a reverse transcriptase and an RNA subunit, which provides the template for DNA synthesis. Despite extensive divergence at the sequence level, telomerase RNAs share several structural features within the catalytic core, suggesting a conserved enzyme mechanism. We have investigated the structure of the core of the human and yeast telomerase RNAs using SHAPE, which interrogates flexibility of each nucleotide. We present improved secondary-structure models, refined by addition of five base triples within the yeast pseudoknot and an alternate pairing within the human-specific element J2a.1 in the human pseudoknot, both of which have implications for thermodynamic stability. We also identified a potentially structured CCC region within the template that may facilitate substrate binding and enzyme mechanism. Overall, the SHAPE findings reveal multiple similarities between the Saccharomyces cerevisiae and Homo sapiens telomerase RNA cores. PMID:25512567

  13. Structural basis for ArfA-RF2-mediated translation termination on mRNAs lacking stop codons.

    PubMed

    Huter, Paul; Müller, Claudia; Beckert, Bertrand; Arenz, Stefan; Berninghausen, Otto; Beckmann, Roland; Wilson, Daniel N

    2017-01-26

    In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems. Although tmRNA-ribosome and ArfB-ribosome structures have been determined, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.

  14. Impact of Oil on Bacterial Community Structure in Bioturbated Sediments

    PubMed Central

    Stauffert, Magalie; Cravo-Laureau, Cristiana; Jézéquel, Ronan; Barantal, Sandra; Cuny, Philippe; Gilbert, Franck; Cagnon, Christine; Militon, Cécile; Amouroux, David; Mahdaoui, Fatima; Bouyssiere, Brice; Stora, Georges; Merlin, François-Xavier; Duran, Robert

    2013-01-01

    Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions – with tidal cycles and natural seawater – was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g−1 wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by

  15. Structural basis for bacterial quorum sensing-mediated oxalogenesis.

    PubMed

    Oh, Juntaek; Goo, Eunhye; Hwang, Ingyu; Rhee, Sangkee

    2014-04-18

    The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)8-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr(322) as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.

  16. Structure of a bacterial cell surface decaheme electron conduit

    USDA-ARS?s Scientific Manuscript database

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits...

  17. The effect of deuteration on the structure of bacterial cellulose

    SciTech Connect

    Bali, Garima; Foston, Marcus; O'Neill, Hugh Michael; Evans, Barbara R; He, Junhong; Ragauskas, Arthur

    2013-01-01

    ABSTRACT In vivo generated deuterated bacterial cellulose, cultivated from 100% deuterated glycerol in D2O medium, was analyzed for deuterium incorporation by ionic liquid dissolution and 2H and 1H nuclear magnetic resonance (NMR). A solution NMR method of the dissolved cellulose was used to determine that this bacterial cellulose had 85 % deuterium incorporation. Acetylation and 1H and 2H NMR of deuterated bacterial cellulose indicated near equal deuteration at all sites of the glucopyranosyl ring except C-6 which was partly deuterated. Despite the high level of deuterium incorporation there were no significant differences in the molecular and morphological properties were observed for the deuterated and protio bacterial cellulose samples. The highly deuterated bacterial cellulose presented here can be used as a model substrate for studying cellulose biopolymer properties via future small angle neutron scattering (SANS) studies.

  18. GTPases involved in bacterial ribosome maturation.

    PubMed

    Goto, Simon; Muto, Akira; Himeno, Hyouta

    2013-05-01

    The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

  19. Bacterial community structure and predicted alginate metabolic pathway in an alginate-degrading bacterial consortium.

    PubMed

    Kita, Akihisa; Miura, Toyokazu; Kawata, Satoshi; Yamaguchi, Takeshi; Okamura, Yoshiko; Aki, Tsunehiro; Matsumura, Yukihiko; Tajima, Takahisa; Kato, Junichi; Nishio, Naomichi; Nakashimada, Yutaka

    2016-03-01

    Methane fermentation is one of the effective approaches for utilization of brown algae; however, this process is limited by the microbial capability to degrade alginate, a main polysaccharide found in these algae. Despite its potential, little is known about anaerobic microbial degradation of alginate. Here we constructed a bacterial consortium able to anaerobically degrade alginate. Taxonomic classification of 16S rRNA gene, based on high-throughput sequencing data, revealed that this consortium included two dominant strains, designated HUA-1 and HUA-2; these strains were related to Clostridiaceae bacterium SK082 (99%) and Dysgonomonas capnocytophagoides (95%), respectively. Alginate lyase activity and metagenomic analyses, based on high-throughput sequencing data, revealed that this bacterial consortium possessed putative genes related to a predicted alginate metabolic pathway. However, HUA-1 and 2 did not grow on agar medium with alginate by using roll-tube method, suggesting the existence of bacterial interactions like symbiosis for anaerobic alginate degradation.

  20. Co-acclimation of bacterial communities under stresses of hydrocarbons with different structures

    NASA Astrophysics Data System (ADS)

    Wang, Hui; Wang, Bin; Dong, Wenwen; Hu, Xiaoke

    2016-10-01

    Crude oil is a complex mixture of hydrocarbons with different structures; its components vary in bioavailability and toxicity. It is important to understand how bacterial communities response to different hydrocarbons and their co-acclimation in the process of degradation. In this study, microcosms with the addition of structurally different hydrocarbons were setup to investigate the successions of bacterial communities and the interactions between different bacterial taxa. Hydrocarbons were effectively degraded in all microcosms after 40 days. High-throughput sequencing offered a great quantity of data for analyzing successions of bacterial communities. The results indicated that the bacterial communities responded dramatically different to various hydrocarbons. KEGG database and PICRUSt were applied to predict functions of individual bacterial taxa and networks were constructed to analyze co-acclimations between functional bacterial groups. Almost all functional genes catalyzing degradation of different hydrocarbons were predicted in bacterial communities. Most of bacterial taxa were believed to conduct biodegradation processes via interactions with each other. This study addressed a few investigated area of bacterial community responses to structurally different organic pollutants and their co-acclimation and interactions in the process of biodegradation. The study could provide useful information to guide the bioremediation of crude oil pollution.

  1. Co-acclimation of bacterial communities under stresses of hydrocarbons with different structures

    PubMed Central

    Wang, Hui; Wang, Bin; Dong, Wenwen; Hu, Xiaoke

    2016-01-01

    Crude oil is a complex mixture of hydrocarbons with different structures; its components vary in bioavailability and toxicity. It is important to understand how bacterial communities response to different hydrocarbons and their co-acclimation in the process of degradation. In this study, microcosms with the addition of structurally different hydrocarbons were setup to investigate the successions of bacterial communities and the interactions between different bacterial taxa. Hydrocarbons were effectively degraded in all microcosms after 40 days. High-throughput sequencing offered a great quantity of data for analyzing successions of bacterial communities. The results indicated that the bacterial communities responded dramatically different to various hydrocarbons. KEGG database and PICRUSt were applied to predict functions of individual bacterial taxa and networks were constructed to analyze co-acclimations between functional bacterial groups. Almost all functional genes catalyzing degradation of different hydrocarbons were predicted in bacterial communities. Most of bacterial taxa were believed to conduct biodegradation processes via interactions with each other. This study addressed a few investigated area of bacterial community responses to structurally different organic pollutants and their co-acclimation and interactions in the process of biodegradation. The study could provide useful information to guide the bioremediation of crude oil pollution. PMID:27698451

  2. Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

    SciTech Connect

    Devarkar, Swapnil C.; Wang, Chen; Miller, Matthew T.; Ramanathan, Anand; Jiang, Fuguo; Khan, Abdul G.; Patel, Smita S.; Marcotrigiano, Joseph

    2016-01-05

    The cytosolic innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I) is the principal detector of pathogenic RNAs carrying a 5'-triphosphate (5'ppp). Self RNAs like mRNAs evade recognition by RIG-I due to posttranscriptional modifications like 5'-end capping with 7-methyl guanosine (m7G) and 2'-O-methylation of 5'-end nucleotides. Viruses have also evolved mechanisms to mimic these modifications, which in part is believed to aid in immune evasion. Currently, it is unclear how these modifications modulate RIG-I recognition. This paper provides structural and mechanistic insights into the roles of the m7G cap and 2'-O-methylation in RIG-I evasion. We show that RIG-I accommodates the m7G base while maintaining the 5'ppp contacts and can recognize Cap-0 RNAs but not Cap-1.

  3. Structural and functional analysis of four non-coding Y RNAs from Chinese hamster cells: identification, molecular dynamics simulations and DNA replication initiation assays.

    PubMed

    de Lima Neto, Quirino Alves; Duarte Junior, Francisco Ferreira; Bueno, Paulo Sérgio Alves; Seixas, Flavio Augusto Vicente; Kowalski, Madzia Pauline; Kheir, Eyemen; Krude, Torsten; Fernandez, Maria Aparecida

    2016-01-05

    The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no information is available about Y RNAs in Chinese hamster cells, which have already been used to detect replication origins and alternative DNA structures around these sites. Here, we report the gene sequences and predicted structural characteristics of the Chinese hamster Y RNAs, and analyze their ability to support the initiation of chromosomal DNA replication in vitro. We identified DNA sequences in the Chinese hamster genome of four Y RNAs (chY1, chY3, chY4 and chY5) with upstream promoter sequences, which are homologous to the four main types of vertebrate Y RNAs. The chY1, chY3 and chY5 genes were highly conserved with their vertebrate counterparts, whilst the chY4 gene showed a relatively high degree of diversification from the other vertebrate Y4 genes. Molecular dynamics simulations suggest that chY4 RNA is structurally stable despite its evolutionarily divergent predicted stem structure. Of the four Y RNA genes present in the hamster genome, we found that only the chY1 and chY3 RNA were strongly expressed in the Chinese hamster GMA32 cell line, while expression of the chY4 and chY5 RNA genes was five orders of magnitude lower, suggesting that they may in fact not be expressed. We synthesized all four chY RNAs and showed that any of these four could support the initiation of DNA replication in an established human cell-free system. These data therefore establish that non-coding chY RNAs are stable structures and can substitute for human Y RNAs in a reconstituted cell-free DNA replication initiation system. The pattern of Y RNA expression and functionality is consistent with Y RNAs of other rodents, including mouse and rat.

  4. Assessment of bacterial and structural dynamics in aerobic granular biofilms.

    PubMed

    Weissbrodt, David G; Neu, Thomas R; Kuhlicke, Ute; Rappaz, Yoan; Holliger, Christof

    2013-01-01

    Aerobic granular sludge (AGS) is based on self-granulated flocs forming mobile biofilms with a gel-like consistence. Bacterial and structural dynamics from flocs to granules were followed in anaerobic-aerobic sequencing batch reactors (SBR) fed with synthetic wastewater, namely a bubble column (BC-SBR) operated under wash-out conditions for fast granulation, and two stirred-tank enrichments of Accumulibacter (PAO-SBR) and Competibacter (GAO-SBR) operated at steady-state. In the BC-SBR, granules formed within 2 weeks by swelling of Zoogloea colonies around flocs, developing subsequently smooth zoogloeal biofilms. However, Zoogloea predominance (37-79%) led to deteriorated nutrient removal during the first months of reactor operation. Upon maturation, improved nitrification (80-100%), nitrogen removal (43-83%), and high but unstable dephosphatation (75-100%) were obtained. Proliferation of dense clusters of nitrifiers, Accumulibacter, and Competibacter from granule cores outwards resulted in heterogeneous bioaggregates, inside which only low abundance Zoogloea (<5%) were detected in biofilm interstices. The presence of different extracellular glycoconjugates detected by fluorescence lectin-binding analysis showed the complex nature of the intracellular matrix of these granules. In the PAO-SBR, granulation occurred within two months with abundant and active Accumulibacter populations (56 ± 10%) that were selected under full anaerobic uptake of volatile fatty acids and that aggregated as dense clusters within heterogeneous granules. Flocs self-granulated in the GAO-SBR after 480 days during a period of over-aeration caused by biofilm growth on the oxygen sensor. Granules were dominated by heterogeneous clusters of Competibacter (37 ± 11%). Zoogloea were never abundant in biomass of both PAO- and GAO-SBRs. This study showed that Zoogloea, Accumulibacter, and Competibacter affiliates can form granules, and that the granulation mechanisms rely on the dominant population

  5. Assessment of bacterial and structural dynamics in aerobic granular biofilms

    PubMed Central

    Weissbrodt, David G.; Neu, Thomas R.; Kuhlicke, Ute; Rappaz, Yoan; Holliger, Christof

    2013-01-01

    Aerobic granular sludge (AGS) is based on self-granulated flocs forming mobile biofilms with a gel-like consistence. Bacterial and structural dynamics from flocs to granules were followed in anaerobic-aerobic sequencing batch reactors (SBR) fed with synthetic wastewater, namely a bubble column (BC-SBR) operated under wash-out conditions for fast granulation, and two stirred-tank enrichments of Accumulibacter (PAO-SBR) and Competibacter (GAO-SBR) operated at steady-state. In the BC-SBR, granules formed within 2 weeks by swelling of Zoogloea colonies around flocs, developing subsequently smooth zoogloeal biofilms. However, Zoogloea predominance (37–79%) led to deteriorated nutrient removal during the first months of reactor operation. Upon maturation, improved nitrification (80–100%), nitrogen removal (43–83%), and high but unstable dephosphatation (75–100%) were obtained. Proliferation of dense clusters of nitrifiers, Accumulibacter, and Competibacter from granule cores outwards resulted in heterogeneous bioaggregates, inside which only low abundance Zoogloea (<5%) were detected in biofilm interstices. The presence of different extracellular glycoconjugates detected by fluorescence lectin-binding analysis showed the complex nature of the intracellular matrix of these granules. In the PAO-SBR, granulation occurred within two months with abundant and active Accumulibacter populations (56 ± 10%) that were selected under full anaerobic uptake of volatile fatty acids and that aggregated as dense clusters within heterogeneous granules. Flocs self-granulated in the GAO-SBR after 480 days during a period of over-aeration caused by biofilm growth on the oxygen sensor. Granules were dominated by heterogeneous clusters of Competibacter (37 ± 11%). Zoogloea were never abundant in biomass of both PAO- and GAO-SBRs. This study showed that Zoogloea, Accumulibacter, and Competibacter affiliates can form granules, and that the granulation mechanisms rely on the dominant

  6. Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices▿

    PubMed Central

    Hartmann, Martin; Widmer, Franco

    2006-01-01

    Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities. PMID:17041161

  7. Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices.

    PubMed

    Hartmann, Martin; Widmer, Franco

    2006-12-01

    Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.

  8. The structure of two subgenomic RNAs from human influenza virus A/PR/8/34.

    PubMed Central

    Winter, G; Fields, S; Ratti, G

    1981-01-01

    The nucleotide sequences of two subgenomic RNA segments from influenza virus A/PR/8/34 have been determined by cloning viral cDNA into the vector M13mp7. Sequence analysis was facilitated by a re-cloning strategy which takes advantage of both wild-type and amber derivatives of the M13 vector. The RNA species (444 and 480 nucleotides) contain the 5' and 3' termini of segment 1 and therefore derive by simple internal deletions of this segment. However, these species are not exact copies of the terminal regions of the progenitor segment but contain a few base changes. These differences suggest that after these RNAs have arisen, their sequences can drift, presumably reflecting a lower selective pressure than on the standard RNA segments. PMID:7335495

  9. Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

    PubMed Central

    Didychuk, Allison L.; Montemayor, Eric J.; Brow, David A.; Butcher, Samuel E.

    2016-01-01

    Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 ‘telestem’ helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2–8 complex, which binds adjacent to the telestem at the 3′ end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA. PMID:26673715

  10. Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly.

    PubMed

    Didychuk, Allison L; Montemayor, Eric J; Brow, David A; Butcher, Samuel E

    2016-02-18

    Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 'telestem' helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2-8 complex, which binds adjacent to the telestem at the 3' end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.

  11. Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

    PubMed

    Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

    2016-06-17

    The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

  12. Non-coding RNAs as antibiotic targets.

    PubMed

    Colameco, Savannah; Elliot, Marie A

    2016-12-22

    Antibiotics inhibit a wide range of essential processes in the bacterial cell, including replication, transcription, translation and cell wall synthesis. In many instances, these antibiotics exert their effects through association with non-coding RNAs. This review highlights many classical antibiotic targets (e.g. rRNAs and the ribosome), explores a number of emerging targets (e.g. tRNAs, RNase P, riboswitches and small RNAs), and discusses the future directions and challenges associated with non-coding RNAs as antibiotic targets.

  13. Shifts in bacterial community structure during succession in a glacier foreland of the High Arctic.

    PubMed

    Kim, Mincheol; Jung, Ji Young; Laffly, Dominique; Kwon, Hye Young; Lee, Yoo Kyung

    2017-01-01

    Primary succession after glacier retreat has been widely studied in plant communities, but bacterial succession is still poorly understood. In particular, few studies of microbial succession have been performed in the Arctic. We investigated the shifts in bacterial community structure and soil physicochemical properties along a successional gradient in a 100-year glacier foreland of the High Arctic. Multivariate analyses revealed that time after glacier retreat played a key role in associated bacterial community structure during succession. However, environmental filtering (i.e. pH and soil temperature) also accounted for a different, but substantial, proportion of the bacterial community structure. Using the functional trait-based approach, we found that average rRNA operon (rrn) copy number of bacterial communities is high in earlier successional stages and decreased over time. This suggests that soil bacterial taxa with higher rrn copy number have a selective advantage in early successional stages due to their ability of rapidly responding to nutrient inputs in newly exposed soils after glacier retreat. Taken together, our results demonstrate that both deglaciation time and environmental filters play key roles in structuring bacterial communities and soil bacterial groups with different ecological strategies occur in different stages of succession in this glacier foreland. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Biosynthesis and structural characterization of silver nanoparticles from bacterial isolates

    SciTech Connect

    Zaki, Sahar; El Kady, M.F.; Abd-El-Haleem, Desouky

    2011-10-15

    Graphical abstract: In this study five bacterial isolates belong to different genera were found to be able to biosynthesize silver nanoparticles. Biosynthesis and spectral characterization are reported here. Highlights: {yields} About 300 bacterial isolates were screened for their ability to produce nanosilvers {yields} Five of them were potential candidates for synthesis of silver nanoparticles {yields} Production of silver nanoparticles was examined using UV-Vis, XRD, SEM and EDS. {yields} The presence of nanoparticles with all five bacterial isolates was confirmed. -- Abstract: This study aimed to develop a green process for biosynthesis of silver nanomaterials by some Egyptian bacterial isolates. This target was achieved by screening an in-house culture collection consists of 300 bacterial isolates for silver nanoparticle formation. Through screening process, it was observed that strains belonging to Escherichia coli (S30, S78), Bacillus megaterium (S52), Acinetobacter sp. (S7) and Stenotrophomonas maltophilia (S54) were potential candidates for synthesis of silver nanoparticles. The extracellular production of silver nanoparticles by positive isolates was investigated by UV-Vis spectroscopy, X-ray diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The results demonstrated that UV-visible spectrum of the aqueous medium containing silver ion showed a peak at 420 nm corresponding to the plasmon absorbance of silver nanoparticles. Scanning electron microscopy micrograph showed formation of silver nanoparticles in the range of 15-50 nm. XRD-spectrum of the silver nanoparticles exhibited 2{theta} values corresponding to the silver nanocrystal that produce in hexagonal and cubic crystal configurations with different plane of orientation. In addition, the signals of the silver atoms were observed by EDS-spectrum analysis that confirms the presence of silver nanoparticles (Ag

  15. Multiple G-quartet structures in pre-edited mRNAs suggest evolutionary driving force for RNA editing in trypanosomes

    PubMed Central

    Leeder, W.-Matthias; Hummel, Niklas F. C.; Göringer, H. Ulrich

    2016-01-01

    Mitochondrial transcript maturation in African trypanosomes requires a U-nucleotide specific RNA editing reaction. In its most extreme form hundreds of U’s are inserted into and deleted from primary transcripts to generate functional mRNAs. Unfortunately, both origin and biological role of the process have remained enigmatic. Here we report a so far unrecognized structural feature of pre-edited mRNAs. We demonstrate that the cryptic pre-mRNAs contain numerous clustered G-nt, which fold into G-quadruplex (GQ) structures. We identified 27 GQ’s in the different pre-mRNAs and demonstrate a positive correlation between the steady state abundance of guide (g)RNAs and the sequence position of GQ-elements. We postulate that the driving force for selecting G-rich sequences lies in the formation of DNA/RNA hybrid G-quadruplex (HQ) structures between the pre-edited transcripts and the non-template strands of mitochondrial DNA. HQ’s are transcription termination/replication initiation sites and thus guarantee an unperturbed replication of the mt-genome. This is of special importance in the insect-stage of the parasite. In the transcription-on state, the identified GQ’s require editing as a GQ-resolving activity indicating a link between replication, transcription and RNA editing. We propose that the different processes have coevolved and suggest the parasite life-cycle and the single mitochondrion as evolutionary driving forces. PMID:27436151

  16. Structural variation and functional importance of a D-loop–T-loop interaction in valine-accepting tRNA-like structures of plant viral RNAs

    PubMed Central

    de Smit, Maarten H.; Gultyaev, Alexander P.; Hilge, Mark; Bink, Hugo H. J.; Barends, Sharief; Kraal, Barend; Pleij, Cornelis W. A.

    2002-01-01

    Valine-accepting tRNA-like structures (TLSs) are found at the 3′ ends of the genomic RNAs of most plant viruses belonging to the genera Tymovirus, Furovirus, Pomovirus and Pecluvirus, and of one Tobamovirus species. Sequence alignment of these TLSs suggests the existence of a tertiary D-loop–T-loop interaction consisting of 2 bp, analogous to those in the elbow region of canonical tRNAs. The conserved G18·Ψ55 pair of regular tRNAs is found to covary in these TLSs between G·U (possibly also modified to G·Ψ) and A·G. We have mutated the relevant bases in turnip yellow mosaic virus (TYMV) and examined the mutants for symptom development on Chinese cabbage plants and for accumulation of genetic reversions. Development of symptoms is shown to rely on the presence of either A·G or G·U in the original mutants or in revertants. This finding supports the existence and functional importance of this tertiary interaction. The fact that only G·U and A·G are accepted at this position appears to result from steric and energetic limitations related to the highly compact nature of the elbow region. We discuss the implications of these findings for the various possible functions of the valine-accepting TLS. PMID:12364602

  17. Bacterial community structure in the drinking water microbiome is governed by filtration processes.

    PubMed

    Pinto, Ameet J; Xi, Chuanwu; Raskin, Lutgarde

    2012-08-21

    The bacterial community structure of a drinking water microbiome was characterized over three seasons using 16S rRNA gene based pyrosequencing of samples obtained from source water (a mix of a groundwater and a surface water), different points in a drinking water plant operated to treat this source water, and in the associated drinking water distribution system. Even though the source water was shown to seed the drinking water microbiome, treatment process operations limit the source water's influence on the distribution system bacterial community. Rather, in this plant, filtration by dual media rapid sand filters played a primary role in shaping the distribution system bacterial community over seasonal time scales as the filters harbored a stable bacterial community that seeded the water treatment processes past filtration. Bacterial taxa that colonized the filter and sloughed off in the filter effluent were able to persist in the distribution system despite disinfection of finished water by chloramination and filter backwashing with chloraminated backwash water. Thus, filter colonization presents a possible ecological survival strategy for bacterial communities in drinking water systems, which presents an opportunity to control the drinking water microbiome by manipulating the filter microbial community. Grouping bacterial taxa based on their association with the filter helped to elucidate relationships between the abundance of bacterial groups and water quality parameters and showed that pH was the strongest regulator of the bacterial community in the sampled drinking water system.

  18. Effect of redox conditions on bacterial community structure in Baltic Sea sediments with contrasting phosphorus fluxes.

    PubMed

    Steenbergh, Anne K; Bodelier, Paul L E; Slomp, Caroline P; Laanbroek, Hendrikus J

    2014-01-01

    Phosphorus release from sediments can exacerbate the effect of eutrophication in coastal marine ecosystems. The flux of phosphorus from marine sediments to the overlying water is highly dependent on the redox conditions at the sediment-water interface. Bacteria are key players in the biological processes that release or retain phosphorus in marine sediments. To gain more insight in the role of bacteria in phosphorus release from sediments, we assessed the effect of redox conditions on the structure of bacterial communities. To do so, we incubated surface sediments from four sampling sites in the Baltic Sea under oxic and anoxic conditions and analyzed the fingerprints of the bacterial community structures in these incubations and the original sediments. This paper describes the effects of redox conditions, sampling station, and sample type (DNA, RNA, or whole-cell sample) on bacterial community structure in sediments. Redox conditions explained only 5% of the variance in community structure, and bacterial communities from contrasting redox conditions showed considerable overlap. We conclude that benthic bacterial communities cannot be classified as being typical for oxic or anoxic conditions based on community structure fingerprints. Our results suggest that the overall structure of the benthic bacterial community has only a limited impact on benthic phosphate fluxes in the Baltic Sea.

  19. Bacterial population structure of the jute-retting environment.

    PubMed

    Munshi, Tulika K; Chattoo, Bharat B

    2008-08-01

    Jute is one of the most versatile bast fibers obtained through the process of retting, which is a result of decomposition of stalks by the indigenous microflora. However, bacterial communities associated with the retting of jute are not well characterized. To investigate the presence of microorganisms during the process of jute retting, full-cycle rRNA approach was followed, and two 16S rRNA gene libraries, from jute-retting locations of Krishnanagar and Barrackpore, were constructed. Phylotypes affiliating to seven bacterial divisions were identified in both libraries. The bulk of clones came from Proteobacteria ( approximately 37, 41%) and a comparatively smaller proportion of clones from the divisions-Firmicutes ( approximately 11, 12%), Cytophaga-Flexibacter-Bacteroidetes group (CFB; approximately 9, 7%), Verrucomicrobia ( approximately 6, 5%), Acidobacteria ( approximately 4, 5%), Chlorobiales ( approximately 5, 5%), and Actinobacteria ( approximately 4, 2%) were identified. Percent coverage value and diversity estimations of phylotype richness, Shannon-Weiner index, and evenness confirmed the diverse nature of both the libraries. Evaluation of the retting waters by whole cell rRNA-targeted flourescent in situ hybridization, as detected by domain- and group-specific probes, we observed a considerable dominance of the beta-Proteobacteria (25.9%) along with the CFB group (24.4%). In addition, 32 bacterial species were isolated on culture media from the two retting environments and identified by 16S rDNA analysis, confirming the presence of phyla, Proteobacteria ( approximately 47%), Firmicutes ( approximately 22%), CFB group ( approximately 19%), and Actinobacteria ( approximately 13%) in the retting niche. Thus, our study presents the first quantification of the dominant and diverse bacterial phylotypes in the retting ponds, which will further help in improving the retting efficiency, and hence the fiber quality.

  20. Higher order structural effects stabilizing the reverse Watson-Crick Guanine-Cytosine base pair in functional RNAs.

    PubMed

    Chawla, Mohit; Abdel-Azeim, Safwat; Oliva, Romina; Cavallo, Luigi

    2014-01-01

    The G:C reverse Watson-Crick (W:W trans) base pair, also known as Levitt base pair in the context of tRNAs, is a structurally and functionally important base pair that contributes to tertiary interactions joining distant domains in functional RNA molecules and also participates in metabolite binding in riboswitches. We previously indicated that the isolated G:C W:W trans base pair is a rather unstable geometry, and that dicationic metal binding to the Guanine base or posttranscriptional modification of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G:C trans base pairs. Interestingly, 66% of the G:C trans base pairs in the PDB are engaged in additional H-bonding interactions with other bases, the RNA backbone or structured water molecules. High level quantum mechanical calculations on a data set of representative crystal structures were performed to shed light on the structural stability and energetics of the various crystallographic motifs. This analysis was extended to the binding of the preQ1 metabolite to a preQ1-II riboswitch.

  1. Higher order structural effects stabilizing the reverse Watson–Crick Guanine-Cytosine base pair in functional RNAs

    PubMed Central

    Chawla, Mohit; Abdel-Azeim, Safwat; Oliva, Romina; Cavallo, Luigi

    2014-01-01

    The G:C reverse Watson–Crick (W:W trans) base pair, also known as Levitt base pair in the context of tRNAs, is a structurally and functionally important base pair that contributes to tertiary interactions joining distant domains in functional RNA molecules and also participates in metabolite binding in riboswitches. We previously indicated that the isolated G:C W:W trans base pair is a rather unstable geometry, and that dicationic metal binding to the Guanine base or posttranscriptional modification of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G:C trans base pairs. Interestingly, 66% of the G:C trans base pairs in the PDB are engaged in additional H-bonding interactions with other bases, the RNA backbone or structured water molecules. High level quantum mechanical calculations on a data set of representative crystal structures were performed to shed light on the structural stability and energetics of the various crystallographic motifs. This analysis was extended to the binding of the preQ1 metabolite to a preQ1-II riboswitch. PMID:24121683

  2. Structure and functions of the bacterial microbiota of plants.

    PubMed

    Bulgarelli, Davide; Schlaeppi, Klaus; Spaepen, Stijn; Ver Loren van Themaat, Emiel; Schulze-Lefert, Paul

    2013-01-01

    Plants host distinct bacterial communities on and inside various plant organs, of which those associated with roots and the leaf surface are best characterized. The phylogenetic composition of these communities is defined by relatively few bacterial phyla, including Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. A synthesis of available data suggests a two-step selection process by which the bacterial microbiota of roots is differentiated from the surrounding soil biome. Rhizodeposition appears to fuel an initial substrate-driven community shift in the rhizosphere, which converges with host genotype-dependent fine-tuning of microbiota profiles in the selection of root endophyte assemblages. Substrate-driven selection also underlies the establishment of phyllosphere communities but takes place solely at the immediate leaf surface. Both the leaf and root microbiota contain bacteria that provide indirect pathogen protection, but root microbiota members appear to serve additional host functions through the acquisition of nutrients from soil for plant growth. Thus, the plant microbiota emerges as a fundamental trait that includes mutualism enabled through diverse biochemical mechanisms, as revealed by studies on plant growth-promoting and plant health-promoting bacteria.

  3. Supraglacial bacterial community structures vary across the Greenland ice sheet.

    PubMed

    Cameron, Karen A; Stibal, Marek; Zarsky, Jakub D; Gözdereliler, Erkin; Schostag, Morten; Jacobsen, Carsten S

    2016-02-01

    The composition and spatial variability of microbial communities that reside within the extensive (>200 000 km(2)) biologically active area encompassing the Greenland ice sheet (GrIS) is hypothesized to be variable. We examined bacterial communities from cryoconite debris and surface ice across the GrIS, using sequence analysis and quantitative PCR of 16S rRNA genes from co-extracted DNA and RNA. Communities were found to differ across the ice sheet, with 82.8% of the total calculated variation attributed to spatial distribution on a scale of tens of kilometers separation. Amplicons related to Sphingobacteriaceae, Pseudanabaenaceae and WPS-2 accounted for the greatest portion of calculated dissimilarities. The bacterial communities of ice and cryoconite were moderately similar (global R = 0.360, P = 0.002) and the sampled surface type (ice versus cryoconite) did not contribute heavily towards community dissimilarities (2.3% of total variability calculated). The majority of dissimilarities found between cryoconite 16S rRNA gene amplicons from DNA and RNA was calculated to be the result of changes in three taxa, Pseudanabaenaceae, Sphingobacteriaceae and WPS-2, which together contributed towards 80.8 ± 12.6% of dissimilarities between samples. Bacterial communities across the GrIS are spatially variable active communities that are likely influenced by localized biological inputs and physicochemical conditions.

  4. Bacterial Community Structure and Diversity in a Century-Old Manure-Treated Agroecosystem

    PubMed Central

    Sun, H. Y.; Deng, S. P.; Raun, W. R.

    2004-01-01

    Changes in soil microbial community structure and diversity may reflect environmental impact. We examined 16S rRNA gene fingerprints of bacterial communities in six agroecosystems by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. These soils were treated with manure for over a century or different fertilizers for over 70 years. Bacterial community structure and diversity were affected by soil management practices, as evidenced by changes in the PCR-DGGE banding patterns. Bacterial community structure in the manure-treated soil was more closely related to the structure in the untreated soil than that in soils treated with inorganic fertilizers. Lime treatment had little effect on bacterial community structure. Soils treated with P and N-P had bacterial community structures more closely related to each other than to those of soils given other treatments. Among the soils tested, a significantly higher number of bacterial ribotypes and a more even distribution of the bacterial community existed in the manure-treated soil. Of the 99 clones obtained from the soil treated with manure for over a century, two (both Pseudomonas spp.) exhibited 100% similarity to sequences in the GenBank database. Two of the clones were possible chimeras. Based on similarity matching, the remaining 97 clones formed six major clusters. Fifty-six out of 97 were assigned taxonomic units which grouped into five major taxa: α-, β-, and γ-Proteobacteria (36 clones), Acidobacteria (16 clones), Bacteroidetes (2 clones), Nitrospirae (1 clone), and Firmicutes (1 clone). Forty-one clones remained unclassified. Results from this study suggested that bacterial community structure was closely related to agroecosystem management practices conducted for over 70 years. PMID:15466526

  5. Elevated Air Humidity Changes Soil Bacterial Community Structure in the Silver Birch Stand

    PubMed Central

    Truu, Marika; Ostonen, Ivika; Preem, Jens-Konrad; Lõhmus, Krista; Nõlvak, Hiie; Ligi, Teele; Rosenvald, Katrin; Parts, Kaarin; Kupper, Priit; Truu, Jaak

    2017-01-01

    Soil microbes play a fundamental role in forest ecosystems and respond rapidly to changes in the environment. Simultaneously with the temperature increase the climate change scenarios also predict an intensified hydrological cycle for the Baltic Sea runoff region. The aim of this study was to assess the effect of elevated air humidity on the top soil microbial community structure of a silver birch (Betula pendula Roth.) stand by using a free air humidity manipulation facility (FAHM). The bacterial community structures of bulk soil and birch rhizosphere were analyzed using high-throughput sequencing of bacteria-specific16S rRNA gene fragments and quantification of denitrification related genes. The increased air humidity altered both bulk soil and rhizosphere bacterial community structures, and changes in the bacterial communities initiated by elevated air humidity were related to modified soil abiotic and biotic variables. Network analysis revealed that variation in soil bacterial community structural units is explained by altered abiotic conditions such as increased pH value in bulk soil, while in rhizosphere the change in absorptive root morphology had a higher effect. Among root morphological traits, the absorptive root diameter was strongest related to the bacterial community structure. The changes in bacterial community structures under elevated air humidity are associated with shifts in C, N, and P turnover as well as mineral weathering processes in soil. Increased air humidity decreased the nir and nosZ gene abundance in the rhizosphere bacterial community. The potential contribution of the denitrification to the N2O emission was not affected by the elevated air humidity in birch stand soil. In addition, the study revealed a strong link between the bacterial community structure, abundance of denitrification related genes, and birch absorptive root morphology in the ecosystem system adaptation to elevated air humidity. PMID:28421053

  6. 78 FR 63220 - Guidance for Industry on Acute Bacterial Skin and Skin Structure Infections: Developing Drugs for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-23

    ... HUMAN SERVICES Food and Drug Administration Guidance for Industry on Acute Bacterial Skin and Skin... guidance for industry entitled ``Acute Bacterial Skin and Skin Structure Infections: Developing Drugs for... drugs to treat acute bacterial skin and skin structure infections (ABSSSI). This guidance finalizes the...

  7. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  8. Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection.

    PubMed

    Carbonell, Alberto; Daròs, José-Antonio

    2016-12-27

    Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn-tasiRNAs to specifically interfere with infections by viroids, small (250-400 nt) non-coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent high-throughput methods for artificial sRNA construct generation and of the Potato spindle tuber viroid (PSTVd)/Nicotiana benthamiana pathosystem allowed for the simple and time-effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (+) or (-) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co-expressed with an infectious PSTVd transcript, as were syn-tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn-tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non-agroinfiltrated leaves where PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syn-tasiRNAs can be used effectively to control viroid infections in plants. This article is protected by copyright. All rights reserved.

  9. New insights into RNA secondary structure in the alternative splicing of pre-mRNAs.

    PubMed

    Jin, Yongfeng; Yang, Yun; Zhang, Peng

    2011-01-01

    Alternative splicing is an important mechanism in generating proteomic diversity, and RNA secondary structure is an important element in splicing regulation. The use of high-throughput sequencing and other approaches has increased the number of known pre-mRNA secondary structures by several orders of magnitude, and we now have new insights into the role of RNA secondary structure in alternative splicing and the mechanisms involved (e.g., physical competition, long-range RNA pairing, the structural splicing code, and co-transcriptional splicing). Furthermore, an RNA pairing-based mechanism ensures the selection of only one of several available exons (e.g., Dscam splicing). Here we review several recent discoveries related to the role of RNA secondary structure in alternative splicing and the underlying mechanisms.

  10. The structure and functions of bacterial communities in an agrocenosis

    NASA Astrophysics Data System (ADS)

    Dobrovol'skaya, T. G.; Khusnetdinova, K. A.; Manucharova, N. A.; Balabko, P. N.

    2016-01-01

    The most significant factor responsible for the specific taxonomic composition of the bacterial communities in the agrocenosis studied was found to be a part or organ of plants (leaves, flowers, roots, fruits). A stage of plant ontogeny also determines changes of taxa. In the course of the plant growth, eccrisotrophic bacteria are replaced by hydrolytic ones that belong to the group of cellulose-decomposing bacteria. Representatives of the proteobacteria genera that are difficult to identify by phenotypic methods were determined using molecular-biological methods. They were revealed only on oat leaves in the moist period. As the vetch-oat mixture was fertilized with BIOUD-1 (foliar application) in the phyllosphere of both oats and vetch, on all the plant organs, representatives of the Rhodococcus genus as dominants were isolated. This fact was related to the capability of bacteria to decompose the complex aromatic compounds that are ingredients of the fertilizers applied. Another positive effect for plants of the bacterial communities forming in agrocenoses is the presence of bacteria that are antagonists of phytopathogenic bacteria. Thus, in agrocenoses, some interrelationships promoting the growth and reproduction of plants are formed in crop plants and bacteria.

  11. Driving forces of soil bacterial community structure, diversity, and function in temperate grasslands and forests

    PubMed Central

    Kaiser, Kristin; Wemheuer, Bernd; Korolkow, Vera; Wemheuer, Franziska; Nacke, Heiko; Schöning, Ingo; Schrumpf, Marion; Daniel, Rolf

    2016-01-01

    Soil bacteria provide a large range of ecosystem services such as nutrient cycling. Despite their important role in soil systems, compositional and functional responses of bacterial communities to different land use and management regimes are not fully understood. Here, we assessed soil bacterial communities in 150 forest and 150 grassland soils derived from three German regions by pyrotag sequencing of 16S rRNA genes. Land use type (forest and grassland) and soil edaphic properties strongly affected bacterial community structure and function, whereas management regime had a minor effect. In addition, a separation of soil bacterial communities by sampling region was encountered. Soil pH was the best predictor for bacterial community structure, diversity and function. The application of multinomial log-linear models revealed distinct responses of abundant bacterial groups towards pH. Predicted functional profiles revealed that differences in land use not only select for distinct bacterial populations but also for specific functional traits. The combination of 16S rRNA data and corresponding functional profiles provided comprehensive insights into compositional and functional adaptations to changing environmental conditions associated with differences in land use and management. PMID:27650273

  12. Driving forces of soil bacterial community structure, diversity, and function in temperate grasslands and forests

    NASA Astrophysics Data System (ADS)

    Kaiser, Kristin; Wemheuer, Bernd; Korolkow, Vera; Wemheuer, Franziska; Nacke, Heiko; Schöning, Ingo; Schrumpf, Marion; Daniel, Rolf

    2016-09-01

    Soil bacteria provide a large range of ecosystem services such as nutrient cycling. Despite their important role in soil systems, compositional and functional responses of bacterial communities to different land use and management regimes are not fully understood. Here, we assessed soil bacterial communities in 150 forest and 150 grassland soils derived from three German regions by pyrotag sequencing of 16S rRNA genes. Land use type (forest and grassland) and soil edaphic properties strongly affected bacterial community structure and function, whereas management regime had a minor effect. In addition, a separation of soil bacterial communities by sampling region was encountered. Soil pH was the best predictor for bacterial community structure, diversity and function. The application of multinomial log-linear models revealed distinct responses of abundant bacterial groups towards pH. Predicted functional profiles revealed that differences in land use not only select for distinct bacterial populations but also for specific functional traits. The combination of 16S rRNA data and corresponding functional profiles provided comprehensive insights into compositional and functional adaptations to changing environmental conditions associated with differences in land use and management.

  13. Wolbachia small noncoding RNAs and their role in cross-kingdom communications.

    PubMed

    Mayoral, Jaime G; Hussain, Mazhar; Joubert, D Albert; Iturbe-Ormaetxe, Iñaki; O'Neill, Scott L; Asgari, Sassan

    2014-12-30

    In prokaryotes, small noncoding RNAs (snRNAs) of 50-500 nt are produced that are important in bacterial virulence and response to environmental stimuli. Here, we identified and characterized snRNAs from the endosymbiotic bacteria, Wolbachia, which are widespread in invertebrates and cause reproductive manipulations. Most importantly, some strains of Wolbachia inhibit replication of several vector-borne pathogens in insects. We demonstrate that two abundant snRNAs, WsnRNA-46 and WsnRNA-49, are expressed in Wolbachia from noncoding RNA transcripts that contain precursors with stem-loop structures. WsnRNAs were detected in Aedes aegypti mosquitoes infected with the wMelPop-CLA strain of Wolbachia and in Drosophila melanogaster and Drosophila simulans infected with wMelPop and wAu strains, respectively, indicating that the WsnRNAs are conserved across species and strains. In addition, we show that the WsnRNAs may potentially regulate host genes and Wolbachia genes. Our findings provide evidence for the production of functional snRNAs by Wolbachia that play roles in cross-kingdom communication between the endosymbiont and the host.

  14. Structures of the RNA-guided surveillance complex from a bacterial immune system

    PubMed Central

    Zhou, Kaihong; Jore, Matthijs M.; Brouns, Stan J. J.; van der Oost, John; Doudna, Jennifer A.; Nogales, Eva

    2014-01-01

    Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors1–6. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger7–12. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages13,14. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5′ end of the crRNA. Base pairing extends along the crRNA, resultingina series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences. PMID:21938068

  15. Systematic discovery of structural elements governing stability of mammalian messenger RNAs.

    PubMed

    Goodarzi, Hani; Najafabadi, Hamed S; Oikonomou, Panos; Greco, Todd M; Fish, Lisa; Salavati, Reza; Cristea, Ileana M; Tavazoie, Saeed

    2012-04-08

    Decoding post-transcriptional regulatory programs in RNA is a critical step towards the larger goal of developing predictive dynamical models of cellular behaviour. Despite recent efforts, the vast landscape of RNA regulatory elements remains largely uncharacterized. A long-standing obstacle is the contribution of local RNA secondary structure to the definition of interaction partners in a variety of regulatory contexts, including--but not limited to--transcript stability, alternative splicing and localization. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (for example, human cardiac troponin T) or affects other aspects of RNA biology. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence. Here we present a computational framework based on context-free grammars and mutual information that systematically explores the immense space of small structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behaviour. By applying this framework to genome-wide human mRNA stability data, we reveal eight highly significant elements with substantial structural information, for the strongest of which we show a major role in global mRNA regulation. Through biochemistry, mass spectrometry and in vivo binding studies, we identified human HNRPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1, also known as HNRNPA2B1) as the key regulator that binds this element and stabilizes a large number of its target genes. We created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach could also be used to reveal the structural elements that modulate other aspects of RNA behaviour.

  16. Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): Characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans

    PubMed Central

    Herget, Stephan; Toukach, Philip V; Ranzinger, René; Hull, William E; Knirel, Yuriy A; von der Lieth, Claus-Wilhelm

    2008-01-01

    Background There are considerable differences between bacterial and mammalian glycans. In contrast to most eukaryotic carbohydrates, bacterial glycans are often composed of repeating units with diverse functions ranging from structural reinforcement to adhesion, colonization and camouflage. Since bacterial glycans are typically displayed at the cell surface, they can interact with the environment and, therefore, have significant biomedical importance. Results The sequence characteristics of glycans (monosaccharide composition, modifications, and linkage patterns) for the higher bacterial taxonomic classes have been examined and compared with the data for mammals, with both similarities and unique features becoming evident. Compared to mammalian glycans, the bacterial glycans deposited in the current databases have a more than ten-fold greater diversity at the monosaccharide level, and the disaccharide pattern space is approximately nine times larger. Specific bacterial subclasses exhibit characteristic glycans which can be distinguished on the basis of distinctive structural features or sequence properties. Conclusion For the first time a systematic database analysis of the bacterial glycome has been performed. This study summarizes the current knowledge of bacterial glycan architecture and diversity and reveals putative targets for the rational design and development of therapeutic intervention strategies by comparing bacterial and mammalian glycans. PMID:18694500

  17. Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans.

    PubMed

    Herget, Stephan; Toukach, Philip V; Ranzinger, René; Hull, William E; Knirel, Yuriy A; von der Lieth, Claus-Wilhelm

    2008-08-11

    There are considerable differences between bacterial and mammalian glycans. In contrast to most eukaryotic carbohydrates, bacterial glycans are often composed of repeating units with diverse functions ranging from structural reinforcement to adhesion, colonization and camouflage. Since bacterial glycans are typically displayed at the cell surface, they can interact with the environment and, therefore, have significant biomedical importance. The sequence characteristics of glycans (monosaccharide composition, modifications, and linkage patterns) for the higher bacterial taxonomic classes have been examined and compared with the data for mammals, with both similarities and unique features becoming evident. Compared to mammalian glycans, the bacterial glycans deposited in the current databases have a more than ten-fold greater diversity at the monosaccharide level, and the disaccharide pattern space is approximately nine times larger. Specific bacterial subclasses exhibit characteristic glycans which can be distinguished on the basis of distinctive structural features or sequence properties. For the first time a systematic database analysis of the bacterial glycome has been performed. This study summarizes the current knowledge of bacterial glycan architecture and diversity and reveals putative targets for the rational design and development of therapeutic intervention strategies by comparing bacterial and mammalian glycans.

  18. Structure-based receptor MIMICS targeted against bacterial superantigen toxins

    DOEpatents

    Gupta, Goutam; Hong-Geller, Elizabeth; Shiflett, Patrick R.; Lehnert, Nancy M.

    2009-08-18

    The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

  19. Using Structural and Evolutionary Information to Detect and Correct Pyrosequencing Errors in Noncoding RNAs

    PubMed Central

    Reinharz, Vladimir

    2013-01-01

    Abstract The analysis of the sequence–structure relationship in RNA molecules is not only essential for evolutionary studies but also for concrete applications such as error-correction in next generation sequencing (NGS) technologies. The prohibitive sizes of the mutational and conformational landscapes, combined with the volume of data to process, require efficient algorithms to compute sequence–structure properties. In this article, we address the correction of NGS errors by calculating which mutations most increase the likelihood of a sequence to a given structure and RNA family. We introduce RNApyro, an efficient, linear time and space inside–outside algorithm that computes exact mutational probabilities under secondary structure and evolutionary constraints given as a multiple sequence alignment with a consensus structure. We develop a scoring scheme combining classical stacking base-pair energies to novel isostericity scores and apply our techniques to correct pointwise errors in 5s and 16s rRNA sequences. Our results suggest that RNApyro is a promising algorithm to complement existing tools in the NGS error-correction pipeline. PMID:24134390

  20. Bacterial community structure is indicative of chemical inputs in the Upper Mississippi River

    PubMed Central

    Staley, Christopher; Gould, Trevor J.; Wang, Ping; Phillips, Jane; Cotner, James B.; Sadowsky, Michael J.

    2014-01-01

    Local and regional associations between bacterial communities and nutrient and chemical concentrations were assessed in the Upper Mississippi River in Minnesota to determine if community structure was associated with discrete types of chemical inputs associated with different land cover. Bacterial communities were characterized by Illumina sequencing of the V6 region of 16S rDNA and compared to >40 chemical and nutrient concentrations. Local bacterial community structure was shaped primarily by associations among bacterial orders. However, order abundances were correlated regionally with nutrient and chemical concentrations, and were also related to major land coverage types. Total organic carbon and total dissolved solids were among the primary abiotic factors associated with local community composition and co-varied with land cover. Escherichia coli concentration was poorly related to community composition or nutrient concentrations. Abundances of 14 bacterial orders were related to land coverage type, and seven showed significant differences in abundance (P ≤ 0.046) between forested or anthropogenically-impacted sites. This study identifies specific bacterial orders that were associated with chemicals and nutrients derived from specific land cover types and may be useful in assessing water quality. Results of this study reveal the need to investigate community dynamics at both the local and regional scales and to identify shifts in taxonomic community structure that may be useful in determining sources of pollution in the Upper Mississippi River. PMID:25339945

  1. Centralized Drinking Water Treatment Operations Shape Bacterial and Fungal Community Structure.

    PubMed

    Ma, Xiao; Vikram, Amit; Casson, Leonard; Bibby, Kyle

    2017-07-05

    Drinking water microbial communities impact opportunistic pathogen colonization and corrosion of water distribution systems, and centralized drinking water treatment represents a potential control for microbial community structure in finished drinking water. In this article, we examine bacterial and fungal abundance and diversity, as well as the microbial community taxonomic structure following each unit operation in a conventional surface water treatment plant. Treatment operations drove the microbial composition more strongly than sampling time. Both bacterial and fungal abundance and diversity decreased following sedimentation and filtration; however, only bacterial abundance and diversity was significantly impacted by free chlorine disinfection. Similarly, each treatment step was found to shift bacterial and fungal community beta-diversity, with the exception of disinfection on the fungal community structure. We observed the enrichment of bacterial and fungal taxa commonly found in drinking water distribution systems through the treatment process, for example, Sphingomonas following filtration and Leptospirillium and Penicillium following disinfection. Study results suggest that centralized drinking water treatment processes shape the final drinking water microbial community via selection of community members and that the bacterial community is primarily driven by disinfection while the eukaryotic community is primarily controlled by physical treatment processes.

  2. Dynamic Effects of Biochar on the Bacterial Community Structure in Soil Contaminated with Polycyclic Aromatic Hydrocarbons.

    PubMed

    Song, Yang; Bian, Yongrong; Wang, Fang; Xu, Min; Ni, Ni; Yang, Xinglun; Gu, Chenggang; Jiang, Xin

    2017-08-16

    Amending soil with biochar is an effective soil remediation strategy for organic contaminants. This study investigated the dynamic effects of wheat straw biochar on the bacterial community structure during remediation by high-throughput sequencing. The wheat straw biochar amended into the soil significantly reduced the bioavailability and toxicity of polycyclic aromatic hydrocarbons (PAHs). Biochar amendment helped to maintain the bacterial diversity in the PAH-contaminated soil. The relationship between the immobilization of PAHs and the soil bacterial diversity fit a quadratic model. Before week 12 of the incubation, the incubation time was the main factor contributing to the changes in the soil bacterial community structure. However, biochar greatly affected the bacterial community structure after 12 weeks of amendment, and the effects were dependent upon the biochar type. Amendment with biochar mainly facilitated the growth of rare bacterial genera (relative abundance of 0.01-1%) in the studied soil. Therefore, the application of wheat straw biochar into PAH-contaminated soil can reduce the environmental risks of PAHs and benefit the soil microbial ecology.

  3. Bacterial community structure and soil properties of a subarctic tundra soil in Council, Alaska.

    PubMed

    Kim, Hye Min; Jung, Ji Young; Yergeau, Etienne; Hwang, Chung Yeon; Hinzman, Larry; Nam, Sungjin; Hong, Soon Gyu; Kim, Ok-Sun; Chun, Jongsik; Lee, Yoo Kyung

    2014-08-01

    The subarctic region is highly responsive and vulnerable to climate change. Understanding the structure of subarctic soil microbial communities is essential for predicting the response of the subarctic soil environment to climate change. To determine the composition of the bacterial community and its relationship with soil properties, we investigated the bacterial community structure and properties of surface soil from the moist acidic tussock tundra in Council, Alaska. We collected 70 soil samples with 25-m intervals between sampling points from 0-10 cm to 10-20 cm depths. The bacterial community was analyzed by pyrosequencing of 16S rRNA genes, and the following soil properties were analyzed: soil moisture content (MC), pH, total carbon (TC), total nitrogen (TN), and inorganic nitrogen (NH4+ and NO3-). The community compositions of the two different depths showed that Alphaproteobacteria decreased with soil depth. Among the soil properties measured, soil pH was the most significant factor correlating with bacterial community in both upper and lower-layer soils. Bacterial community similarity based on jackknifed unweighted unifrac distance showed greater similarity across horizontal layers than through the vertical depth. This study showed that soil depth and pH were the most important soil properties determining bacterial community structure of the subarctic tundra soil in Council, Alaska.

  4. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression.

    PubMed

    Derrien, Thomas; Johnson, Rory; Bussotti, Giovanni; Tanzer, Andrea; Djebali, Sarah; Tilgner, Hagen; Guernec, Gregory; Martin, David; Merkel, Angelika; Knowles, David G; Lagarde, Julien; Veeravalli, Lavanya; Ruan, Xiaoan; Ruan, Yijun; Lassmann, Timo; Carninci, Piero; Brown, James B; Lipovich, Leonard; Gonzalez, Jose M; Thomas, Mark; Davis, Carrie A; Shiekhattar, Ramin; Gingeras, Thomas R; Hubbard, Tim J; Notredame, Cedric; Harrow, Jennifer; Guigó, Roderic

    2012-09-01

    The human genome contains many thousands of long noncoding RNAs (lncRNAs). While several studies have demonstrated compelling biological and disease roles for individual examples, analytical and experimental approaches to investigate these genes have been hampered by the lack of comprehensive lncRNA annotation. Here, we present and analyze the most complete human lncRNA annotation to date, produced by the GENCODE consortium within the framework of the ENCODE project and comprising 9277 manually annotated genes producing 14,880 transcripts. Our analyses indicate that lncRNAs are generated through pathways similar to that of protein-coding genes, with similar histone-modification profiles, splicing signals, and exon/intron lengths. In contrast to protein-coding genes, however, lncRNAs display a striking bias toward two-exon transcripts, they are predominantly localized in the chromatin and nucleus, and a fraction appear to be preferentially processed into small RNAs. They are under stronger selective pressure than neutrally evolving sequences-particularly in their promoter regions, which display levels of selection comparable to protein-coding genes. Importantly, about one-third seem to have arisen within the primate lineage. Comprehensive analysis of their expression in multiple human organs and brain regions shows that lncRNAs are generally lower expressed than protein-coding genes, and display more tissue-specific expression patterns, with a large fraction of tissue-specific lncRNAs expressed in the brain. Expression correlation analysis indicates that lncRNAs show particularly striking positive correlation with the expression of antisense coding genes. This GENCODE annotation represents a valuable resource for future studies of lncRNAs.

  5. Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage

    PubMed Central

    Göpel, Yvonne; Khan, Muna Ayesha; Görke, Boris

    2016-01-01

    In E. coli, small RNA GlmZ activates the glmS mRNA by base-pairing in an Hfq dependent manner. When not required, GlmZ is bound by adaptor protein RapZ and recruited to RNase E, which cleaves GlmZ in its base-pairing sequence. Small RNA GlmY counteracts cleavage of GlmZ by sequestration of RapZ. Although both sRNAs are highly homologous, only GlmZ specifically binds Hfq and undergoes cleavage by RNase E. We used domain swapping to identify the responsible modules. Two elements, the 3′ terminal oligo(U) stretch and the base-pairing region enable GlmZ to interact with Hfq. Accordingly, Hfq inhibits cleavage of GlmZ, directing it to base-pairing. Intriguingly, the central stem loop of GlmZ is decisive for cleavage, whereas the sequence comprising the actual cleavage site is dispensable. Assisted by RapZ, RNase E cleaves any RNA fused to the 3′ end of this module. These results suggest a novel mode for RNase E recognition, in which one of the required handholds in the substrate is replaced by an RNA binding protein. This device can generate RNAs of interest in their 5′ monophosphorylated form on demand. As these species are rapidly degraded, this tool allows to regulate gene expression post-transcriptionally by modulation of RapZ levels. PMID:26531825

  6. Bacterial and Fungal Community Structures in Loess Plateau Grasslands with Different Grazing Intensities

    PubMed Central

    Huhe; Chen, Xianjiang; Hou, Fujiang; Wu, Yanpei; Cheng, Yunxiang

    2017-01-01

    The Loess Plateau of China is one of the most fragile ecosystems worldwide; thus, human production activities need to be conducted very cautiously. In this study, MiSeq high-throughput sequencing was applied to assess the relationship between bacterial and fungal community structures and changes in vegetation and soil physical and chemical properties induced by grazing, in four grasslands with different levels of grazing intensity (0, 2.67, 5.33, and 8.67 sheep/ha) in the semiarid region of the Loess Plateau. The relative abundances of the bacterial community in the grasslands with 2.67 and 5.33 sheep/ha were significantly higher than those in grasslands with 0 and 8.67 sheep/ha, and the fungal diversity was significantly lower for grasslands with 2.67 sheep/ha than for the other grasslands. Redundancy analysis (RDA) showed that plant biomass, nitrate, and total nitrogen have significant effects on bacterial community structure, whereas nitrate and total nitrogen also significantly affect fungal community structure. Variation partitioning showed that soil and plant characteristics influence the bacterial and fungal community structures; these characteristics explained 51.9 and 52.9% of the variation, respectively. Thus, bacterial and fungal community structures are very sensitive to grazing activity and change to different extents with different grazing intensities. Based on our findings, a grazing intensity of about 2.67 sheep/ha is considered the most appropriate in semiarid grassland of the Loess Plateau. PMID:28439265

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

    PubMed

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

    2014-06-01

    A large number of collagen-like proteins have been identified in bacteria during the past 10years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in Escherichia coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35-39°C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions.

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

    PubMed Central

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

    2014-01-01

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

  9. Bacterial and Fungal Community Structures in Loess Plateau Grasslands with Different Grazing Intensities.

    PubMed

    Huhe; Chen, Xianjiang; Hou, Fujiang; Wu, Yanpei; Cheng, Yunxiang

    2017-01-01

    The Loess Plateau of China is one of the most fragile ecosystems worldwide; thus, human production activities need to be conducted very cautiously. In this study, MiSeq high-throughput sequencing was applied to assess the relationship between bacterial and fungal community structures and changes in vegetation and soil physical and chemical properties induced by grazing, in four grasslands with different levels of grazing intensity (0, 2.67, 5.33, and 8.67 sheep/ha) in the semiarid region of the Loess Plateau. The relative abundances of the bacterial community in the grasslands with 2.67 and 5.33 sheep/ha were significantly higher than those in grasslands with 0 and 8.67 sheep/ha, and the fungal diversity was significantly lower for grasslands with 2.67 sheep/ha than for the other grasslands. Redundancy analysis (RDA) showed that plant biomass, nitrate, and total nitrogen have significant effects on bacterial community structure, whereas nitrate and total nitrogen also significantly affect fungal community structure. Variation partitioning showed that soil and plant characteristics influence the bacterial and fungal community structures; these characteristics explained 51.9 and 52.9% of the variation, respectively. Thus, bacterial and fungal community structures are very sensitive to grazing activity and change to different extents with different grazing intensities. Based on our findings, a grazing intensity of about 2.67 sheep/ha is considered the most appropriate in semiarid grassland of the Loess Plateau.

  10. Hydrologic linkages drive spatial structuring of bacterial assemblages and functioning in alpine floodplains.

    PubMed

    Freimann, Remo; Bürgmann, Helmut; Findlay, Stuart E G; Robinson, Christopher T

    2015-01-01

    Microbial community assembly and microbial functions are affected by a number of different but coupled drivers such as local habitat characteristics, dispersal rates, and species interactions. In groundwater systems, hydrological flow can introduce spatial structure and directional dependencies among these drivers. We examined the importance of hydrology in structuring bacterial communities and their function within two alpine floodplains during different hydrological states. Piezometers were installed in stream sediments and surrounding riparian zones to assess hydrological flows and also were used as incubation chambers to examine bacterial community structures and enzymatic functions along hydrological flow paths. Spatial eigenvector models in conjunction with models based on physico-chemical groundwater characteristics were used to evaluate the importance of hydrologically-driven processes influencing bacterial assemblages and their enzymatic activities. Our results suggest a strong influence (up to 40% explained variation) of hydrological connectivity on enzymatic activities. The effect of hydrology on bacterial community structure was considerably less strong, suggesting that assemblages demonstrate large functional plasticity/redundancy. Effect size varied between hydrological periods but flow-related mechanisms always had the most power in explaining both bacterial structure and functioning. Changes in hydrology should be considered in models predicting ecosystem functioning and integrated into ecosystem management strategies for floodplains.

  11. Hydrologic linkages drive spatial structuring of bacterial assemblages and functioning in alpine floodplains

    PubMed Central

    Freimann, Remo; Bürgmann, Helmut; Findlay, Stuart E. G.; Robinson, Christopher T.

    2015-01-01

    Microbial community assembly and microbial functions are affected by a number of different but coupled drivers such as local habitat characteristics, dispersal rates, and species interactions. In groundwater systems, hydrological flow can introduce spatial structure and directional dependencies among these drivers. We examined the importance of hydrology in structuring bacterial communities and their function within two alpine floodplains during different hydrological states. Piezometers were installed in stream sediments and surrounding riparian zones to assess hydrological flows and also were used as incubation chambers to examine bacterial community structures and enzymatic functions along hydrological flow paths. Spatial eigenvector models in conjunction with models based on physico-chemical groundwater characteristics were used to evaluate the importance of hydrologically-driven processes influencing bacterial assemblages and their enzymatic activities. Our results suggest a strong influence (up to 40% explained variation) of hydrological connectivity on enzymatic activities. The effect of hydrology on bacterial community structure was considerably less strong, suggesting that assemblages demonstrate large functional plasticity/redundancy. Effect size varied between hydrological periods but flow-related mechanisms always had the most power in explaining both bacterial structure and functioning. Changes in hydrology should be considered in models predicting ecosystem functioning and integrated into ecosystem management strategies for floodplains. PMID:26579113

  12. Evaluating bacterial gene-finding HMM structures as probabilistic logic programs.

    PubMed

    Mørk, Søren; Holmes, Ian

    2012-03-01

    Probabilistic logic programming offers a powerful way to describe and evaluate structured statistical models. To investigate the practicality of probabilistic logic programming for structure learning in bioinformatics, we undertook a simplified bacterial gene-finding benchmark in PRISM, a probabilistic dialect of Prolog. We evaluate Hidden Markov Model structures for bacterial protein-coding gene potential, including a simple null model structure, three structures based on existing bacterial gene finders and two novel model structures. We test standard versions as well as ADPH length modeling and three-state versions of the five model structures. The models are all represented as probabilistic logic programs and evaluated using the PRISM machine learning system in terms of statistical information criteria and gene-finding prediction accuracy, in two bacterial genomes. Neither of our implementations of the two currently most used model structures are best performing in terms of statistical information criteria or prediction performances, suggesting that better-fitting models might be achievable. The source code of all PRISM models, data and additional scripts are freely available for download at: http://github.com/somork/codonhmm. Supplementary data are available at Bioinformatics online.

  13. Target activation by regulatory RNAs in bacteria

    PubMed Central

    Papenfort, Kai; Vanderpool, Carin K.

    2015-01-01

    Bacterial small regulatory RNAs (sRNAs) are commonly known to repress gene expression by base pairing to target mRNAs. In many cases, sRNAs base pair with and sequester mRNA ribosome-binding sites, resulting in translational repression and accelerated transcript decay. In contrast, a growing number of examples of translational activation and mRNA stabilization by sRNAs have now been documented. A given sRNA often employs a conserved region to interact with and regulate both repressed and activated targets. However, the mechanisms underlying activation differ substantially from repression. Base pairing resulting in target activation can involve sRNA interactions with the 5′ untranslated region (UTR), the coding sequence or the 3′ UTR of the target mRNAs. Frequently, the activities of protein factors such as cellular ribonucleases and the RNA chaperone Hfq are required for activation. Bacterial sRNAs, including those that function as activators, frequently control stress response pathways or virulence-associated functions required for immediate responses to changing environments. This review aims to summarize recent advances in knowledge regarding target mRNA activation by bacterial sRNAs, highlighting the molecular mechanisms and biological relevance of regulation. PMID:25934124

  14. tRNAs as Antibiotic Targets

    PubMed Central

    Chopra, Shaileja; Reader, John

    2014-01-01

    Transfer RNAs (tRNAs) are central players in the protein translation machinery and as such are prominent targets for a large number of natural and synthetic antibiotics. This review focuses on the role of tRNAs in bacterial antibiosis. We will discuss examples of antibiotics that target multiple stages in tRNA biology from tRNA biogenesis and modification, mature tRNAs, aminoacylation of tRNA as well as prevention of proper tRNA function by small molecules binding to the ribosome. Finally, the role of deacylated tRNAs in the bacterial “stringent response” mechanism that can lead to bacteria displaying antibiotic persistence phenotypes will be discussed. PMID:25547494

  15. Molecular Dynamics Simulations for Deciphering the Structural Basis of Recognition of Pre-let-7 miRNAs by LIN28.

    PubMed

    Sharma, Chhaya; Mohanty, Debasisa

    2017-02-07

    LIN28 protein inhibits biogenesis of miRNAs belonging to the let-7 family by binding to precursor forms of miRNAs. Overexpression of LIN28 and low levels of let-7 miRNAs are associated with several forms of cancer cells. We have performed multiple explicit solvent molecular dynamics simulations ranging from 200 to 500 ns in length on different isoforms of preE-let-7 in complex with LIN28 and also in isolation to identify structural features and key specificity-determining residues (SDRs) that are important for the inhibitory role of LIN28. Our simulations suggest that a conserved structural feature of the loop regions of preE-let-7 miRNAs is more important for LIN28 recognition than sequence conservation among members of the let-7 family or the presence of the GGAG motif in the 3' region. The loop region consisting of a minimum of five nucleotides helps pre-miRNAs to acquire a conformation ideal for binding to LIN28, but pre-let-7c-2 prefers a conformation with a three-nucleotide loop. Thus, our simulations provide a theoretical rationale for the recent experimental observation of the escape of LIN28-mediated repression by pre-let-7c-2. The essential structural and sequence features highlighted in this study might aid in designing synthetic small molecule inhibitors for modulating LIN28-let-7 interaction in malignant cells. We have also identified crucial SDRs of the LIN28-preE-let-7 complex involving 13 residues of LIN28 and 10 residues of the pre-miRNA. On the basis of the conservation profile of these 13 SDRs, we have identified 10 novel proteins that are not annotated as LIN28 like but are similar in sequence, domain, or fold level to LIN28.

  16. Study on the Coordination Structure of Pt Sorbed on Bacterial Cells Using X-Ray Absorption Fine Structure Spectroscopy

    PubMed Central

    Tanaka, Kazuya; Watanabe, Naoko

    2015-01-01

    Biosorption has been intensively investigated as a promising technology for the recovery of precious metals from solution. However, the detailed mechanism responsible for the biosorption of Pt on a biomass is not fully understood because of a lack of spectroscopic studies. We applied X-ray absorption fine structure spectroscopy to elucidate the coordination structure of Pt sorbed on bacterial cells. We examined the sorption of Pt(II) and Pt(IV) species on bacterial cells of Bacillus subtilis and Shewanella putrefaciens in NaCl solutions. X-ray absorption near-edge structure and extended X-ray absorption fine structure (EXAFS) of Pt-sorbed bacteria suggested that Pt(IV) was reduced to Pt(II) on the cell’s surface, even in the absence of an organic material as an exogenous electron donor. EXAFS spectra demonstrated that Pt sorbed on bacterial cells has a fourfold coordination of chlorine ions, similar to PtCl42-, which indicated that sorption on the protonated amine groups of the bacterial cells. This work clearly demonstrated the coordination structure of Pt sorbed on bacterial cells. The findings of this study will contribute to the understanding of Pt biosorption on biomass, and facilitate the development of recovery methods for rare metals using biosorbent materials. PMID:25996945

  17. Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions.

    PubMed

    Baltar, Federico; Palovaara, Joakim; Unrein, Fernando; Catala, Philippe; Horňák, Karel; Šimek, Karel; Vaqué, Dolors; Massana, Ramon; Gasol, Josep M; Pinhassi, Jarone

    2016-03-01

    To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 10(6) cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 10(4) cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 10(6) bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.

  18. A longitudinal study of vaginal douching and bacterial vaginosis--a marginal structural modeling analysis.

    PubMed

    Brotman, Rebecca M; Klebanoff, Mark A; Nansel, Tonja R; Andrews, William W; Schwebke, Jane R; Zhang, Jun; Yu, Kai F; Zenilman, Jonathan M; Scharfstein, Daniel O

    2008-07-15

    The etiology of bacterial vaginosis is unknown, and there are no long-term therapies for preventing this frequently recurring condition. Vaginal douching has been reported to be associated with bacterial vaginosis in observational studies. However, this association may be due to confounding by indication--that is, confounding by women douching in response to vaginal symptoms associated with bacterial vaginosis. The authors used marginal structural modeling to estimate the causal effect of douching on bacterial vaginosis risk while controlling for this confounding effect. In 1999-2002, nonpregnant women (n = 3,620) were recruited into a prospective study when they visited one of 12 public health clinics in Birmingham, Alabama, for routine care. Participants were assessed quarterly for 1 year. Bacterial vaginosis was based on a Nugent's Gram stain score of 7 or higher. Thirty-two percent of participants douched in every study interval, and 43.0% never douched. Of the 12,349 study visits, 40.2% were classified as involving bacterial vaginosis. The relative risk for regular douching as compared with no douching was 1.21 (95% confidence interval: 1.08, 1.38). These findings indicate that douching confers increased risk of disruption of vaginal flora. In the absence of a large randomized trial, these findings provide the best evidence to date for a risk of bacterial vaginosis associated with douching.

  19. Shift of bacterial community structure along different coastal reclamation histories in Jiangsu, Eastern China.

    PubMed

    Hua, Jianfeng; Feng, Youzhi; Jiang, Qian; Bao, Xuewen; Yin, Yunlong

    2017-08-30

    Tideland reclamation has drastic effects on coastal ecosystem involved in soil microorganisms. However, the knowledge regarding temporal variations of microbial community along reclamation chronosequence and their environmental variable predictor is still poorly known. Using Illumina sequencing, we qualified bacterial community composition in soils collected from one tideland and four reclamation stages, i.e. 2-year, 7-year, 19-year and 39-year in Jiangsu, Eastern China. Across all samples, the dominant groups were Proteobacteria, Bacteroidete, Acidobacteria, Planctomycetes and Chloroflexi. Reclamation activity and its histories greatly altered bacterial community structure, and only 0.28% of phylotypes were shared by five soils. Specially, some typical marine bacteria (Gaetulibacter, Alcanivorax …) disappeared in reclamation soils, while other groups (Niabella, Flavisolibacter…) were gradually eminent. Generally, bacterial diversity and richness increased with reclamation histories. Bacterial community was correlated with most of soil physico-chemical properties. Amongst, mean weight diameter of soil aggregates (MWD) was detected as a primary factor predicting bacterial community composition. Together, our results indicated that effects of reclamation on bacterial community varied with diked histories, and MWD was a major factor predicting bacterial community during progressive reclamation. These findings offer predicting case study for understanding the impact of reclamation and its histories on microbial community in a coastal ecosystem.

  20. Bacterial community structure and function shift across a northern boreal forest fire chronosequence

    PubMed Central

    Sun, Hui; Santalahti, Minna; Pumpanen, Jukka; Köster, Kajar; Berninger, Frank; Raffaello, Tommaso; Asiegbu, Fred O.; Heinonsalo, Jussi

    2016-01-01

    Soil microbial responses to fire are likely to change over the course of forest recovery. Investigations on long-term changes in bacterial dynamics following fire are rare. We characterized the soil bacterial communities across three different times post fire in a 2 to 152-year fire chronosequence by Illumina MiSeq sequencing, coupled with a functional gene array (GeoChip). The results showed that the bacterial diversity did not differ between the recently and older burned areas, suggesting a concomitant recovery in the bacterial diversity after fire. The differences in bacterial communities over time were mainly driven by the rare operational taxonomic units (OTUs < 0.1%). Proteobacteria (39%), Acidobacteria (34%) and Actinobacteria (17%) were the most abundant phyla across all sites. Genes involved in C and N cycling pathways were present in all sites showing high redundancy in the gene profiles. However, hierarchical cluster analysis using gene signal intensity revealed that the sites with different fire histories formed separate clusters, suggesting potential differences in maintaining essential biogeochemical soil processes. Soil temperature, pH and water contents were the most important factors in shaping the bacterial community structures and function. This study provides functional insight on the impact of fire disturbance on soil bacterial community. PMID:27573440

  1. A Longitudinal Study of Vaginal Douching and Bacterial Vaginosis—A Marginal Structural Modeling Analysis

    PubMed Central

    Klebanoff, Mark A.; Nansel, Tonja R.; Andrews, William W.; Schwebke, Jane R.; Zhang, Jun; Yu, Kai F.; Zenilman, Jonathan M.; Scharfstein, Daniel O.

    2008-01-01

    The etiology of bacterial vaginosis is unknown, and there are no long-term therapies for preventing this frequently recurring condition. Vaginal douching has been reported to be associated with bacterial vaginosis in observational studies. However, this association may be due to confounding by indication—that is, confounding by women douching in response to vaginal symptoms associated with bacterial vaginosis. The authors used marginal structural modeling to estimate the causal effect of douching on bacterial vaginosis risk while controlling for this confounding effect. In 1999–2002, nonpregnant women (n = 3,620) were recruited into a prospective study when they visited one of 12 public health clinics in Birmingham, Alabama, for routine care. Participants were assessed quarterly for 1 year. Bacterial vaginosis was based on a Nugent's Gram stain score of 7 or higher. Thirty-two percent of participants douched in every study interval, and 43.0% never douched. Of the 12,349 study visits, 40.2% were classified as involving bacterial vaginosis. The relative risk for regular douching as compared with no douching was 1.21 (95% confidence interval: 1.08, 1.38). These findings indicate that douching confers increased risk of disruption of vaginal flora. In the absence of a large randomized trial, these findings provide the best evidence to date for a risk of bacterial vaginosis associated with douching. PMID:18503038

  2. Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions

    PubMed Central

    Baltar, Federico; Palovaara, Joakim; Unrein, Fernando; Catala, Philippe; Horňák, Karel; Šimek, Karel; Vaqué, Dolors; Massana, Ramon; Gasol, Josep M; Pinhassi, Jarone

    2016-01-01

    To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 106 cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 104 cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 106 bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities. PMID:26262814

  3. Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes

    PubMed Central

    Zhang, Xing; Lai, Mason; Chang, Winston; Yu, Iris; Ding, Ke; Mrazek, Jan; Ng, Hwee L.; Yang, Otto O.; Maslov, Dmitri A.; Zhou, Z. Hong

    2016-01-01

    The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome. PMID:27752045

  4. Intrinsic factors of Peltigera lichens influence the structure of the associated soil bacterial microbiota.

    PubMed

    Leiva, Diego; Clavero-León, Claudia; Carú, Margarita; Orlando, Julieta

    2016-11-01

    Definition of lichens has evolved from bi(tri)partite associations to multi-species symbioses, where bacteria would play essential roles. Besides, although soil bacterial communities are known to be affected by edaphic factors, when lichens grow upon them these could become less preponderant. We hypothesized that the structure of both the lichen microbiota and the microbiota in the soil underneath lichens is shaped by lichen intrinsic and extrinsic factors. In this work, intrinsic factors corresponded to mycobiont and cyanobiont identities of Peltigera lichens, metabolite diversity and phenoloxidase activity and extrinsic factors involved the site of the forest where lichens grow. Likewise, the genetic and metabolic structure of the lichen and soil bacterial communities were analyzed by fingerprinting. Among the results, metabolite diversity was inversely related to the genetic structure of bacterial communities of lichens and soils, highlighting the far-reaching effect of these substances; while phenoloxidase activity was inversely related to the metabolic structure only of the lichen bacterial microbiota, presuming a more limited effect of the products of these enzymes. Soil bacterial microbiota was different depending on the site and, strikingly, according to the cyanobiont present in the lichen over them, which could indicate an influence of the photobiont metabolism on the availability of soil nutrients. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Surface-structured bacterial cellulose with guided assembly-based biolithography (GAB).

    PubMed

    Bottan, Simone; Robotti, Francesco; Jayathissa, Prageeth; Hegglin, Alicia; Bahamonde, Nicolas; Heredia-Guerrero, José A; Bayer, Ilker S; Scarpellini, Alice; Merker, Hannes; Lindenblatt, Nicole; Poulikakos, Dimos; Ferrari, Aldo

    2015-01-27

    A powerful replica molding methodology to transfer on-demand functional topographies to the surface of bacterial cellulose nanofiber textures is presented. With this method, termed guided assembly-based biolithography (GAB), a surface-structured polydimethylsiloxane (PDMS) mold is introduced at the gas-liquid interface of an Acetobacter xylinum culture. Upon bacterial fermentation, the generated bacterial cellulose nanofibers are assembled in a three-dimensional network reproducing the geometric shape imposed by the mold. Additionally, GAB yields directional alignment of individual nanofibers and memory of the transferred geometrical features upon dehydration and rehydration of the substrates. Scanning electron and atomic force microscopy are used to establish the good fidelity of this facile and affordable method. Interaction of surface-structured bacterial cellulose substrates with human fibroblasts and keratinocytes illustrates the efficient control of cellular activities which are fundamental in skin wound healing and tissue regeneration. The deployment of surface-structured bacterial cellulose substrates in model animals as skin wound dressing or body implant further proves the high durability and low inflammatory response to the material over a period of 21 days, demonstrating beneficial effects of surface structure on skin regeneration.

  6. Mineral composition and charcoal determine the bacterial community structure in artificial soils.

    PubMed

    Ding, Guo-Chun; Pronk, Geertje Johanna; Babin, Doreen; Heuer, Holger; Heister, Katja; Kögel-Knabner, Ingrid; Smalla, Kornelia

    2013-10-01

    To study the influence of the clay minerals montmorillonite (M) and illite (I), the metal oxides ferrihydrite (F) and aluminum hydroxide (A), and charcoal (C) on soil bacterial communities, seven artificial soils with identical texture provided by quartz (Q) were mixed with sterilized manure as organic carbon source before adding a microbial inoculant derived from a Cambisol. Bacterial communities established in artificial soils after 90 days of incubation were compared by DGGE analysis of bacterial and taxon-specific 16S rRNA gene amplicons. The bacterial community structure of charcoal-containing soils highly differed from the other soils at all taxonomic levels studied. Effects of montmorillonite and illite were observed for Bacteria and Betaproteobacteria, but not for Actinobacteria or Alphaproteobacteria. A weak influence of metal oxides on Betaproteobacteria was found. Barcoded pyrosequencing of 16S rRNA gene amplicons done for QM, QI, QIF, and QMC revealed a high bacterial diversity in the artificial soils. The composition of the artificial soils was different from the inoculant, and the structure of the bacterial communities established in QMC soil was most different from the other soils, suggesting that charcoal provided distinct microenvironments and biogeochemical interfaces formed. Several populations with discriminative relative abundance between artificial soils were identified.

  7. Comparison of the structural basis for thermal stability between archaeal and bacterial proteins.

    PubMed

    Ding, Yanrui; Cai, Yujie; Han, Yonggang; Zhao, Bingqiang

    2012-01-01

    In this study, the structural basis for thermal stability in archaeal and bacterial proteins was investigated. There were many common factors that confer resistance to high temperature in both archaeal and bacterial proteins. These factors include increases in the Lys content, the bends and blanks of secondary structure, the Glu content of salt bridge; decreases in the number of main-side chain hydrogen bond and exposed surface area, and changes in the bends and blanks of amino acids. Certainly, the utilization of charged amino acids to form salt bridges is a primary factor. In both heat-resistant archaeal and bacterial proteins, most Glu and Asp participate in the formation of salt bridges. Other factors may influence either archaeal or bacterial protein thermostability, which includes the more frequent occurrence of shorter 3(10)-helices and increased hydrophobicity in heat-resistant archaeal proteins. However, there were increases in average helix length, the Glu content in salt bridges, temperature factors and decreases in the number of main-side chain hydrogen bonds, uncharged-uncharged hydrogen bonds, hydrophobicity, and buried and exposed polar surface area in heat-resistant bacterial proteins. Evidently, there are few similarities and many disparities between the heat-resistant mechanisms of archaeal and bacterial proteins.

  8. Assessment of bacterial community structure in nitrifying biofilm under inorganic carbon-sufficient and -limited conditions.

    PubMed

    Bae, Hyokwan; Chung, Yun-Chul; Yang, Heejeong; Lee, Changsoo; Aryapratama, Rio; Yoo, Young J; Lee, Seockheon

    2015-01-01

    In this work, nitrification and changes in the composition of the total bacterial community under inorganic carbon (IC)-limited conditions, in a nitrifying moving bed biofilm reactor, was investigated. A culture-independent analysis of cloning and sequencing based on the 16S rRNA gene was applied to quantify the bacterial diversity and to determine bacterial taxonomic assignment. IC concentrations had significant effects on the stability of ammonia-oxidation as indicated by the reduction of the nitrogen conversion rate with high NH4(+)-N loadings. The predominance of Nitrosomonas europaea was maintained in spite of changes in the IC concentration. In contrast, heterotrophic bacterial species contributed to a high bacterial diversity, and to a dynamic shift in the bacterial community structure, under IC-limited conditions. In this study, individual functions of heterotrophic bacteria were estimated based on taxonomic information. Possible key roles of coexisting heterotrophic bacteria are the assimilation of organic compounds of extracellular polymeric substances produced by nitrifiers, and biofilm formation by providing a filamentous structure and aggregation properties.

  9. Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment.

    PubMed

    Paissé, Sandrine; Coulon, Frédéric; Goñi-Urriza, Marisol; Peperzak, Louis; McGenity, Terry J; Duran, Robert

    2008-11-01

    The bacterial diversity of a chronically oil-polluted retention basin sediment located in the Berre lagoon (Etang-de-Berre, France) was investigated. This study combines chemical and molecular approaches in order to define how the in situ petroleum hydrocarbon contamination level affects the bacterial community structure of a subsurface sediment. Hydrocarbon content analysis clearly revealed a gradient of hydrocarbon contamination in both the water and the sediment following the basin periphery from the pollution input to the lagoon water. The nC17 and pristane concentrations suggested alkane biodegradation in the sediments. These results, combined with those of terminal-restriction fragment length polymorphism analysis of the 16S rRNA genes, indicated that bacterial community structure was obviously associated with the gradient of oil contamination. The analysis of bacterial community composition revealed dominance of bacteria related to the Proteobacteria phylum (Gamma-, Delta-, Alpha-, Epsilon- and Betaproteobacteria), Bacteroidetes and Verrucomicrobium groups and Spirochaetes, Actinobacteria and Cyanobacteria phyla. The adaptation of the bacterial community to oil contamination was not characterized by dominance of known oil-degrading bacteria, because a predominance of populations associated to the sulphur cycle was observed. The input station presented particular bacterial community composition associated with a low oil concentration in the sediment, indicating the adaptation of this community to the oil contamination.

  10. An experimental model for the spatial structuring and selection of bacterial communities.

    PubMed

    Thomas, Torsten; Kindinger, Ilona; Yu, Dan; Esvaran, Meera; Blackall, Linda; Forehead, Hugh; Johnson, Craig R; Manefield, Mike

    2011-11-01

    Community-level selection is an important concept in evolutionary biology and has been predicted to arise in systems that are spatially structured. Here we develop an experimental model for spatially-structured bacterial communities based on coaggregating strains and test their relative fitness under a defined selection pressure. As selection we apply protozoan grazing in a defined, continuous culturing system. We demonstrate that a slow-growing bacterial strain Blastomonas natatoria 2.1, which forms coaggregates with Micrococcus luteus, can outcompete a fast-growing, closely related strain Blastomonas natatoria 2.8 under conditions of protozoan grazing. The competitive benefit provided by spatial structuring has implications for the evolution of natural bacterial communities in the environment.

  11. Population pharmacokinetics of ceftaroline in patients with acute bacterial skin and skin structure infections or community-acquired bacterial pneumonia.

    PubMed

    Van Wart, Scott A; Forrest, Alan; Khariton, Tatiana; Rubino, Christopher M; Bhavnani, Sujata M; Reynolds, Daniel K; Riccobene, Todd; Ambrose, Paul G

    2013-11-01

    Ceftaroline, the active form of ceftaroline fosamil, is a broad-spectrum cephalosporin antibiotic. A population pharmacokinetic (PPK) model for ceftaroline was developed in NONMEM® using data from 185 healthy subjects and 92 patients with acute bacterial skin and skin structure infection (ABSSSI). Data from 128 patients with community-acquired bacterial pneumonia (CABP) were used for external model validation. Healthy subjects received 50-2,000 mg ceftaroline fosamil via intravenous (IV) infusion over 1 hour or intramuscular (IM) injection q12h or q24h. ABSSSI and CABP patients received 600 mg of ceftaroline fosamil IV over 1 hour q12h. A three-compartment model with zero-order IV or parallel first-order IM input and first-order elimination described ceftaroline fosamil PK. A two-compartment model with first-order conversion of prodrug to ceftaroline and parallel linear and saturable elimination described ceftaroline PK. Creatinine clearance was the primary determinant of ceftaroline exposure. Good agreement between the observed data and both population (r(2)  = 0.93) and individual post-hoc (r(2)  = 0.98) predictions suggests the PPK model can adequately approximate ceftaroline PK using covariate information. Such a PPK model can evaluate dose adjustments for patients with renal impairment and generate ceftaroline exposures for use in pharmacokinetic-pharmacodynamic assessments of efficacy in patients with ABSSSI or CABP.

  12. Multiple oligomeric structures of a bacterial small heat shock protein

    PubMed Central

    Mani, Nandini; Bhandari, Spraha; Moreno, Rodolfo; Hu, Liya; Prasad, B. V. Venkataram; Suguna, Kaza

    2016-01-01

    Small heat shock proteins are ubiquitous molecular chaperones that form the first line of defence against the detrimental effects of cellular stress. Under conditions of stress they undergo drastic conformational rearrangements in order to bind to misfolded substrate proteins and prevent cellular protein aggregation. Owing to the dynamic nature of small heat shock protein oligomers, elucidating the structural basis of chaperone action and oligomerization still remains a challenge. In order to understand the organization of sHSP oligomers, we have determined crystal structures of a small heat shock protein from Salmonella typhimurium in a dimeric form and two higher oligomeric forms: an 18-mer and a 24-mer. Though the core dimer structure is conserved in all the forms, structural heterogeneity arises due to variation in the terminal regions. PMID:27053150

  13. Structural basis for bacterial transcription-coupled DNA repair.

    PubMed

    Deaconescu, Alexandra M; Chambers, Anna L; Smith, Abigail J; Nickels, Bryce E; Hochschild, Ann; Savery, Nigel J; Darst, Seth A

    2006-02-10

    Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

  14. eIF4AIII enhances translation of nuclear cap-binding complex-bound mRNAs by promoting disruption of secondary structures in 5'UTR.

    PubMed

    Choe, Junho; Ryu, Incheol; Park, Ok Hyun; Park, Joori; Cho, Hana; Yoo, Jin Seon; Chi, Sung Wook; Kim, Min Kyung; Song, Hyun Kyu; Kim, Yoon Ki

    2014-10-28

    It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5'-end of mRNAs bound by the CBC by direct interaction with the CBC-dependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5'UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.

  15. The Bacterial Helicase-Primase Interaction: A Common Structural/Functional Module

    PubMed Central

    Soultanas, Panos

    2011-01-01

    The lack of a high-resolution structure for the bacterial helicase-primase complex and the fragmented structural information for the individual proteins have been hindering our detailed understanding of this crucial binary protein interaction. Two new structures for the helicase-interacting domain of the bacterial primases from Escherichia coli and Bacillus stearothermophilus have recently been solved and both revealed a unique and surprising structural similarity to the amino-terminal domain of the helicase itself. In this minireview, the current data are discussed and important new structural and functional aspects of the helicase-primase interaction are highlighted. An attractive structural model with direct biological significance for the function of this complex and also for the development of new antibacterial compounds is examined. PMID:15939015

  16. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGES

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  17. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  18. Exploring bacterial community structure and function associated with atrazine biodegradation in repeatedly treated soils.

    PubMed

    Fang, Hua; Lian, Jianjun; Wang, Huifang; Cai, Lin; Yu, Yunlong

    2015-04-09

    Substantial application of the herbicide atrazine in agriculture leads to persistent contamination, which may damage the succeeding crops and pose potential threats to soil ecology and environmental health. Here, the degradation characteristics of atrazine and dynamic change of soil bacterial community structure and function as well as their relations were studied during three repeated treatments at the recommended, double, and five-fold doses. The results showed that the degradation half-life of atrazine obviously decreased with increased treatment frequency. Soil microbial functional diversity displayed a variation trend of suppression-recovery-stimulation, which was associated with increased degradation rate of atrazine. 16S amplicon sequencing was conducted to explore bacterial community structure and correlate the genus to potential atrazine degradation. A total of seven potentially atrazine-degrading bacterial genera were found including Nocardioides, Arthrobacter, Bradyrhizobium, Burkholderia, Methylobacterium, Mycobacterium, and Clostridium. These bacterial genera showed almost complete atrazine degradation pathways including dechlorination, dealkylation, hydroxylation, and ring cleavage. Furthermore, the relative abundance of four of them (i.e., Nocardioides, Arthrobacter, Methylobacterium, and Bradyrhizobium) increased with treatment frequency and atrazine concentration, suggesting that they may participate in atrazine degradation during repeated treatments. Our findings reveal the potential relationship between atrazine degradation and soil bacterial community structure in repeatedly treated soils. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Spatial and Vertical Variability in Bacterial Community Structure in the Sediment of the South China Sea

    NASA Astrophysics Data System (ADS)

    Wang, P.; Xie, W.; Chen, S.; Zhang, C. L.

    2014-12-01

    The ocean subsurface contains one of the largest pools of reactive carbon and nitrogen on earth, and thus serves as the largest realm for microbial life. However, the microbial communities that drive deep-subsurface geochemical processes are vastly unexplored. In this study, the bacterial community structure in the subsurface of the South China Sea were examined using sediment cores collected from shelf (water depth 667 m) to slope (water depth 3840 m). High-throughput sequencing of the bacterial 16S rRNA genes from the sediment samples resulted in a total of 270,000 sequences with each sample averaging about 10,000 sequences. In all sediment cores, the 16S rRNA gene copies of bacteria were highest in the surface sediment and decreased with the core depth. The bacterial community was dominated by Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. In most of the sediment cores, Proteobacteria dominated surface sediment samples and decreased with depth. The community structure showed no significant difference among the stations at different water depths, which indicates that bacterial distribution in the sediment is not influenced by the water column above. However, stations along the transect from Pearl River canyon to the deep basin were grouped together by cluster analysis, which indicates that bacterial community structure at these stations may bear the same consequence of sedimentary processes of the deep South China Sea.

  20. Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

    SciTech Connect

    Jansson, Janet; Edlund, Anna; Hardeman, Fredrik; Jansson, Janet K.; Sjoling, Sara

    2008-05-15

    Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179 mV, -64 mV and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labeled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labeled DNA and DNA from reverse transcription PCR (rt-PCR) showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

  1. Structural diversity of bacterial communities in a heavy metal mineralized granite outcrop.

    PubMed

    Gleeson, Deirdre; McDermott, Frank; Clipson, Nicholas

    2006-03-01

    This laboratory study of a variably mineralized and hydrothermally altered granite outcrop investigated the influences of rock-surface chemistry and heavy metal content on resident bacterial populations. Results indicated that elevated heavy metal concentrations had a profound impact on bacterial community structure, with strong relationships found between certain ribotypes and particular chemical/heavy metal elements. Automated ribosomal intergenic sequence analysis (ARISA) was used to assess the nature and extent of bacterial diversity, and this was combined with chemical analysis and multivariate statistics to identify the main geochemical factors influencing bacterial community structure. A randomization test revealed significant changes in bacterial structure between samples, while canonical correspondence analysis (CCA) related each individual ARISA profile to linear combinations of the chemical variables (mineralogy, major element and heavy metal concentrations) revealing the geochemical factors that correlated with changes in the ARISA data. anova was performed to further explore interactions between individual ribotypes and chemical/heavy metal composition, and revealed that a high proportion of ribotypes correlated significantly with heavy metals.

  2. The impact of natural transformation on adaptation in spatially structured bacterial populations.

    PubMed

    Moradigaravand, Danesh; Engelstädter, Jan

    2014-06-20

    Recent studies have demonstrated that natural transformation and the formation of highly structured populations in bacteria are interconnected. In spite of growing evidence about this connection, little is known about the dynamics of natural transformation in spatially structured bacterial populations. In this work, we model the interdependency between the dynamics of the bacterial gene pool and those of environmental DNA in space to dissect the effect of transformation on adaptation. Our model reveals that even with only a single locus under consideration, transformation with a free DNA fragment pool results in complex adaptation dynamics that do not emerge in previous models focusing only on the gene shuffling effect of transformation at multiple loci. We demonstrate how spatial restriction on population growth and DNA diffusion in the environment affect the impact of transformation on adaptation. We found that in structured bacterial populations intermediate DNA diffusion rates predominantly cause transformation to impede adaptation by spreading deleterious alleles in the population. Overall, our model highlights distinctive evolutionary consequences of bacterial transformation in spatially restricted compared to planktonic bacterial populations.

  3. Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils

    PubMed Central

    Delgado-Balbuena, Laura; Bello-López, Juan M.; Navarro-Noya, Yendi E.; Rodríguez-Valentín, Analine; Luna-Guido, Marco L.; Dendooven, Luc

    2016-01-01

    Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the

  4. Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils.

    PubMed

    Delgado-Balbuena, Laura; Bello-López, Juan M; Navarro-Noya, Yendi E; Rodríguez-Valentín, Analine; Luna-Guido, Marco L; Dendooven, Luc

    2016-01-01

    Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the

  5. Bacterial structures and ecosystem functions in glaciated floodplains: contemporary states and potential future shifts

    PubMed Central

    Freimann, Remo; Bürgmann, Helmut; Findlay, Stuart EG; Robinson, Christopher T

    2013-01-01

    Glaciated alpine floodplains are responding quickly to climate change through shrinking ice masses. Given the expected future changes in their physicochemical environment, we anticipated variable shifts in structure and ecosystem functioning of hyporheic microbial communities in proglacial alpine streams, depending on present community characteristics and landscape structures. We examined microbial structure and functioning during different hydrologic periods in glacial (kryal) streams and, as contrasting systems, groundwater-fed (krenal) streams. Three catchments were chosen to cover an array of landscape features, including interconnected lakes, differences in local geology and degree of deglaciation. Community structure was assessed by automated ribosomal intergenic spacer analysis and microbial function by potential enzyme activities. We found each catchment to contain a distinct bacterial community structure and different degrees of separation in structure and functioning that were linked to the physicochemical properties of the waters within each catchment. Bacterial communities showed high functional plasticity, although achieved by different strategies in each system. Typical kryal communities showed a strong linkage of structure and function that indicated a major prevalence of specialists, whereas krenal sediments were dominated by generalists. With the rapid retreat of glaciers and therefore altered ecohydrological characteristics, lotic microbial structure and functioning are likely to change substantially in proglacial floodplains in the future. The trajectory of these changes will vary depending on contemporary bacterial community characteristics and landscape structures that ultimately determine the sustainability of ecosystem functioning. PMID:23842653

  6. Bacterial structures and ecosystem functions in glaciated floodplains: contemporary states and potential future shifts.

    PubMed

    Freimann, Remo; Bürgmann, Helmut; Findlay, Stuart E G; Robinson, Christopher T

    2013-12-01

    Glaciated alpine floodplains are responding quickly to climate change through shrinking ice masses. Given the expected future changes in their physicochemical environment, we anticipated variable shifts in structure and ecosystem functioning of hyporheic microbial communities in proglacial alpine streams, depending on present community characteristics and landscape structures. We examined microbial structure and functioning during different hydrologic periods in glacial (kryal) streams and, as contrasting systems, groundwater-fed (krenal) streams. Three catchments were chosen to cover an array of landscape features, including interconnected lakes, differences in local geology and degree of deglaciation. Community structure was assessed by automated ribosomal intergenic spacer analysis and microbial function by potential enzyme activities. We found each catchment to contain a distinct bacterial community structure and different degrees of separation in structure and functioning that were linked to the physicochemical properties of the waters within each catchment. Bacterial communities showed high functional plasticity, although achieved by different strategies in each system. Typical kryal communities showed a strong linkage of structure and function that indicated a major prevalence of specialists, whereas krenal sediments were dominated by generalists. With the rapid retreat of glaciers and therefore altered ecohydrological characteristics, lotic microbial structure and functioning are likely to change substantially in proglacial floodplains in the future. The trajectory of these changes will vary depending on contemporary bacterial community characteristics and landscape structures that ultimately determine the sustainability of ecosystem functioning.

  7. DASHR: database of small human noncoding RNAs

    PubMed Central

    Leung, Yuk Yee; Kuksa, Pavel P.; Amlie-Wolf, Alexandre; Valladares, Otto; Ungar, Lyle H.; Kannan, Sampath; Gregory, Brian D.; Wang, Li-San

    2016-01-01

    Small non-coding RNAs (sncRNAs) are highly abundant RNAs, typically <100 nucleotides long, that act as key regulators of diverse cellular processes. Although thousands of sncRNA genes are known to exist in the human genome, no single database provides searchable, unified annotation, and expression information for full sncRNA transcripts and mature RNA products derived from these larger RNAs. Here, we present the Database of small human noncoding RNAs (DASHR). DASHR contains the most comprehensive information to date on human sncRNA genes and mature sncRNA products. DASHR provides a simple user interface for researchers to view sequence and secondary structure, compare expression levels, and evidence of specific processing across all sncRNA genes and mature sncRNA products in various human tissues. DASHR annotation and expression data covers all major classes of sncRNAs including microRNAs (miRNAs), Piwi-interacting (piRNAs), small nuclear, nucleolar, cytoplasmic (sn-, sno-, scRNAs, respectively), transfer (tRNAs), and ribosomal RNAs (rRNAs). Currently, DASHR (v1.0) integrates 187 smRNA high-throughput sequencing (smRNA-seq) datasets with over 2.5 billion reads and annotation data from multiple public sources. DASHR contains annotations for ∼48 000 human sncRNA genes and mature sncRNA products, 82% of which are expressed in one or more of the curated tissues. DASHR is available at http://lisanwanglab.org/DASHR. PMID:26553799

  8. Structure of the basal components of a bacterial transporter

    SciTech Connect

    Meisner, Jeffrey; Maehigashi, Tatsuya; André, Ingemar; Dunham, Christine M.; Moran, Jr., Charles P.

    2012-12-10

    Proteins SpoIIQ and SpoIIIAH interact through two membranes to connect the forespore and the mother cell during endospore development in the bacterium Bacillus subtilis. SpoIIIAH consists of a transmembrane segment and an extracellular domain with similarity to YscJ proteins. YscJ proteins form large multimeric rings that are the structural scaffolds for the assembly of type III secretion systems in Gram-negative bacteria. The predicted ring-forming motif of SpoIIIAH and other evidence led to the model that SpoIIQ and SpoIIIAH form the core components of a channel or transporter through which the mother cell nurtures forespore development. Therefore, to understand the roles of SpoIIIAH and SpoIIQ in channel formation, it is critical to determine whether SpoIIIAH adopts a ring-forming structural motif, and whether interaction of SpoIIIAH with SpoIIQ would preclude ring formation. We report a 2.8-{angstrom} resolution structure of a complex of SpoIIQ and SpoIIIAH. SpoIIIAH folds into the ring-building structural motif, and modeling shows that the structure of the SpoIIQ-SpoIIIAH complex is compatible with forming a symmetrical oligomer that is similar to those in type III systems. The inner diameters of the two most likely ring models are large enough to accommodate several copies of other integral membrane proteins. SpoIIQ contains a LytM domain, which is found in metalloendopeptidases, but lacks residues important for metalloprotease activity. Other LytM domains appear to be involved in protein-protein interactions. We found that the LytM domain of SpoIIQ contains an accessory region that interacts with SpoIIIAH.

  9. Microbiological functioning, diversity, and structure of bacterial communities in ultramafic soils from a tropical savanna.

    PubMed

    Pessoa-Filho, Marco; Barreto, Cristine Chaves; dos Reis Junior, Fábio Bueno; Fragoso, Rodrigo Rocha; Costa, Flávio Silva; de Carvalho Mendes, Ieda; de Andrade, Leide Rovênia Miranda

    2015-04-01

    Ultramafic soils are characterized by high levels of metals, and have been studied because of their geochemistry and its relation to their biological component. This study evaluated soil microbiological functioning (SMF), richness, diversity, and structure of bacterial communities from two ultramafic soils and from a non-ultramafic soil in the Brazilian Cerrado, a tropical savanna. SMF was represented according to simultaneous analysis of microbial biomass C (MBC) and activities of the enzymes β-glucosidase, acid phosphomonoesterase and arylsulfatase, linked to the C, P and S cycles. Bacterial community diversity and structure were studied by sequencing of 16S rRNA gene clone libraries. MBC and enzyme activities were not affected by high Ni contents. Changes in SMF were more related to the organic matter content of soils (SOM) than to their available Ni. Phylogeny-based methods detected qualitative and quantitative differences in pairwise comparisons of bacterial community structures of the three sites. However, no correlations between community structure differences and SOM or SMF were detected. We believe this work presents benchmark information on SMF, diversity, and structure of bacterial communities for a unique type of environment within the Cerrado biome.

  10. Characterisation of the bacterial community structures in the intestine of Lampetra morii.

    PubMed

    Li, Yingying; Xie, Wenfang; Li, Qingwei

    2016-07-01

    The metagenomic analysis and 16S rDNA sequencing method were used to investigate the bacterial community in the intestines of Lampetra morii. The bacterial community structure in L. morii intestine was relatively simple. Eight different operational taxonomic units were observed. Chitinophagaceae_unclassified (26.5 %) and Aeromonas spp. (69.6 %) were detected as dominant members at the genus level. The non-dominant genera were as follows: Acinetobacter spp. (1.4 %), Candidatus Bacilloplasma (2.5 %), Enterobacteria spp. (1.5 %), Shewanella spp. (0.04 %), Vibrio spp. (0.09 %), and Yersinia spp. (1.8 %). The Shannon-Wiener (H) and Simpson (1-D) indexes were 0.782339 and 0.5546, respectively. The rarefaction curve representing the bacterial community richness and Shannon-Wiener curve representing the bacterial community diversity reached asymptote, which indicated that the sequence depth were sufficient to represent the majority of species richness and bacterial community diversity. The number of Aeromonas in lamprey intestine was two times higher after stimulation by lipopolysaccharide than PBS. This study provides data for understanding the bacterial community harboured in lamprey intestines and exploring potential key intestinal symbiotic bacteria essential for the L. morii immune response.

  11. Soil bacterial community structure responses to precipitation reduction and forest management in forest ecosystems across Germany.

    PubMed

    Felsmann, Katja; Baudis, Mathias; Gimbel, Katharina; Kayler, Zachary E; Ellerbrock, Ruth; Bruelheide, Helge; Bruehlheide, Helge; Bruckhoff, Johannes; Welk, Erik; Puhlmann, Heike; Weiler, Markus; Gessler, Arthur; Ulrich, Andreas

    2015-01-01

    Soil microbial communities play an important role in forest ecosystem functioning, but how climate change will affect the community composition and consequently bacterial functions is poorly understood. We assessed the effects of reduced precipitation with the aim of simulating realistic future drought conditions for one growing season on the bacterial community and its relation to soil properties and forest management. We manipulated precipitation in beech and conifer forest plots managed at different levels of intensity in three different regions across Germany. The precipitation reduction decreased soil water content across the growing season by between 2 to 8% depending on plot and region. T-RFLP analysis and pyrosequencing of the 16S rRNA gene were used to study the total soil bacterial community and its active members after six months of precipitation reduction. The effect of reduced precipitation on the total bacterial community structure was negligible while significant effects could be observed for the active bacteria. However, the effect was secondary to the stronger influence of specific soil characteristics across the three regions and management selection of overstorey tree species and their respective understorey vegetation. The impact of reduced precipitation differed between the studied plots; however, we could not determine the particular parameters being able to modify the response of the active bacterial community among plots. We conclude that the moderate drought induced by the precipitation manipulation treatment started to affect the active but not the total bacterial community, which points to an adequate resistance of the soil microbial system over one growing season.

  12. Relationships between soil organic matter, nutrients, bacterial community structure, and the performance of microbial fuel cells.

    PubMed

    Dunaj, Sara J; Vallino, Joseph J; Hines, Mark E; Gay, Marcus; Kobyljanec, Christine; Rooney-Varga, Juliette N

    2012-02-07

    Microbial fuel cells (MFCs) offer the potential for generating electricity, mitigating greenhouse gas emissions, and bioremediating pollutants through utilization of a plentiful renewable resource: soil organic carbon. We analyzed bacterial community structure, MFC performance, and soil characteristics in different microhabitats within MFCs constructed from agricultural or forest soils in order to determine how soil type and bacterial dynamics influence MFC performance. Our results indicated that MFCs constructed from agricultural soil had power output about 17 times that of forest soil-based MFCs and respiration rates about 10 times higher than forest soil MFCs. Agricultural soil MFCs had lower C:N ratios, polyphenol content, and acetate concentrations than forest soil MFCs. Bacterial community profile data indicate that the bacterial communities at the anode of the high power MFCs were less diverse than in low power MFCs and were dominated by Deltaproteobacteria, Geobacter, and to a lesser extent, Clostridia, while low-power MFC anode communities were dominated by Clostridia. These results suggest that the presence of organic carbon substrate (acetate) was not the major limiting factor in selecting for highly electrogenic bacterial communities, while the quality of available organic matter may have played a significant role in supporting high performing bacterial communities.

  13. A Dissolved Oxygen Threshold for Shifts in Bacterial Community Structure in a Seasonally Hypoxic Estuary

    PubMed Central

    Spietz, Rachel L.; Williams, Cheryl M.; Rocap, Gabrielle; Horner-Devine, M. Claire

    2015-01-01

    Pelagic ecosystems can become depleted of dissolved oxygen as a result of both natural processes and anthropogenic effects. As dissolved oxygen concentration decreases, energy shifts from macrofauna to microorganisms, which persist in these hypoxic zones. Oxygen-limited regions are rapidly expanding globally; however, patterns of microbial communities associated with dissolved oxygen gradients are not yet well understood. To assess the effects of decreasing dissolved oxygen on bacteria, we examined shifts in bacterial community structure over space and time in Hood Canal, Washington, USA−a glacial fjord-like water body that experiences seasonal low dissolved oxygen levels known to be detrimental to fish and other marine organisms. We found a strong negative association between bacterial richness and dissolved oxygen. Bacterial community composition across all samples was also strongly associated with the dissolved oxygen gradient, and significant changes in bacterial community composition occurred at a dissolved oxygen concentration between 5.18 and 7.12 mg O2 L-1. This threshold value of dissolved oxygen is higher than classic definitions of hypoxia (<2.0 mg O2 L-1), suggesting that changes in bacterial communities may precede the detrimental effects on ecologically and economically important macrofauna. Furthermore, bacterial taxa responsible for driving whole community changes across the oxygen gradient are commonly detected in other oxygen-stressed ecosystems, suggesting that the patterns we uncovered in Hood Canal may be relevant in other low oxygen ecosystems. PMID:26270047

  14. A Dissolved Oxygen Threshold for Shifts in Bacterial Community Structure in a Seasonally Hypoxic Estuary.

    PubMed

    Spietz, Rachel L; Williams, Cheryl M; Rocap, Gabrielle; Horner-Devine, M Claire

    2015-01-01

    Pelagic ecosystems can become depleted of dissolved oxygen as a result of both natural processes and anthropogenic effects. As dissolved oxygen concentration decreases, energy shifts from macrofauna to microorganisms, which persist in these hypoxic zones. Oxygen-limited regions are rapidly expanding globally; however, patterns of microbial communities associated with dissolved oxygen gradients are not yet well understood. To assess the effects of decreasing dissolved oxygen on bacteria, we examined shifts in bacterial community structure over space and time in Hood Canal, Washington, USA-a glacial fjord-like water body that experiences seasonal low dissolved oxygen levels known to be detrimental to fish and other marine organisms. We found a strong negative association between bacterial richness and dissolved oxygen. Bacterial community composition across all samples was also strongly associated with the dissolved oxygen gradient, and significant changes in bacterial community composition occurred at a dissolved oxygen concentration between 5.18 and 7.12 mg O2 L(-1). This threshold value of dissolved oxygen is higher than classic definitions of hypoxia (<2.0 mg O2 L(-1)), suggesting that changes in bacterial communities may precede the detrimental effects on ecologically and economically important macrofauna. Furthermore, bacterial taxa responsible for driving whole community changes across the oxygen gradient are commonly detected in other oxygen-stressed ecosystems, suggesting that the patterns we uncovered in Hood Canal may be relevant in other low oxygen ecosystems.

  15. [Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

    PubMed

    Minamino, Tohru

    2015-01-01

    The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

  16. Nematode sbRNAs: homologs of vertebrate Y RNAs.

    PubMed

    Boria, Ilenia; Gruber, Andreas R; Tanzer, Andrea; Bernhart, Stephan H; Lorenz, Ronny; Mueller, Michael M; Hofacker, Ivo L; Stadler, Peter F

    2010-04-01

    Stem-bulge RNAs (sbRNAs) are a group of small, functionally yet uncharacterized noncoding RNAs first described in C. elegans, with a few homologous sequences postulated in C. briggsae. In this study, we report on a comprehensive survey of this ncRNA family in the phylum Nematoda. Employing homology search strategies based on both sequence and secondary structure models and a computational promoter screen we identified a total of 240 new sbRNA homologs. For the majority of these loci we identified both promoter regions and transcription termination signals characteristic for pol-III transcripts. Sequence and structure comparison with known RNA families revealed that sbRNAs are homologs of vertebrate Y RNAs. Most of the sbRNAs show the characteristic Ro protein binding motif, and contain a region highly similar to a functionally required motif for DNA replication previously thought to be unique to vertebrate Y RNAs. The single Y RNA that was previously described in C. elegans, however, does not show this motif, and in general bears the hallmarks of a highly derived family member.

  17. The 3D Structures of Some Diarrheal Causing Bacterial Toxins

    DTIC Science & Technology

    1990-01-02

    The determination of the crystal and molecular structure of diarrzeal causing enterotoxin produced by S. aureus is the overall goal of this research...mitogenic and antigenic properties and activities of S. aureus toxins and enterotoxins have been studied extensively during t,-4 last two decades...interest in S. aureus enterotoxins has resurfaced because of their ability to stimulatj -T6 -proliferation. The arzswer to the question of how this

  18. The 3D Structure of Some Diarrheal Causing Bacterial Toxins

    DTIC Science & Technology

    1988-07-01

    structure analysis were grown by methods which we published recently (1). X-ray data have been collected from the native protein and from heavy atom...by chromatofocusing into individual bands which could be crystallized. Later on better separation was attained with isoelectric focusing. Two of the...Three possibl-a methods are under consideration: SIRAS, ISIR, and the direct ixthod with single isomorphous replacement. (2) Work -n the other toxins

  19. The 3D Structure of Some Diarrheal Causing Bacterial Toxins

    DTIC Science & Technology

    1992-07-23

    of intoxication resulting from food poisoning by •. anreus and for elucidating stereochemical details in the mechanism of superantigencity. The method ...of approach was to use classical methods of protein crystallography to determine the 3D structures of the SEs. Our strategy consisted of solving the...successful. At this stage purification by chromatofocusing was tried. The IEF gel clearly showed two or three bands which we were able to separate

  20. Structure and specificity of a permissive bacterial C-prenyltransferase.

    PubMed

    Elshahawi, Sherif I; Cao, Hongnan; Shaaban, Khaled A; Ponomareva, Larissa V; Subramanian, Thangaiah; Farman, Mark L; Spielmann, H Peter; Phillips, George N; Thorson, Jon S; Singh, Shanteri

    2017-04-01

    This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug.

  1. Structural and molecular comparison of bacterial and eukaryotic trigger factors.

    PubMed

    Ries, Fabian; Carius, Yvonne; Rohr, Marina; Gries, Karin; Keller, Sandro; Lancaster, C Roy D; Willmund, Felix

    2017-09-06

    A considerably small fraction of approximately 60-100 proteins of all chloroplast proteins are encoded by the plastid genome. Many of these proteins are major subunits of complexes with central functions within plastids. In comparison with other subcellular compartments and bacteria, many steps of chloroplast protein biogenesis are not well understood. We report here on the first study of chloroplast-localised trigger factor. In bacteria, this molecular chaperone is known to associate with translating ribosomes to facilitate the folding of newly synthesized proteins. Chloroplast trigger factors of the unicellular green algae Chlamydomonas reinhardtii and the vascular land plant Arabidopsis thaliana were characterized by biophysical and structural methods and compared to the Escherichia coli isoform. We show that chloroplast trigger factor is mainly monomeric and displays only moderate stability against thermal unfolding even under mild heat-stress conditions. The global shape and conformation of these proteins were determined in solution by small-angle X-ray scattering and subsequent ab initio modelling. As observed for bacteria, plastidic trigger factors have a dragon-like structure, albeit with slightly altered domain arrangement and flexibility. This structural conservation despite low amino acid sequence homology illustrates a remarkable evolutionary robustness of chaperone conformations across various kingdoms of life.

  2. Structural basis of interprotein electron transfer in bacterial sulfite oxidation

    PubMed Central

    McGrath, Aaron P; Laming, Elise L; Casas Garcia, G Patricia; Kvansakul, Marc; Guss, J Mitchell; Trewhella, Jill; Calmes, Benoit; Bernhardt, Paul V; Kappler, Ulrike; Maher, Megan J

    2015-01-01

    Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction show the complex is dynamic in solution, and that the proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT and SorU, both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU, and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly. DOI: http://dx.doi.org/10.7554/eLife.09066.001 PMID:26687009

  3. Crystal structure of the RNA component of bacterial ribonuclease P.

    PubMed

    Torres-Larios, Alfredo; Swinger, Kerren K; Krasilnikov, Andrey S; Pan, Tao; Mondragón, Alfonso

    2005-09-22

    Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 A resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.

  4. Structural basis for transcription elongation by bacterial RNA polymerase.

    PubMed

    Vassylyev, Dmitry G; Vassylyeva, Marina N; Perederina, Anna; Tahirov, Tahir H; Artsimovitch, Irina

    2007-07-12

    The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

  5. Home Life: Factors Structuring the Bacterial Diversity Found within and between Homes

    PubMed Central

    2013-01-01

    Most of our time is spent indoors where we are exposed to a wide array of different microorganisms living on surfaces and in the air of our homes. Despite their ubiquity and abundance, we have a limited understanding of the microbial diversity found within homes and how the composition and diversity of microbial communities change across different locations within the home. Here we examined the diversity of bacterial communities found in nine distinct locations within each of forty homes in the Raleigh-Durham area of North Carolina, USA, using high-throughput sequencing of the bacterial 16S rRNA gene. We found that each of the sampled locations harbored bacterial communities that were distinct from one another with surfaces that are regularly cleaned typically harboring lower levels of diversity than surfaces that are cleaned infrequently. These location-specific differences in bacterial communities could be directly related to usage patterns and differences in the likely sources of bacteria dispersed onto these locations. Finally, we examined whether the variability across homes in bacterial diversity could be attributed to outdoor environmental factors, indoor habitat structure, or the occupants of the home. We found that the presence of dogs had a significant effect on bacterial community composition in multiple locations within homes as the homes occupied by dogs harbored more diverse communities and higher relative abundances of dog-associated bacterial taxa. Furthermore, we found a significant correlation between the types of bacteria deposited on surfaces outside the home and those found inside the home, highlighting that microbes from outside the home can have a direct effect on the microbial communities living on surfaces within our homes. Together this work provides the first comprehensive analysis of the microbial communities found in the home and the factors that shape the structure of these communities both within and between homes. PMID:23717552

  6. Developmental trajectories of amphibian microbiota: response to bacterial therapy depends on initial community structure.

    PubMed

    Davis, Leyla R; Bigler, Laurent; Woodhams, Douglas C

    2017-02-22

    Improving host health through microbial manipulation requires untangling factors that shape the microbiome. There is currently little understanding of how initial community structure may drive the microbiota trajectory across host development or influence bacterial therapy outcomes. Probiotic baths of surface symbionts, Pseudomonas fluorescens and Flavobacterium johnsoniae were administered to 240 tadpoles of the midwife toad, Alytes obstetricans in semi-natural outdoor mesocosms originating from geographically and genetically distinct populations in Switzerland. Host bacterial and fungal assemblages were compared in tadpoles from the pond of origin, across metamorphosis, and in toadlets via microbial fingerprinting. Bacterial and fungal community structures differed significantly among populations and a microbial population signature persisted from the tadpole stage, through metamorphosis, and following probiotic treatment. A minimal core surface microbiota is described by persistence through development and by shared membership across populations. The impact of F. johnsoniae on the tadpole surface microbiome was assessed with shotgun metagenomics. Bacterial therapy reduced abundance, diversity, and functional repertoire compared to untreated controls. A correlation between host skin peptides and microbiota suggests a mechanism of host-directed symbiosis throughout development. Early developmental stages are ideal targets for amphibian bacterial therapy that can govern a microbiome trajectory at critical timepoints and may impact susceptibility to disease.

  7. Bacterial community structure in cooling water and biofilm in an industrial recirculating cooling water system.

    PubMed

    Wang, Jinmei; Liu, Min; Xiao, Huijie; Wu, Wei; Xie, Meijuan; Sun, Mengjia; Zhu, Chenglin; Li, Pengfu

    2013-01-01

    Microbial fouling is a serious problem in open recirculating cooling water systems. The bacterial communities that cause it have not been fully understood. In this study, we analyzed the community structure of free-living bacteria and particle-attached bacteria in cooling water, and bacteria in biofilm collected from the wall of the water reservoir in an industrial recirculating cooling water system by construction of a 16S rRNA gene clone library. Based on amplified ribosomal DNA restriction analysis, clones of all three libraries were clustered into 45 operational taxonomic units (OTUs). Thirteen OTUs displaying 91-96% sequence similarity to a type strain might be novel bacterial species. Noted differences in community structure were observed among the three libraries. The relative species richness of the free-living bacteria in cooling water was much lower than that of particle-attached bacteria and bacteria in biofilm. The majority of the free-living bacterial community (99.0%) was Betaproteobacteria. The predominant bacteria in the particle-attached bacterial community were Alphaproteobacteria (20.5%), Betaproteobacteria (27.8%) and Planctomycetes (42.0%), while those in the biofilm bacterial community were Alphaproteobacteria (47.9%), Betaproteobacteria (11.7%), Acidobacteria (13.1%) and Gemmatimonadetes (11.3%). To control microbial fouling in industrial recirculating cooling water systems, additional physiological and ecological studies of these species will be essential.

  8. Insights into Metalloregulation by M-box Riboswitch RNAs via Structural Analysis of Manganese-Bound Complexes

    SciTech Connect

    Ramesh, Arati; Wakeman, Catherine A.; Winkler, Wade C.

    2011-12-09

    The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 {angstrom} by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.

  9. Crystal structure of a bacterial signal Peptide peptidase.

    PubMed

    Kim, Apollos C; Oliver, David C; Paetzel, Mark

    2008-02-15

    Signal peptide peptidase (Spp) is the enzyme responsible for cleaving the remnant signal peptides left behind in the membrane following Sec-dependent protein secretion. Spp activity appears to be present in all cell types, eukaryotic, prokaryotic and archaeal. Here we report the first structure of a signal peptide peptidase, that of the Escherichia coli SppA (SppA(EC)). SppA(EC) forms a tetrameric assembly with a novel bowl-shaped architecture. The bowl has a dramatically hydrophobic interior and contains four separate active sites that utilize a Ser/Lys catalytic dyad mechanism. Our structural analysis of SppA reveals that while in many Gram-negative bacteria as well as characterized plant variants, a tandem duplication in the protein fold creates an intact active site at the interface between the repeated domains, other species, particularly Gram-positive and archaeal organisms, encode half-size, unduplicated SppA variants that could form similar oligomers to their duplicated counterparts, but using an octamer arrangement and with the catalytic residues provided by neighboring monomers. The structure reveals a similarity in the protein fold between the domains in the periplasmic Ser/Lys protease SppA and the monomers seen in the cytoplasmic Ser/His/Asp protease ClpP. We propose that SppA may, in addition to its role in signal peptide hydrolysis, have a role in the quality assurance of periplasmic and membrane-bound proteins, similar to the role that ClpP plays for cytoplasmic proteins.

  10. Crystal Structure of a Bacterial Signal Peptide Peptidase

    SciTech Connect

    Kim,A.; Oliver, D.; Paetzel, M.

    2008-01-01

    Signal peptide peptidase (Spp) is the enzyme responsible for cleaving the remnant signal peptides left behind in the membrane following Sec-dependent protein secretion. Spp activity appears to be present in all cell types, eukaryotic, prokaryotic and archaeal. Here we report the first structure of a signal peptide peptidase, that of the Escherichia coli SppA (SppAEC). SppAEC forms a tetrameric assembly with a novel bowl-shaped architecture. The bowl has a dramatically hydrophobic interior and contains four separate active sites that utilize a Ser/Lys catalytic dyad mechanism. Our structural analysis of SppA reveals that while in many Gram-negative bacteria as well as characterized plant variants, a tandem duplication in the protein fold creates an intact active site at the interface between the repeated domains, other species, particularly Gram-positive and archaeal organisms, encode half-size, unduplicated SppA variants that could form similar oligomers to their duplicated counterparts, but using an octamer arrangement and with the catalytic residues provided by neighboring monomers. The structure reveals a similarity in the protein fold between the domains in the periplasmic Ser/Lys protease SppA and the monomers seen in the cytoplasmic Ser/His/Asp protease ClpP. We propose that SppA may, in addition to its role in signal peptide hydrolysis, have a role in the quality assurance of periplasmic and membrane-bound proteins, similar to the role that ClpP plays for cytoplasmic proteins.

  11. The ribonuclease polynucleotide phosphorylase can interact with small regulatory RNAs in both protective and degradative modes

    PubMed Central

    Bandyra, Katarzyna J.; Sinha, Dhriti; Syrjanen, Johanna; Luisi, Ben F.; De Lay, Nicholas R.

    2016-01-01

    In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3′ ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action. PMID:26759452

  12. Structure and Specificity of a Permissive Bacterial C-Prenyltransferase

    PubMed Central

    Elshahawi, Sherif I.; Cao, Hongnan; Shaaban, Khaled A.; Ponomareva, Larissa V.; Subramanian, Thangaiah; Farman, Mark L.; Spielmann, H. Peter; Phillips, George N.; Thorson, Jon S.; Singh, Shanteri

    2016-01-01

    This study highlights the biochemical and structural characterization of the L-tryptophan C-6 C-prenyltransferase PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including the antibiotic daptomycin (Cubicin®). This study also highlights two additional PTs (FgaPT2 and CdpNPT) as catalysts for daptomycin prenylation where novel prenylated daptomycins also displayed improved antibacterial activities over the parent drug. PMID:28166207

  13. Characterizing changes in soil bacterial community structure in response to short-term warming.

    PubMed

    Xiong, Jinbo; Sun, Huaibo; Peng, Fei; Zhang, Huayong; Xue, Xian; Gibbons, Sean M; Gilbert, Jack A; Chu, Haiyan

    2014-08-01

    High altitude alpine meadows are experiencing considerably greater than average increases in soil surface temperature, potentially as a result of ongoing climate change. The effects of warming on plant productivity and soil edaphic variables have been established previously, but the influence of warming on soil microbial community structure has not been well characterized. Here, the impact of 15 months of soil warming (both +1 and +2 °C) on bacterial community structure was examined in a field experiment on a Tibetan plateau alpine meadow using bar-coded pyrosequencing. Warming significantly changed (P < 0.05) the structure of the soil bacterial community, but the alpha diversity was not dramatically affected. Changes in the abundance of the Actinobacteria and Alphaproteobacteria were found to contribute the most to differences between ambient (AT) and artificially warmed conditions. A variance partitioning analysis (VPA) showed that warming directly explained 7.15% variation in bacterial community structure, while warming-induced changes in soil edaphic and plant phenotypic properties indirectly accounted for 28.3% and 20.6% of the community variance, respectively. Interestingly, certain taxa showed an inconsistent response to the two warming treatments, for example Deltaproteobacteria showed a decreased relative abundance at +1 °C, but a return to AT control relative abundance at +2 °C. This suggests complex microbial dynamics that could result from conditional dependencies between bacterial taxa.

  14. Citrus huanglongbing shapes the structure of bacterial community associated with citrus roots

    USDA-ARS?s Scientific Manuscript database

    To examine the effect of pathogen on the diversity and structure of plant associated bacterial community, we carried out a molecular based analysis using citrus and huanglongbing as host-disease model. 16S rDNA clone library analysis of the citrus roots revealed shifts in the microbial diversity in ...

  15. Characterizing changes in soil bacterial community structure in response to short-term warming

    SciTech Connect

    Gibbons, Sean M.; sun, Huaibo; Xiong, Jinbo; Gilbert, Jack A.; Peng, Fei; Chu, Haiyan; Chu, Haiyan

    2014-08-01

    High altitude alpine meadows are experiencing considerably greater than average increases in soil surface temperature, potentially as a result of ongoing climate change. The effects of warming on plant productivity and soil edaphic variables have been established previously, but the influence of warming on soil microbial community structure has not been well characterized. Here, the impact of 15 months of soil warming (both + 1 and + 2 degrees C) on bacterial community structure was examined in a field experiment on a Tibetan plateau alpine meadow using bar-coded pyrosequencing. Warming significantly changed (P < 0.05) the structure of the soil bacterial community, but the alpha diversity was not dramatically affected. Changes in the abundance of the Actinobacteria and Alphaproteobacteria were found to contribute the most to differences between ambient (AT) and artificially warmed conditions. A variance partitioning analysis (VPA) showed that warming directly explained 7.15% variation in bacterial community structure, while warming-induced changes in soil edaphic and plant phenotypic properties indirectly accounted for 28.3% and 20.6% of the community variance, respectively. Interestingly, certain taxa showed an inconsistent response to the two warming treatments, for example Deltaproteobacteria showed a decreased relative abundance at + 1 degrees C, but a return to AT control relative abundance at + 2 degrees C. This suggests complex microbial dynamics that could result from conditional dependencies between bacterial taxa.

  16. Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors

    PubMed Central

    Frossard, Aline; Gerull, Linda; Mutz, Michael; Gessner, Mark O

    2012-01-01

    A fundamental issue in microbial and general ecology is the question to what extent environmental conditions dictate the structure of communities and the linkages with functional properties of ecosystems (that is, ecosystem function). We approached this question by taking advantage of environmental gradients established in soil and sediments of small stream corridors in a recently created, early successional catchment. Specifically, we determined spatial and temporal patterns of bacterial community structure and their linkages with potential microbial enzyme activities along the hydrological flow paths of the catchment. Soil and sediments were sampled in a total of 15 sites on four occasions spread throughout a year. Denaturing gradient gel electrophoresis (DGGE) was used to characterize bacterial communities, and substrate analogs linked to fluorescent molecules served to track 10 different enzymes as specific measures of ecosystem function. Potential enzyme activities varied little among sites, despite contrasting environmental conditions, especially in terms of water availability. Temporal changes, in contrast, were pronounced and remarkably variable among the enzymes tested. This suggests much greater importance of temporal dynamics than spatial heterogeneity in affecting specific ecosystem functions. Most strikingly, bacterial community structure revealed neither temporal nor spatial patterns. The resulting disconnect between bacterial community structure and potential enzyme activities indicates high functional redundancy within microbial communities even in the physically and biologically simplified stream corridors of early successional landscapes. PMID:22030674

  17. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    SciTech Connect

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; Landick, Robert; Darst, Seth A.

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

  18. Structural insights into the bacterial carbon-phosphorus lyase machinery

    PubMed Central

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J.; Passmore, Lori A.; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E.

    2015-01-01

    Summary Phosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown. PMID:26280334

  19. Structural insights into the bacterial carbon-phosphorus lyase machinery.

    PubMed

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J; Passmore, Lori A; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E

    2015-09-03

    Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

  20. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    DOE PAGES

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

  1. Graded Structural Polymorphism in a Bacterial Thermosensor Protein.

    PubMed

    Narayan, Abhishek; Campos, Luis A; Bhatia, Sandhya; Fushman, David; Naganathan, Athi N

    2017-01-18

    Thermosensing is critical for the expression of virulence genes in pathogenic bacteria that infect warm-blooded hosts. Proteins of the Hha-family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of a large number of genes upon temperature shifts. However, there is little mechanistic evidence at the molecular level as to how changes in temperature are transduced into structural changes and hence the functional outcome. In this study, we delineate the conformational behavior of Cnu, a putative molecular thermosensor, employing diverse spectroscopic, calorimetric and hydrodynamic measurements. We find that Cnu displays probe-dependent unfolding in equilibrium, graded increase in structural fluctuations and temperature-dependent swelling of the dimensions of its native ensemble within the physiological range of temperatures, features that are indicative of a highly malleable native ensemble. Site-specific fluorescence and NMR experiments in combination with multiple computational approaches-statistical mechanical model, coarse-grained and all-atom MD simulations-reveal that the fourth helix of Cnu acts as a unique thermosensing module displaying varying degrees of order and orientation in response to temperature modulations while undergoing a continuous unfolding transition. Our combined experimental-computational study unravels the folding-functional landscape of a natural thermosensor protein, the molecular origins of its unfolding complexity, highlights the role of functional constraints in determining folding-mechanistic behaviors, and the design principles orchestrating the signal transduction roles of the Hha protein family.

  2. The Crystal Structures of EAP Domains from Staphylococcus aureus Reveal an Unexpected Homology to Bacterial Superantigens

    SciTech Connect

    Geisbrecht, B V; Hamaoka, B Y; Perman, B; Zemla, A; Leahy, D J

    2005-10-14

    The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 {angstrom} resolution, respectively. These structures reveal a core fold that is comprised of an {alpha}-helix lying diagonally across a five-stranded, mixed {beta}-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the {beta}-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

  3. Structure and function of the bacterial Sec translocon.

    PubMed

    Gold, Vicki A M; Duong, Franck; Collinson, Ian

    2007-01-01

    Bacteria and archaea possess a protein complex in the plasma membrane that governs protein secretion and membrane protein insertion. Eukaryotes carry homologues in the endoplasmic reticulum (ER) where they direct the same reaction. A combination of experiments conducted on the systems found in all three domains of life has revealed a great deal about protein translocation. The channel provides a route for proteins to pass through the hydrophobic barrier of the membrane, assisted by various partner proteins which maintain an unfolded state of the substrate, target it to the channel and provide the energy and mechanical drive required for transport. In bacteria, the post-translational reaction utilizes an ATPase that couples the free energy of ATP binding and hydrolysis to move the substrate through the protein pore. This review will draw on genetic, biochemical and structural findings in an account of our current understanding of this mechanism.

  4. Structure, mechanism and cooperation of bacterial multidrug transporters.

    PubMed

    Du, Dijun; van Veen, Hendrik W; Murakami, Satoshi; Pos, Klaas M; Luisi, Ben F

    2015-08-01

    Cells from all domains of life encode energy-dependent trans-membrane transporters that can expel harmful substances including clinically applied therapeutic agents. As a collective body, these transporters perform as a super-system that confers tolerance to an enormous range of harmful compounds and consequently aid survival in hazardous environments. In the Gram-negative bacteria, some of these transporters serve as energy-transducing components of tripartite assemblies that actively efflux drugs and other harmful compounds, as well as deliver virulence agents across the entire cell envelope. We draw together recent structural and functional data to present the current models for the transport mechanisms for the main classes of multi-drug transporters and their higher-order assemblies. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Effects of transient temperature conditions on the divergence of activated sludge bacterial community structure and function.

    PubMed

    Nadarajah, Nalina; Allen, D Grant; Fulthorpe, Roberta R

    2007-06-01

    The effect of temperature fluctuations on bacterial community structure and function in lab-scale sequencing batch reactors treating bleached kraft mill effluent was investigated. An increase in temperature from 30 to 45 degrees C caused shifts in both bacterial community structure and function. Triplicate reactors were highly similar for 40 days following startup. After the temperature shift, their community structure and function started to diverge from each other and from the control. A multi-response permutation procedure confirmed that the variability in community structure between transient and control reactors were greater than that among the triplicate transient reactors. The fact that these disturbances manifest themselves in different ways in apparently identical reactors suggests a high degree of variability between replicate systems.

  6. The role of enzymology in a structure-based drug discovery program: bacterial DNA gyrase.

    PubMed

    Cunningham, Mark L

    2012-01-01

    The capability to accurately, rapidly, and reproducibly determine the affinity of a ligand for a target protein or enzyme is a vital component for a successful structure-based drug design effort. In order to successfully drive a structure-based drug design (SBDD) project forward, multiple distinct assays, each with particular strengths and weaknesses, need to be employed. Using bacterial DNA gyrase as an example, a range of assays are described that will fully support an SBDD program.

  7. Structure of the translocator domain of a bacterial autotransporter

    PubMed Central

    Oomen, Clasien J; van Ulsen, Peter; Van Gelder, Patrick; Feijen, Maya; Tommassen, Jan; Gros, Piet

    2004-01-01

    Autotransporters are virulence-related proteins of Gram-negative bacteria that are secreted via an outer-membrane-based C-terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N-terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro-folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12-stranded β-barrel with a hydrophilic pore of 10 × 12.5 Å that is filled by an N-terminal α-helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger-domain transport through the hydrophilic channel within the β-barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger-domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer-membrane proteins, on which autotransporters are dependent. PMID:15014442

  8. Structure of a cellulose degrading bacterial community during anaerobic digestion.

    PubMed

    O'Sullivan, Cathryn A; Burrell, Paul C; Clarke, William P; Blackall, Linda L

    2005-12-30

    It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.

  9. Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

    SciTech Connect

    J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

    2011-12-31

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  10. Structural basis for alginate secretion across the bacterial outer membrane

    SciTech Connect

    Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

    2011-08-09

    Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

  11. Interactive effects of solar radiation and dissolved organic matter on bacterial activity and community structure

    PubMed Central

    Pérez, María Teresa; Sommaruga, Ruben

    2007-01-01

    We studied the interactive effects of dissolved organic matter (DOM) and solar radiation on the activity and community structure of bacteria from an alpine lake. Activity was assessed both at the community level as leucine incorporation rates and at the single-cell level by microautoradiography. Fluorescent in situ hybridization and signal amplification by catalysed reporter deposition (CARD-FISH) was used to track changes in the bacterial community composition. Bacteria-free filtrates of different DOM sources (lake, algae or soil) were incubated either in the dark or exposed to solar radiation. Afterwards, the natural bacterial assemblage was inoculated and the cultures incubated in the dark for 24–48 h. Bacterial activity was enhanced in the first 24 h in the soil and algal DOM amendments kept in the dark. After 48 h, the enhancement effect was greatly reduced. The initial bacterial community was dominated by Betaproteobacteria followed by Actinobacteria. The relative abundance (expressed as a percentage of DAPI-stained cells) of Betaproteobacteria increased first in dark incubated DOM amendments, but after 48 h no significant differences were detected among treatments. In contrast, the relative abundance of Actinobacteria increased in pre-irradiated DOM treatments. Although Betaproteobacteria dominated at the end of the experiment, the relative abundance of their R-BT subgroup differed among treatments. Changes in bacterial community activity were significantly correlated with those of the relative abundance and activity of Betaproteobacteria, whereas the contribution of Actinobacteria to the bulk activity was very modest. Our results indicate a negative effect of DOM photoalteration on the bulk bacterial activity. The magnitude of this effect was time-dependent and related to rapid changes in the bacterial assemblage composition. PMID:17686018

  12. Structure and function of the bacterial communities during rhizoremediation of hexachlorobenzene in constructed wetlands.

    PubMed

    Zhang, Cuiping; Wang, Bei; Dai, Xiaoyan; Li, Shuying; Lu, Guangqiu; Zhou, Yuanqing

    2017-04-01

    Vertical flow constructed wetlands (VF CWs) are considered to be effective for treating organic pollutants. The rhizosphere of macrophytes such as Phragmites sp., Typha sp. serves as an active and dynamic zone for the microbial degradation of organic pollutants. However, it is still not clear how soil bacterial communities respond to macrophytes and pollutants during the process. For this purpose, the seedlings of Phragmites australis and Typha angustifolia were planted respectively in the VF CWs added with HCB at a dose of 2 mg/kg. During 96 days of cultivation, we monitored hexachlorobenzene (HCB) removal efficiency by GC/MS and the structure of the rhizosphere bacterial communities in the different VF CWs by denaturing gradient gel electrophoresis (DGGE), and constructed bacterial clone library based on PCR-amplified 16S rRNA gene. As expected, the rhizosphere bacterial communities also remained insensitive to HCB exposure in the wetland soil. The diversity of these microbes presented two stages, from the varied up and down to equilibrium in the entire experimental period. Molecular analysis revealed that the phylum Firmicutes dominated over the bacterial communities. The genera that increased under HCB stress included the well-known HCB-degrading bacteria (Pseudomonas sp. and Alcaligenes sp.) and other common bacteria found in contaminated soil but with lesser known practical functions (Burkholderia sp., Lysinibacillus fusiformis, and Bacillus cereus). Furthermore, there was a certain variance in the relative abundances of the bacterial phyla and HCB removal efficiency among different VF CW treatments. The degradation of HCB in T. angustifolia microcosms was faster than that in P. australis and unvegetated wetlands, and the highest bacterial diversity and richness was found in the VF CWs comprising T. angustifolia.

  13. Influence of oyster culture on biogeochemistry and bacterial community structure at the sediment-water interface.

    PubMed

    Azandégbé, Afi; Poly, Franck; Andrieux-Loyer, Françoise; Kérouel, Roger; Philippon, Xavier; Nicolas, Jean-Louis

    2012-10-01

    Bacterial community structure and some biogeochemical parameters were studied in the sediment of two Pacific oyster farming sites, Aber Benoît (AB) and Rivière d'Auray (RA) in Brittany (France), to examine the ecological impact of oysters and to evaluate the emission of sulfide and ammonia from sediment. At AB, the organic matter accumulated in the sediment beneath the oyster tables was rapidly mineralized, with strong fluxes of ammonia and sulfide that reached 1014 and 215 μmol m(-2) h(-1), respectively, in June 2007. At RA, the fluxes were about half as strong on average and better distributed through the year. The ammonia and sulfide concentrations in the overlying water never reached levels that would be toxic to oysters in either site, nor did hypoxia occur. Total culturable bacteria (TCB) varied greatly according to the temperature: from 1.6 × 10(4) to 9.4 × 10(7) cell g(-1) sediment. Inversely, the bacterial community structure remained surprising stable through the seasons, marginally influenced by the presence of oysters and by temperature. Bacterial communities appeared to be characteristic of the sites, with only one common phylotype, Vibrio aestuarianus, a potential oyster pathogen. These data refine the hypothesis of seawater toxicity to oysters because of ammonia and sulfide fluxes and show that the measured environmental factors had only a weak influence on bacterial community structure.

  14. Bacterial Diversity and Community Structure in Two Bornean Nepenthes Species with Differences in Nitrogen Acquisition Strategies.

    PubMed

    Sickel, Wiebke; Grafe, T Ulmar; Meuche, Ivonne; Steffan-Dewenter, Ingolf; Keller, Alexander

    2016-05-01

    Carnivorous plants of the genus Nepenthes have been studied for over a century, but surprisingly little is known about associations with microorganisms. The two species Nepenthes rafflesiana and Nepenthes hemsleyana differ in their pitcher-mediated nutrient sources, sequestering nitrogen from arthropod prey and arthropods as well as bat faeces, respectively. We expected bacterial communities living in the pitchers to resemble this diet difference. Samples were taken from different parts of the pitchers (leaf, peristome, inside, outside, digestive fluid) of both species. Bacterial communities were determined using culture-independent high-throughput amplicon sequencing. Bacterial richness and community structure were similar in leaves, peristomes, inside and outside walls of both plant species. Regarding digestive fluids, bacterial richness was higher in N. hemsleyana than in N. rafflesiana. Additionally, digestive fluid communities were highly variable in structure, with strain-specific differences in community composition between replicates. Acidophilic taxa were mostly of low abundance, except the genus Acidocella, which strikingly reached extremely high levels in two N. rafflesiana fluids. In N. hemsleyana fluid, some taxa classified as vertebrate gut symbionts as well as saprophytes were enriched compared to N. rafflesiana, with saprophytes constituting potential competitors for nutrients. The high variation in community structure might be caused by a number of biotic and abiotic factors. Nitrogen-fixing bacteria were present in both study species, which might provide essential nutrients to the plant at times of low prey capture and/or rare encounters with bats.

  15. Impact of lime, nitrogen and plant species on bacterial community structure in grassland microcosms.

    PubMed

    Kennedy, Nabla; Brodie, Eoin; Connolly, John; Clipson, Nicholas

    2004-10-01

    A microcosm-based approach was used to study impacts of plant and chemical factors on the bacterial community structure of an upland acidic grassland soil. Seven perennial plant species typical of both natural, unimproved (Nardus stricta, Agrostis capillaris, Festuca ovina and F. rubra) and fertilized, improved (Holcus lanatus, Lolium perenne and Trifolium repens) grasslands were either left unamended or treated with lime, nitrogen, or lime plus nitrogen in a 75-day glasshouse experiment. Lime and nitrogen amendment were shown to have a greater effect on microbial activity, biomass and bacterial ribotype number than plant species. Liming increased soil pH, microbial activity and biomass, while decreasing ribotype number. Nitrogen addition decreased soil pH, microbial activity and ribotype number. Addition of lime plus nitrogen had intermediate effects, which appeared to be driven more by lime than nitrogen. Terminal restriction fragment length polymorphism (TRFLP) analysis revealed that lime and nitrogen addition altered soil bacterial community structure, while plant species had little effect. These results were further confirmed by multivariate redundancy analysis, and suggest that soil lime and nitrogen status are more important controllers of bacterial community structure than plant rhizosphere effects.

  16. Representative sampling of natural biofilms: influence of substratum type on the bacterial and fungal communities structure.

    PubMed

    Hellal, Jennifer; Michel, Caroline; Barsotti, Vanessa; Laperche, Valérie; Garrido, Francis; Joulian, Catherine

    2016-01-01

    In situ biofilm sampling is a key step for the study of natural biofilms and using methodologies that reflect natural diversity is necessary to guarantee representative sampling. Here, we focalise on the impact of the type of substrata on which biofilms grow on bacterial and fungal communities' structure. The indirect molecular approach, Denaturing Gel Gradient Electrophoresis (DGGE) of a gene fragment coding for either 16S rRNA or 28S rRNA, for bacteria or fungi respectively, was used to evaluate the variability of microbial community structures among different biofilm substrata: natural (pebbles, live plants, wood and sediment), or artificial (glass, Plexiglas(®) and sterile wood), in a small river (the Loiret, France). Multivariate statistics, band richness and diversity indexes (Shannon and Simpson) were used to highlight variations in community structure between substrata. Results showed variations of bacterial and fungal diversity between different substrata according to substratum properties/origin (natural or artificial, organic or inorganic) but there was no optimal substratum for sampling, and artificial substrata were not significantly less applicable than natural substrata. Pooling 4 different substrata types allowed a higher bacterial and fungal biodiversity recovery. Point contact sampling may thus gain in robustness by increasing the number of substrata considered. Fungal species richness was similar to the bacterial one on most substrata which suggested they should be more frequently considered in riverine biofilm studies.

  17. Hijacking the Host Ubiquitin Pathway: Structural Strategies of Bacterial E3 Ubiquitin Ligases

    PubMed Central

    Hicks, Stuart W.; Galán, Jorge E.

    2009-01-01

    Summary Ubiquitinylation of proteins is a critical mechanism in regulating numerous eukaryotic cellular processes including cell cycle progression, inflammatory response, and vesicular trafficking. Given the importance of ubiquitinylation, it is not surprising that several pathogenic bacteria have developed strategies to exploit various stages of the ubiquitin pathway for their own benefit. One such strategy is the delivery of bacterial ‘effector’ proteins into the host cell cytosol, which mimic the activities of components of the host ubiquitin pathway. Recent studies have highlighted a number of bacterial effectors that functionally mimic the activity of eukaryotic E3 ubiquitin ligases, including a novel structural class of bacterial E3 ligases that provides a striking example of convergent evolution. PMID:20036613

  18. Small nuclear RNAs in the ciliate Tetrahymena.

    PubMed Central

    Pedersen, N; Hellung-Larsen, P; Engberg, J

    1985-01-01

    We have isolated and partially characterized a family of small nuclear RNAs (snRNAs) from three different species of the protozoan Tetrahymena. We find six distinct snRNAs ranging in size from 100 to 250 nucleotides. The two largest snRNAs, as well as an abundant, heterogenous group of smaller snRNAs are found in the nucleolar RNA fraction. None of the snRNAs are transcription products of the ribosomal RNA gene or its flanking regions, as shown by hybridization tests. The snRNAs are metabolically stable as determined by pulse/chase experiments and several of them contain a number of modified nuclotides. The snRNAs from Tetrahymena all have slightly different sizes from mammalian snRNAs. The cap structure of the snRNAs from Tetrahymena differs from that of the snRNAs from mammalian cells, but has not yet been fully characterized. The relative amount of snRNAs to total RNA is less in Tetrahymena (greater than 0.1%) than in mammalian cells (2%). Images PMID:2409533

  19. Experimental approaches to identify small RNAs and their diverse roles in bacteria--what we have learnt in one decade of MicA research.

    PubMed

    Van Puyvelde, Sandra; Vanderleyden, Jozef; De Keersmaecker, Sigrid C J

    2015-10-01

    Nowadays the identification of small RNAs (sRNAs) and characterization of their role within regulatory networks takes a prominent place in deciphering complex bacterial phenotypes. Compared to the study of other components of bacterial cells, this is a relatively new but fast-growing research field. Although reports on new sRNAs appear regularly, some sRNAs are already subject of research for a longer time. One of such sRNAs is MicA, a sRNA best described for its role in outer membrane remodeling, but probably having a much broader function than anticipated. An overview of what we have learnt from MicA led to the conclusion that even for this well-described sRNA, we still do not have the overall picture. More general, the story of MicA might become an experimental lead for unraveling the many sRNAs with unknown functions. In this review, three important topics in the sRNA field are covered, exemplified from the perspective of MicA: (i) identification of new sRNAs, (ii) target identification and unraveling the biological function, (iii) structural analysis. The complex mechanisms of action of MicA deliver some original insights in the sRNA field which includes the existence of dimer formation or simultaneous cis and trans regulation, and might further inspire the understanding of the function of other sRNAs.

  20. Archaeal and bacterial community structures in the anoxic sediment of Antarctic meromictic lake Nurume-Ike

    NASA Astrophysics Data System (ADS)

    Kurosawa, Norio; Sato, Shota; Kawarabayasi, Yutaka; Imura, Satoshi; Naganuma, Takeshi

    2010-08-01

    Prokaryotic community structures in the anoxic sediment of the Antarctic meromictic Lake Nurume-Ike were revealed by sequence analysis of 16S rRNA gene clones. The archaeal clones obtained (205 total) consisted of only three phylotypes, and were dominantly affiliated with uncultured euryarchaeotes. Specifically, 93% of the clones were identified as marine benthic group-D archaeal phylotype. In contrast to the limited archaeal diversity, 53 phylotypes were detected within 312 bacterial clones. Major bacterial phylotypes were affiliated with α-Proteobacteria (20% of clones), d-Proteobacteria (9%), Planctmycetales (7%), and Cyanobacteria (7%). A small numbers of clones belonging to γ-Proteobacteria, Actinobacteria, Spirochaetes, Flavobacteria, and Verrucomicrobia were also found. A total of 53% of the bacterial clones, consisting of 13 phylotypes, could not be classified into any known group. These results indicated that the bacterial community of Lake Nurume-Ike sediment consisted of numerous phylogenetic groups and had a diversity comparable to the diversity of other Antarctic lakes communities previously reported. Interestingly, however, there were very few phylotypes shared between the communities of lakes Nurume-Ike and five other lakes located in the Vestfold Hills area. This is the first comprehensive study to analyze more than 500 16S rDNA clones for microbial community analysis of an Antarctic lake sediment sample, and the results significantly expand current views of bacterial diversity in Antarctic lakes.

  1. Diversity and morphological structure of bacterial communities inhabiting the Diana-Hygieia Thermal Spring (Budapest, Hungary).

    PubMed

    Anda, Dóra; Büki, Gabriella; Krett, Gergely; Makk, Judit; Márialigeti, Károly; Erőss, Anita; Mádl-Szőnyi, Judit; Borsodi, Andrea K

    2014-09-01

    The Buda Thermal Karst System is an active hypogenic karst area that offers possibility for the analysis of biogenic cave formation. The aim of the present study was to gain information about morphological structure and genetic diversity of bacterial communities inhabiting the Diana-Hygieia Thermal Spring (DHTS). Using scanning electron microscopy, metal accumulating and unusual reticulated filaments were detected in large numbers in the DHTS biofilm samples. The phyla Actinobacteria, Firmicutes and Proteobacteria were represented by both bacterial strains and molecular clones but phyla Acidobacteria, Chlorobi, Chlorofexi, Gemmatimonadetes, Nitrospirae and Thermotogae only by molecular clones which showed the highest similarity to uncultured clone sequences originating from different environmental sources. The biofilm bacterial community proved to be somewhat more diverse than that of the water sample and the distribution of the dominant bacterial clones was different between biofilm and water samples. The majority of biofilm clones was affiliated with Deltaproteobacteria and Nitrospirae while the largest group of water clones was related to Betaproteobacteria. Considering the metabolic properties of known species related to the strains and molecular clones from DHTS, it can be assumed that these bacterial communities may participate in the local sulphur and iron cycles, and contribute to biogenic cave formation.

  2. Characterization of bacterial community structure in a hydrocarbon-contaminated tropical African soil.

    PubMed

    Salam, Lateef B; Ilori, Mathew O; Amund, Olukayode O; LiiMien, Yee; Nojiri, Hideaki

    2017-04-26

    The bacterial community structure in a hydrocarbon-contaminated Mechanical Engineering Workshop (MWO) soil was deciphered using 16S rRNA gene clone library analysis. Four hundred and thirty-seven clones cutting across 13 bacterial phyla were recovered from the soil. The representative bacterial phyla identified from MWO soil are Proteobacteria, Bacteroidetes, Chloroflexi, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, Ignavibacteriae, Spirochaetes, Chlamydiae, Candidatus Saccharibacteria and Parcubacteria. Proteobacteria is preponderant in the contaminated soil (51.2%) with all classes except Epsilonproteobacteria duly represented. Rarefaction analysis indicates 42%, 52% and 77% of the clone library is covered at the species, genus and family/class delineations with Shannon diversity (H') and Chao1 richness indices of 5.59 and 1126, respectively. A sizeable number of bacterial phylotypes in the clone library shared high similarities with strains previously described to be involved in hydrocarbon biodegradation. Novel uncultured genera were identified that have not been previously reported from tropical African soil to be associated with natural attenuation of hydrocarbon pollutants. This study establishes the involvement of a wide array of physiologically diverse bacterial groups in natural attenuation of hydrocarbon pollutants in soil.

  3. Fertilization Shapes Bacterial Community Structure by Alteration of Soil pH.

    PubMed

    Zhang, Yuting; Shen, Hong; He, Xinhua; Thomas, Ben W; Lupwayi, Newton Z; Hao, Xiying; Thomas, Matthew C; Shi, Xiaojun

    2017-01-01

    Application of chemical fertilizer or manure can affect soil microorganisms directly by supplying nutrients and indirectly by altering soil pH. However, it remains uncertain which effect mostly shapes microbial community structure. We determined soil bacterial diversity and community structure by 454 pyrosequencing the V1-V3 regions of 16S rRNA genes after 7-years (2007-2014) of applying chemical nitrogen, phosphorus and potassium (NPK) fertilizers, composted manure or their combination to acidic (pH 5.8), near-neutral (pH 6.8) or alkaline (pH 8.4) Eutric Regosol soil in a maize-vegetable rotation in southwest China. In alkaline soil, nutrient sources did not affect bacterial Operational Taxonomic Unit (OTU) richness or Shannon diversity index, despite higher available N, P, K, and soil organic carbon in fertilized than in unfertilized soil. In contrast, bacterial OTU richness and Shannon diversity index were significantly lower in acidic and near-neutral soils under NPK than under manure or their combination, which corresponded with changes in soil pH. Permutational multivariate analysis of variance showed that bacterial community structure was significantly affected across these three soils, but the PCoA ordination patterns indicated the effect was less distinct among nutrient sources in alkaline than in acidic and near-neural soils. Distance-based redundancy analysis showed that bacterial community structures were significantly altered by soil pH in acidic and near-neutral soils, but not by any soil chemical properties in alkaline soil. The relative abundance (%) of most bacterial phyla was higher in near-neutral than in acidic or alkaline soils. The most dominant phyla were Proteobacteria (24.6%), Actinobacteria (19.7%), Chloroflexi (15.3%) and Acidobacteria (12.6%); the medium dominant phyla were Bacterioidetes (5.3%), Planctomycetes (4.8%), Gemmatimonadetes (4.5%), Firmicutes (3.4%), Cyanobacteria (2.1%), Nitrospirae (1.8%), and candidate division TM7 (1

  4. Host species and developmental stage, but not host social structure, affects bacterial community structure in socially polymorphic bees.

    PubMed

    McFrederick, Quinn S; Wcislo, William T; Hout, Michael C; Mueller, Ulrich G

    2014-05-01

    Social transmission and host developmental stage are thought to profoundly affect the structure of bacterial communities associated with honey bees and bumble bees, but these ideas have not been explored in other bee species. The halictid bees Megalopta centralis and M. genalis exhibit intrapopulation social polymorphism, which we exploit to test whether bacterial communities differ by host social structure, developmental stage, or host species. We collected social and solitary Megalopta nests and sampled bees and nest contents from all stages of host development. To survey these bacterial communities, we used 16S rRNA gene 454 pyrosequencing. We found no effect of social structure, but found differences by host species and developmental stage. Wolbachia prevalence differed between the two host species. Bacterial communities associated with different developmental stages appeared to be driven by environmentally acquired bacteria. A Lactobacillus kunkeei clade bacterium that is consistently associated with other bee species was dominant in pollen provisions and larval samples, but less abundant in mature larvae and pupae. Foraging adults appeared to often reacquire L. kunkeei clade bacteria, likely while foraging at flowers. Environmental transmission appears to be more important than social transmission for Megalopta bees at the cusp between social and solitary behavior.

  5. microRNAs in lupus

    PubMed Central

    ZAN, HONG; TAT, CONNIE; CASALI, PAOLO

    2014-01-01

    Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies, which plays an important role in disease development and progression. Lupus preferentially affects women during their reproductive years. The pathogenesis of lupus is contributed by both genetic factors and epigenetic modifications that arise from exposure to the environment. Epigenetic marks, including DNA methylation, histone post-translational modifications and microRNAs (miRNAs), interact with genetic programs to regulate immune responses. Epigenetic modifications influence gene expression and modulate B cell functions, such as class switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation, thereby informing the antibody response. Epigenetic dysregulation can result in aberrant antibody responses to exogenous antigens or self-antigens, such as chromatin, histones and dsDNA in lupus. miRNAs play key roles in the post-transcriptional regulation of most gene-regulatory pathways and regulate both the innate and the adaptive immune responses. In mice, dysregulation of miRNAs leads to aberrant immune responses and development of systemic autoimmunity. Altered miRNA expression has been reported in human autoimmune diseases, including lupus. The dysregulation of miRNAs in lupus could be the result of multiple environmental factors, such as sex hormones and viral or bacterial infection. Modulation of miRNA is a potential therapeutic strategy for lupus. PMID:24826805

  6. Phospholipid-derived fatty acids and quinones as markers for bacterial biomass and community structure in marine sediments.

    PubMed

    Kunihiro, Tadao; Veuger, Bart; Vasquez-Cardenas, Diana; Pozzato, Lara; Le Guitton, Marie; Moriya, Kazuyoshi; Kuwae, Michinobu; Omori, Koji; Boschker, Henricus T S; van Oevelen, Dick

    2014-01-01

    Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure.

  7. Phospholipid-Derived Fatty Acids and Quinones as Markers for Bacterial Biomass and Community Structure in Marine Sediments

    PubMed Central

    Kunihiro, Tadao; Veuger, Bart; Vasquez-Cardenas, Diana; Pozzato, Lara; Le Guitton, Marie; Moriya, Kazuyoshi; Kuwae, Michinobu; Omori, Koji; Boschker, Henricus T. S.; van Oevelen, Dick

    2014-01-01

    Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure. PMID:24769853

  8. Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

    PubMed

    Chung, Ben C; Zhao, Jinshi; Gillespie, Robert A; Kwon, Do-Yeon; Guan, Ziqiang; Hong, Jiyong; Zhou, Pei; Lee, Seok-Yong

    2013-08-30

    MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme.

  9. Crystal Structure of MraY, an Essential Membrane Enzyme for Bacterial Cell Wall Synthesis

    PubMed Central

    Chung, Ben C.; Kwon, Do-Yeon; Guan, Ziqiang; Hong, Jiyong; Zhou, Pei; Lee, Seok-Yong

    2013-01-01

    MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg2+ within the active site, and provide a structural basis of catalysis for this class of enzyme. PMID:23990562

  10. Ceftaroline fosamil in the treatment of community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections.

    PubMed

    Lodise, Thomas P; Low, Donald E

    2012-07-30

    Ceftaroline fosamil is a cephalosporin antibacterial approved by the US Food and Drug Administration (FDA) for use in the treatment of acute bacterial skin and skin structure infections (ABSSSI) and community-acquired bacterial pneumonia (CABP). After intravenous administration, ceftaroline fosamil is rapidly converted to its bioactive metabolite, ceftaroline. Ceftaroline has broad-spectrum in vitro activity against Gram-positive and Gram-negative bacteria, including contemporary resistant Gram-positive phenotypes, such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Streptococcus pneumoniae. Because of its unique spectrum of activity, the Clinical and Laboratory Standards Institute (CLSI) designated ceftaroline as a member of a new subclass of β-lactam antimicrobials, cephalosporins with anti-MRSA activity. The activity of ceftaroline against S. aureus extends to heteroresistant vancomycin-intermediate, vancomycin-intermediate, vancomycin-resistant and daptomycin-nonsusceptible isolates. Ceftaroline has low minimum inhibitory concentrations (MICs) for all tested species of streptococci, and has potent activity against S. pneumoniae isolates with varying degrees of penicillin resistance. The activity of ceftaroline is limited against Enterococcus faecalis and Enterococcus faecium and against anaerobes such as Bacteroides fragilis. The in vitro activity of ceftaroline includes many Gram-negative pathogens, but does not extend to bacteria that produce extended-spectrum β-lactamases, class B metallo-β-lactamases or AmpC cephalosporinases, or to most nonfermentative Gram-negative bacilli. Ceftaroline fosamil has been studied for the treatment of complicated skin and skin structure infections (cSSSI) and community-acquired pneumonia (CAP) in phase III randomized, double-blind, international, multicentre noninferiority clinical trials. Two identical trials (CANVAS 1 and CANVAS 2) compared the efficacy of ceftaroline fosamil with that of

  11. Light structures phototroph, bacterial and fungal communities at the soil surface.

    PubMed

    Davies, Lawrence O; Schäfer, Hendrik; Marshall, Samantha; Bramke, Irene; Oliver, Robin G; Bending, Gary D

    2013-01-01

    The upper few millimeters of soil harbour photosynthetic microbial communities that are structurally distinct from those of underlying bulk soil due to the presence of light. Previous studies in arid zones have demonstrated functional importance of these communities in reducing soil erosion, and enhancing carbon and nitrogen fixation. Despite being widely distributed, comparative understanding of the biodiversity of the soil surface and underlying soil is lacking, particularly in temperate zones. We investigated the establishment of soil surface communities on pasture soil in microcosms exposed to light or dark conditions, focusing on changes in phototroph, bacterial and fungal communities at the soil surface (0-3 mm) and bulk soil (3-12 mm) using ribosomal marker gene analyses. Microbial community structure changed with time and structurally similar phototrophic communities were found at the soil surface and in bulk soil in the light exposed microcosms suggesting that light can influence phototroph community structure even in the underlying bulk soil. 454 pyrosequencing showed a significant selection for diazotrophic cyanobacteria such as Nostoc punctiforme and Anabaena spp., in addition to the green alga Scenedesmus obliquus. The soil surface also harboured distinct heterotrophic bacterial and fungal communities in the presence of light, in particular, the selection for the phylum Firmicutes. However, these light driven changes in bacterial community structure did not extend to the underlying soil suggesting a discrete zone of influence, analogous to the rhizosphere.

  12. Fire modifies the phylogenetic structure of soil bacterial co-occurrence networks.

    PubMed

    Pérez-Valera, Eduardo; Goberna, Marta; Faust, Karoline; Raes, Jeroen; García, Carlos; Verdú, Miguel

    2017-01-01

    Fire alters ecosystems by changing the composition and community structure of soil microbes. The phylogenetic structure of a community provides clues about its main assembling mechanisms. While environmental filtering tends to reduce the community phylogenetic diversity by selecting for functionally (and hence phylogenetically) similar species, processes like competitive exclusion by limiting similarity tend to increase it by preventing the coexistence of functionally (and phylogenetically) similar species. We used co-occurrence networks to detect co-presence (bacteria that co-occur) or exclusion (bacteria that do not co-occur) links indicative of the ecological interactions structuring the community. We propose that inspecting the phylogenetic structure of co-presence or exclusion links allows to detect the main processes simultaneously assembling the community. We monitored a soil bacterial community after an experimental fire and found that fire altered its composition, richness and phylogenetic diversity. Both co-presence and exclusion links were more phylogenetically related than expected by chance. We interpret such a phylogenetic clustering in co-presence links as a result of environmental filtering, while that in exclusion links reflects competitive exclusion by limiting similarity. This suggests that environmental filtering and limiting similarity operate simultaneously to assemble soil bacterial communities, widening the traditional view that only environmental filtering structures bacterial communities. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  13. Light Structures Phototroph, Bacterial and Fungal Communities at the Soil Surface

    PubMed Central

    Davies, Lawrence O.; Schäfer, Hendrik; Marshall, Samantha; Bramke, Irene; Oliver, Robin G.; Bending, Gary D.

    2013-01-01

    The upper few millimeters of soil harbour photosynthetic microbial communities that are structurally distinct from those of underlying bulk soil due to the presence of light. Previous studies in arid zones have demonstrated functional importance of these communities in reducing soil erosion, and enhancing carbon and nitrogen fixation. Despite being widely distributed, comparative understanding of the biodiversity of the soil surface and underlying soil is lacking, particularly in temperate zones. We investigated the establishment of soil surface communities on pasture soil in microcosms exposed to light or dark conditions, focusing on changes in phototroph, bacterial and fungal communities at the soil surface (0–3 mm) and bulk soil (3–12 mm) using ribosomal marker gene analyses. Microbial community structure changed with time and structurally similar phototrophic communities were found at the soil surface and in bulk soil in the light exposed microcosms suggesting that light can influence phototroph community structure even in the underlying bulk soil. 454 pyrosequencing showed a significant selection for diazotrophic cyanobacteria such as Nostoc punctiforme and Anabaena spp., in addition to the green alga Scenedesmus obliquus. The soil surface also harboured distinct heterotrophic bacterial and fungal communities in the presence of light, in particular, the selection for the phylum Firmicutes. However, these light driven changes in bacterial community structure did not extend to the underlying soil suggesting a discrete zone of influence, analogous to the rhizosphere. PMID:23894406

  14. Effect of copper exposure on bacterial community structure and function in the sediments of Jiaozhou Bay, China.

    PubMed

    Zhao, Yang-Guo; Feng, Gong; Bai, Jie; Chen, Min; Maqbool, Farhana

    2014-07-01

    Microcosms were setup to investigate the possible impact of copper exposure on bacterial community structure and function in sediments of Jiaozhou Bay, China, by culture-independent microbial ecological techniques and community-level physiological profiling. Bacterial 16S rDNA libraries indicated that proportion of the bacteria in phyla Chloroflexi and Acidobacteria decreased, but that of Gammaproteobacteria and Planctomycetes slightly increased in copper-treated sediment. Denaturing gradient gel profiles showed that bacterial communities in control and copper exposed sediments developed into different directions, while the copper exposure did not change the pattern of ammonia oxidizing bacterial community. Microbial community-level physiological profiling revealed an obvious response to copper dosage. The copper pollution caused an acute decrease of carbon utilizing ability as well as bacterial functional diversity; the number of culturable heterotrophic bacteria was reduced by 90%. This study demonstrated that high copper input would obviously reduce culturable bacterial counts and seriously impact bacterial community function in marine sediments.

  15. Acquisition and structuring of midgut bacterial communities in gypsy moth (Lepidoptera: Erebidae) larvae.

    PubMed

    Mason, Charles J; Raffa, Kenneth F

    2014-06-01

    Insects are associated with a diversity of bacteria that colonize their midguts. The extent to which these communities reflect maternal transmission, environmental acquisition, and subsequent structuring by the extreme conditions within the insect gut are poorly understood in many species. We used gypsy moth (Lymantria dispar L.) as a model to investigate interactions between egg mass and environmental sources of bacteria on larval midgut communities. Egg masses were collected from several wild and laboratory populations, and the effects of diet, initial egg mass community, and internal host environment were evaluated using 454 16S-rRNA gene pyrosequencing. Wild populations were highly diverse, while laboratory-maintained egg masses were associated with few operational taxonomic units. As larvae developed, their midgut bacterial communities became more similar to each other and the consumed diet despite initial differences in egg mass-associated bacteria. Subsequent experiments revealed that while midgut membership was more similar to bacteria associated with diet than with egg mass-associated bacteria, we were unable to detect distinct, persistent differences attributable to specific host plants. The differences between foliar communities and midgut communities of larvae that ingested them were owing to relative changes in populations of several bacteria phylotypes. We conclude that gypsy moth has a relatively characteristic midgut bacterial community that is reflective of, but ultimately distinct from, its foliar diet. This work demonstrates that environmental acquisition of diverse microbes can lead to similar midgut bacterial assemblages, underscoring the importance of host physiological environment in structuring bacterial communities.

  16. Effects of Heavy Fuel Oil on the Bacterial Community Structure of a Pristine Microbial Mat▿

    PubMed Central

    Bordenave, Sylvain; Goñi-Urriza, María Soledad; Caumette, Pierre; Duran, Robert

    2007-01-01

    The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by “Bacilli” and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution. PMID:17704271

  17. Interactions between accumulated copper, bacterial community structure and histamine levels in crayfish meat during storage.

    PubMed

    Soedarini, Bernadeta; van Gestel, Cornelis A M; van Straalen, Nico M; Widianarko, Budi; Röling, Wilfred F M

    2014-08-01

    Pollution in aquaculture areas may negatively impact edible species and threaten seafood quality and safety. The aim of this study was to determine the interaction between copper and bacteria in the aquatic habitat and their impact upon crustaceans. Marbled crayfish was chosen as a model of aquatic crustaceans and the influence of metal contamination on bacterial community structure in water used to culture crayfish and in crayfish themselves was investigated. Histamine, an allergen commonly formed by certain groups of bacteria in crustacean edible tissue during storage, was also determined. Copper exposure increased its concentration in crayfish meat by 17.4%, but the copper concentration remained within acceptable food safety limits. Elevated copper levels affected the bacterial community both in the water used to cultivate crayfish and in the marbled crayfish themselves. Cluster analysis of 16S rRNA-gene based microbial community fingerprints revealed that copper impacted the bacterial community in the water and in the crayfish meat. However, copper exposure reduced the formation of histamine in crayfish meat during storage by 66.3%. Copper from the habitat appears to reduce histamine accumulation in crayfish meat during storage by affecting the bacterial community structure of the cultivation water and most likely also in the intestine of the crayfish. From a food safety point of view, copper treatment during the aqua culturing of crustaceans has a positive impact on the postharvest stage. © 2013 Society of Chemical Industry.

  18. Effects of heavy fuel oil on the bacterial community structure of a pristine microbial mat.

    PubMed

    Bordenave, Sylvain; Goñi-Urriza, María Soledad; Caumette, Pierre; Duran, Robert

    2007-10-01

    The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by "Bacilli" and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution.

  19. Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase-an Essential Enzyme of the Polyamine Metabolism.

    PubMed

    Krossa, Sebastian; Faust, Annette; Ober, Dietrich; Scheidig, Axel J

    2016-01-18

    The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states.

  20. Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase–an Essential Enzyme of the Polyamine Metabolism

    PubMed Central

    Krossa, Sebastian; Faust, Annette; Ober, Dietrich; Scheidig, Axel J.

    2016-01-01

    The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states. PMID:26776105

  1. Characterization of soil bacterial community structure and physicochemical properties in created and natural wetlands.

    PubMed

    Peralta, Rita M; Ahn, Changwoo; Gillevet, Patrick M

    2013-01-15

    We used multi-tag pyrosequencing of 16S ribosomal DNA to characterize bacterial communities of wetland soils collected from created and natural wetlands located in the Virginia piedmont. Soils were also evaluated for their physicochemical properties [i.e., percent moisture, pH, soil organic matter (SOM), total organic carbon (TOC), total nitrogen (TN), and C:N ratio]. Soil moisture varied from 15% up to 55% among the wetlands. Soil pH ranged between 4.2 and 5.8, showing the typical characteristic of acidic soils in the Piedmont region. Soil organic matter contents ranged from 3% up to 6%. Soil bacterial community structures and their differences between the wetlands were distinguished by pyrosequencing. Soil bacterial communities in the created wetlands were less dissimilar to each other than to those of either natural wetland, with little difference in diversity (Shannon's H') between created and natural wetlands, except one natural wetland consistently showing a lower H'. The greatest difference of bacterial community structure was observed between the two natural wetlands (R=0.937, p<0.05), suggesting these two natural wetlands were actually quite different reflecting differences in their soil physicochemistry. The major phylogenic groups of all soils included Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatinomadetes, Nitrospira, and Proteobacteria with Proteobacteria being the majority of the community composition. Acidobacteria group was more abundant in natural wetlands than in created wetlands. We found a significant association between bacterial community structures and physicochemical properties of soils such as C:N ratio (ρ=0.43, p<0.01) and pH (ρ=0.39, p<0.01). The outcomes of the study show that the development of ecological functions, mostly mediated by microbial communities, is connected with the development of soil properties in created wetlands. Soil properties should be carefully monitored to examine the progress of

  2. Application of Bioorganic Fertilizer Significantly Increased Apple Yields and Shaped Bacterial Community Structure in Orchard Soil.

    PubMed

    Wang, Lei; Li, Jing; Yang, Fang; E, Yaoyao; Raza, Waseem; Huang, Qiwei; Shen, Qirong

    2017-02-01

    Application of bioorganic fertilizers has been reported to improve crop yields and change soil bacterial community structure; however, little work has been done in apple orchard soils where the biological properties of the soils are being degraded due to long-term application of chemical fertilizers. In this study, we used Illumina-based sequencing approach to characterize the bacterial community in the 0-60-cm soil profile under different fertilizer regimes in the Loess Plateau. The experiment includes three treatments: (1) control without fertilization (CK); (2) application of chemical fertilizer (CF); and (3) application of bioorganic fertilizer and organic-inorganic mixed fertilizer (BOF). The results showed that the treatment BOF increased the apple yields by 114 and 67 % compared to the CK and CF treatments, respectively. The treatment BOF also increased the soil organic matter (SOM) by 22 and 16 % compared to the CK and CF treatments, respectively. The Illumina-based sequencing showed that Acidobacteria and Proteobacteria were the predominant phyla and Alphaproteobacteria and Gammaproteobacteria were the most abundant classes in the soil profile. The bacterial richness for ACE was increased after the addition of BOF. Compared to CK and CF treatments, BOF-treated soil revealed higher abundance of Proteobacteria, Alphaproteobacteria and Gammaproteobacteria, Rhizobiales, and Xanthomonadales while Acidobacteria, Gp7, Gp17, and Sphaerobacter were found in lower abundance throughout the soil profile. Bacterial community structure varied with soil depth under different fertilizer treatments, e.g., the bacterial richness, diversity, and the relative abundance of Verruccomicrobia, Candidatus Brocadiales, and Skermanella were decreased with the soil depth in all three treatments. Permutational multivariate analysis showed that the fertilizer regime was the major factor than soil depth in the variations of the bacterial community composition. Two groups, Lysobacter

  3. Structure and function of the bacterial decapping enzyme NudC.

    PubMed

    Höfer, Katharina; Li, Sisi; Abele, Florian; Frindert, Jens; Schlotthauer, Jasmin; Grawenhoff, Julia; Du, Jiamu; Patel, Dinshaw J; Jäschke, Andres

    2016-09-01

    RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD-decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric, as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5'-terminal nucleotide. The enzyme strongly prefers NAD-linked RNA (NAD-RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.

  4. Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae.

    PubMed Central

    Cross, S L; Smith, M M

    1988-01-01

    The haploid genome of Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 gene pairs, termed the copy I and copy II loci. The structures of the mRNA transcripts from each of these four genes were examined by nuclease protection and primer extension mapping. For each gene, several species of mRNAs were identified that differed in the lengths of their 5' and 3' untranslated regions. The cell cycle accumulation pattern of the H3 and H4 mRNAs was determined in cells from early-exponential-growth cultures fractionated by centrifugal elutriation. The RNA transcripts from all four genes were regulated with the cell division cycle, and transcripts from the nonallelic gene copies showed tight temporal coordination. Cell cycle regulation did not depend on selection of a particular histone mRNA transcript since the ratio of the multiple species from each gene remained the same across the division cycle. Quantitative measurements showed significant differences in the amounts of mRNA expressed from the two nonallelic gene sets. The mRNAs from the copy II H3 and H4 genes were five to seven times more abundant than the mRNAs from the copy I genes. There was no dosage compensation in the steady-state levels of mRNA when either set of genes was deleted. In particular, there was no increase in the amount of copy I H3 or H4 transcripts in cells in which the high-abundance copy II genes were deleted. Images PMID:3280973

  5. TMV mutants with poly(A) tracts of different lengths demonstrate structural variations in 3′UTR affecting viral RNAs accumulation and symptom expression

    PubMed Central

    Guo, Song; Kierzek, Elzbieta; Chen, Gang; Zhou, Yi-Jun; Wong, Sek-Man

    2015-01-01

    The upstream pseudoknots domain (UPD) of Tobacco mosaic virus (TMV) is located at the 3′-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3′-UTR, and the effects of introduced RNA elements in TMV 3′-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3′-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in N. benthamiana protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3′-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in N. benthamiana. In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3′UTR lead to structural variations that affect virus accumulation and symptom expression. PMID:26678425

  6. TMV mutants with poly(A) tracts of different lengths demonstrate structural variations in 3'UTR affecting viral RNAs accumulation and symptom expression.

    PubMed

    Guo, Song; Kierzek, Elzbieta; Chen, Gang; Zhou, Yi-Jun; Wong, Sek-Man

    2015-12-18

    The upstream pseudoknots domain (UPD) of Tobacco mosaic virus (TMV) is located at the 3'-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3'-UTR, and the effects of introduced RNA elements in TMV 3'-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3'-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in N. benthamiana protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3'-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in N. benthamiana. In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3'UTR lead to structural variations that affect virus accumulation and symptom expression.

  7. Carbohydrate structure database merged from bacterial, archaeal, plant and fungal parts

    PubMed Central

    Toukach, Philip V.; Egorova, Ksenia S.

    2016-01-01

    The Carbohydrate Structure Databases (CSDBs, http://csdb.glycoscience.ru) store structural, bibliographic, taxonomic, NMR spectroscopic, and other data on natural carbohydrates and their derivatives published in the scientific literature. The CSDB project was launched in 2005 for bacterial saccharides (as BCSDB). Currently, it includes two parts, the Bacterial CSDB and the Plant&Fungal CSDB. In March 2015, these databases were merged to the single CSDB. The combined CSDB includes information on bacterial and archaeal glycans and derivatives (the coverage is close to complete), as well as on plant and fungal glycans and glycoconjugates (almost all structures published up to 1998). CSDB is regularly updated via manual expert annotation of original publications. Both newly annotated data and data imported from other databases are manually curated. The CSDB data are exportable in a number of modern formats, such as GlycoRDF. CSDB provides additional services for simulation of 1H, 13C and 2D NMR spectra of saccharides, NMR-based structure prediction, glycan-based taxon clustering and other. PMID:26286194

  8. Carbohydrate structure database merged from bacterial, archaeal, plant and fungal parts.

    PubMed

    Toukach, Philip V; Egorova, Ksenia S

    2016-01-04

    The Carbohydrate Structure Databases (CSDBs, http://csdb.glycoscience.ru) store structural, bibliographic, taxonomic, NMR spectroscopic, and other data on natural carbohydrates and their derivatives published in the scientific literature. The CSDB project was launched in 2005 for bacterial saccharides (as BCSDB). Currently, it includes two parts, the Bacterial CSDB and the Plant&Fungal CSDB. In March 2015, these databases were merged to the single CSDB. The combined CSDB includes information on bacterial and archaeal glycans and derivatives (the coverage is close to complete), as well as on plant and fungal glycans and glycoconjugates (almost all structures published up to 1998). CSDB is regularly updated via manual expert annotation of original publications. Both newly annotated data and data imported from other databases are manually curated. The CSDB data are exportable in a number of modern formats, such as GlycoRDF. CSDB provides additional services for simulation of (1)H, (13)C and 2D NMR spectra of saccharides, NMR-based structure prediction, glycan-based taxon clustering and other.

  9. Soil Bacterial Community Structure Responses to Precipitation Reduction and Forest Management in Forest Ecosystems across Germany

    PubMed Central

    Felsmann, Katja; Baudis, Mathias; Gimbel, Katharina; Kayler, Zachary E.; Ellerbrock, Ruth; Bruehlheide, Helge; Bruckhoff, Johannes; Welk, Erik; Puhlmann, Heike; Weiler, Markus; Gessler, Arthur; Ulrich, Andreas

    2015-01-01

    Soil microbial communities play an important role in forest ecosystem functioning, but how climate change will affect the community composition and consequently bacterial functions is poorly understood. We assessed the effects of reduced precipitation with the aim of simulating realistic future drought conditions for one growing season on the bacterial community and its relation to soil properties and forest management. We manipulated precipitation in beech and conifer forest plots managed at different levels of intensity in three different regions across Germany. The precipitation reduction decreased soil water content across the growing season by between 2 to 8% depending on plot and region. T-RFLP analysis and pyrosequencing of the 16S rRNA gene were used to study the total soil bacterial community and its active members after six months of precipitation reduction. The effect of reduced precipitation on the total bacterial community structure was negligible while significant effects could be observed for the active bacteria. However, the effect was secondary to the stronger influence of specific soil characteristics across the three regions and management selection of overstorey tree species and their respective understorey vegetation. The impact of reduced precipitation differed between the studied plots; however, we could not determine the particular parameters being able to modify the response of the active bacterial community among plots. We conclude that the moderate drought induced by the precipitation manipulation treatment started to affect the active but not the total bacterial community, which points to an adequate resistance of the soil microbial system over one growing season. PMID:25875835

  10. Mineral Types and Tree Species Determine the Functional and Taxonomic Structures of Forest Soil Bacterial Communities

    PubMed Central

    Colin, Y.; Nicolitch, O.; Turpault, M.-P.

    2016-01-01

    ABSTRACT Although minerals represent important soil constituents, their impact on the diversity and structure of soil microbial communities remains poorly documented. In this study, pure mineral particles with various chemistries (i.e., obsidian, apatite, and calcite) were considered. Each mineral type was conditioned in mesh bags and incubated in soil below different tree stands (beech, coppice with standards, and Corsican pine) for 2.5 years to determine the relative impacts of mineralogy and mineral weatherability on the taxonomic and functional diversities of mineral-associated bacterial communities. After this incubation period, the minerals and the surrounding bulk soil were collected to determine mass loss and to perform soil analyses, enzymatic assays, and cultivation-dependent and -independent analyses. Notably, our 16S rRNA gene pyrosequencing analyses revealed that after the 2.5-year incubation period, the mineral-associated bacterial communities strongly differed from those of the surrounding bulk soil for all tree stands considered. When focusing only on minerals, our analyses showed that the bacterial communities associated with calcite, the less recalcitrant mineral type, significantly differed from those that colonized obsidian and apatite minerals. The cultivation-dependent analysis revealed significantly higher abundances of effective mineral-weathering bacteria on the most recalcitrant minerals (i.e., apatite and obsidian). Together, our data showed an enrichment of Betaproteobacteria and effective mineral-weathering bacteria related to the Burkholderia and Collimonas genera on the minerals, suggesting a key role for these taxa in mineral weathering and nutrient cycling in nutrient-poor forest ecosystems. IMPORTANCE Forests are usually developed on nutrient-poor and rocky soils, while nutrient-rich soils have been dedicated to agriculture. In this context, nutrient recycling and nutrient access are key processes in such environments. Deciphering

  11. Mineral Types and Tree Species Determine the Functional and Taxonomic Structures of Forest Soil Bacterial Communities.

    PubMed

    Colin, Y; Nicolitch, O; Turpault, M-P; Uroz, S

    2017-03-01

    Although minerals represent important soil constituents, their impact on the diversity and structure of soil microbial communities remains poorly documented. In this study, pure mineral particles with various chemistries (i.e., obsidian, apatite, and calcite) were considered. Each mineral type was conditioned in mesh bags and incubated in soil below different tree stands (beech, coppice with standards, and Corsican pine) for 2.5 years to determine the relative impacts of mineralogy and mineral weatherability on the taxonomic and functional diversities of mineral-associated bacterial communities. After this incubation period, the minerals and the surrounding bulk soil were collected to determine mass loss and to perform soil analyses, enzymatic assays, and cultivation-dependent and -independent analyses. Notably, our 16S rRNA gene pyrosequencing analyses revealed that after the 2.5-year incubation period, the mineral-associated bacterial communities strongly differed from those of the surrounding bulk soil for all tree stands considered. When focusing only on minerals, our analyses showed that the bacterial communities associated with calcite, the less recalcitrant mineral type, significantly differed from those that colonized obsidian and apatite minerals. The cultivation-dependent analysis revealed significantly higher abundances of effective mineral-weathering bacteria on the most recalcitrant minerals (i.e., apatite and obsidian). Together, our data showed an enrichment of Betaproteobacteria and effective mineral-weathering bacteria related to the Burkholderia and Collimonas genera on the minerals, suggesting a key role for these taxa in mineral weathering and nutrient cycling in nutrient-poor forest ecosystems.IMPORTANCE Forests are usually developed on nutrient-poor and rocky soils, while nutrient-rich soils have been dedicated to agriculture. In this context, nutrient recycling and nutrient access are key processes in such environments. Deciphering how soil

  12. Rewiring two-component signal transduction with small RNAs.

    PubMed

    Göpel, Yvonne; Görke, Boris

    2012-04-01

    Bacterial two-component systems (TCSs) and small regulatory RNAs (sRNAs) form densely interconnected networks that integrate and transduce information from the environment into fine-tuned changes of gene expression. Many TCSs control target genes indirectly through regulation of sRNAs, which in turn regulate gene expression by base-pairing with mRNAs or targeting a protein. Conversely, sRNAs may control TCS synthesis, thereby recruiting the TCS regulon to other regulatory networks. Several TCSs control expression of multiple homologous sRNAs providing the regulatory networks with further flexibility. These sRNAs act redundantly, additively or hierarchically on targets. The regulatory speed of sRNAs and their unique features in gene regulation make them ideal players extending the flexibility, dynamic range or timing of TCS signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

    PubMed Central

    Nakashima, Nobutaka; Miyazaki, Kentaro

    2014-01-01

    Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence) target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering. PMID:24552876

  14. From structure to function of bacterial chromosomes: Evolutionary perspectives and ideas for new experiments.

    PubMed

    Lagomarsino, Marco Cosentino; Espéli, Olivier; Junier, Ivan

    2015-10-07

    The link between chromosome structure and function is a challenging open question because chromosomes in vivo are highly dynamic and arduous to manipulate. Here, we examine several promising approaches to tackle this question specifically in bacteria, by integrating knowledge from different sources. Toward this end, we first provide a brief overview of experimental tools that have provided insights into the description of the bacterial chromosome, including genetic, biochemical and fluorescence microscopy techniques. We then explore the possibility of using comparative genomics to isolate functionally important features of chromosome organization, exploiting the fact that features shared between phylogenetically distant bacterial species reflect functional significance. Finally, we discuss possible future perspectives from the field of experimental evolution. Specifically, we propose novel experiments in which bacteria could be screened and selected on the basis of the structural properties of their chromosomes. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Biodegradation of antibiotic ciprofloxacin: pathways, influential factors, and bacterial community structure.

    PubMed

    Liao, Xiaobin; Li, Bingxin; Zou, Rusen; Dai, Yu; Xie, Shuguang; Yuan, Baoling

    2016-04-01

    Antibiotic ciprofloxacin is ubiquitous in the environment. However, little is known about ciprofloxacin dissipation by microbial community. The present study investigated the biodegradation potential of ciprofloxacin by mixed culture and the influential factors and depicted the structure of ciprofloxacin-degrading microbial community. Both the original microbiota from drinking water biofilter and the microbiota previously acclimated to high levels of ciprofloxacin could utilize ciprofloxacin as sole carbon and nitrogen sources, while the acclimated microbiota had a much stronger removal capacity. Temperature rise and the presence of carbon or nitrogen sources favored ciprofloxacin biodegradation. Many novel biotransformation products were identified, and four different metabolic pathways for ciprofloxacin were proposed. Bacterial community structure illustrated a profound shift with ciprofloxacin biodegradation. The ciprofloxacin-degrading bacterial community was mainly composed of classes Gammaproteobacteria, Bacteroidia, and Betaproteobacteria. Microorganisms from genera Pseudoxanthomonas, Stenotrophomonas, Phenylobacterium, and Leucobacter might have links with the dissipation of ciprofloxacin. This work can provide some new insights towards ciprofloxacin biodegradation.

  16. Algal exudates and stream organic matter influence the structure and function of denitrifying bacterial communities.

    PubMed

    Kalscheur, Kathryn N; Rojas, Miguel; Peterson, Christopher G; Kelly, John J; Gray, Kimberly A

    2012-11-01

    Within aquatic ecosystems, periphytic biofilms can be hot spots of denitrification, and previous work has suggested that algal taxa within periphyton can influence the species composition and activity of resident denitrifying bacteria. This study tested the hypothesis that algal species composition within biofilms influences the structure and function of associated denitrifying bacterial communities through the composition of organic exudates. A mixed population of bacteria was incubated with organic carbon isolated from one of seven algal species or from one of two streams that differed in anthropogenic inputs. Pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) revealed differences in the organic composition of algal exudates and stream waters, which, in turn, selected for distinct bacterial communities. Organic carbon source had a significant effect on potential denitrification rates (DNP) of the communities, with organics isolated from a stream with high anthropogenic inputs resulting in a bacterial community with the highest DNP. There was no correlation between DNP and numbers of denitrifiers (based on nirS copy numbers), but there was a strong relationship between the species composition of denitrifier communities (as indicated by tag pyrosequencing of nosZ genes) and DNP. Specifically, the relative abundance of Pseudomonas stutzeri-like nosZ sequences across treatments correlated significantly with DNP, and bacterial communities incubated with organic carbon from the stream with high anthropogenic inputs had the highest relative abundance of P. stutzeri-like nosZ sequences. These results demonstrate a significant relationship between bacterial community composition and function and provide evidence of the potential impacts of anthropogenic inputs on the structure and function of stream microbial communities.

  17. Structural Modifications of Bacterial Lipopolysaccharide that Facilitate Gram-Negative Bacteria Evasion of Host Innate Immunity

    PubMed Central

    Matsuura, Motohiro

    2013-01-01

    Bacterial lipopolysaccharide (LPS), a cell wall component characteristic of Gram-negative bacteria, is a representative pathogen-associated molecular pattern that allows mammalian cells to recognize bacterial invasion and trigger innate immune responses. The polysaccharide moiety of LPS primary plays protective roles for bacteria such as prevention from complement attacks or camouflage with common host carbohydrate residues. The lipid moiety, termed lipid A, is recognized by the Toll-like receptor 4 (TLR4)/MD-2 complex, which transduces signals for activation of host innate immunity. The basic structure of lipid A is a glucosamine disaccharide substituted by phosphate groups and acyl groups. Lipid A with six acyl groups (hexa-acylated form) has been indicated to be a strong stimulator of the TLR4/MD-2 complex. This type of lipid A is conserved among a wide variety of Gram-negative bacteria, and those bacteria are easily recognized by host cells for activation of defensive innate immune responses. Modifications of the lipid A structure to less-acylated forms have been observed in some bacterial species, and those forms are poor stimulators of the TLR4/MD-2 complex. Such modifications are thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. This hypothesis is supported by studies of Yersinia pestis LPS, which contains hexa-acylated lipid A when the bacterium grows at 27°C (the temperature of the vector flea), and shifts to contain less-acylated forms when grown at the human body temperature of 37°C. This alteration of lipid A forms following transmission of Y. pestis from fleas to humans contributes predominantly to the virulence of this bacterium over other virulence factors. A similar role for less-acylated lipid A forms has been indicated in some other bacterial species, such as Francisella tularensis, Helicobacter pylori, and Porphyromonas gingivalis, and further studies to explore this concept are expected. PMID

  18. Structural Modifications of Bacterial Lipopolysaccharide that Facilitate Gram-Negative Bacteria Evasion of Host Innate Immunity.

    PubMed

    Matsuura, Motohiro

    2013-01-01

    Bacterial lipopolysaccharide (LPS), a cell wall component characteristic of Gram-negative bacteria, is a representative pathogen-associated molecular pattern that allows mammalian cells to recognize bacterial invasion and trigger innate immune responses. The polysaccharide moiety of LPS primary plays protective roles for bacteria such as prevention from complement attacks or camouflage with common host carbohydrate residues. The lipid moiety, termed lipid A, is recognized by the Toll-like receptor 4 (TLR4)/MD-2 complex, which transduces signals for activation of host innate immunity. The basic structure of lipid A is a glucosamine disaccharide substituted by phosphate groups and acyl groups. Lipid A with six acyl groups (hexa-acylated form) has been indicated to be a strong stimulator of the TLR4/MD-2 complex. This type of lipid A is conserved among a wide variety of Gram-negative bacteria, and those bacteria are easily recognized by host cells for activation of defensive innate immune responses. Modifications of the lipid A structure to less-acylated forms have been observed in some bacterial species, and those forms are poor stimulators of the TLR4/MD-2 complex. Such modifications are thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. This hypothesis is supported by studies of Yersinia pestis LPS, which contains hexa-acylated lipid A when the bacterium grows at 27°C (the temperature of the vector flea), and shifts to contain less-acylated forms when grown at the human body temperature of 37°C. This alteration of lipid A forms following transmission of Y. pestis from fleas to humans contributes predominantly to the virulence of this bacterium over other virulence factors. A similar role for less-acylated lipid A forms has been indicated in some other bacterial species, such as Francisella tularensis, Helicobacter pylori, and Porphyromonas gingivalis, and further studies to explore this concept are expected.

  19. Bacterial community structure and functional potential in the northeastern Chukchi Sea

    NASA Astrophysics Data System (ADS)

    McFarlin, Kelly M.; Questel, Jennifer M.; Hopcroft, Russell R.; Leigh, Mary Beth

    2017-03-01

    We performed a molecular microbial ecological analysis in the northeastern Chukchi Sea in order to characterize bacterial community structure and genetic potential for biogeochemical cycling and oil biodegradation in a region targeted for oil and gas exploration (Burger lease area). Samples were collected from the surface, middle (20 m), and bottom (2-3 m above seafloor) of the water column during the open-water season of August and September 2012 at 17 different locations. We determined bacterial community structure with 16S rRNA genes sequencing and detected functional genes, including an array of oil biodegradation and biogeochemical cycling (carbon, nitrogen and phosphorus cycling) genes, using the GeoChip 5.0 microarray, and then correlated molecular data to contextual physical and biogeochemical factors. Bacterial community structure differed significantly by depth (surface water vs. bottom water) and between sampling dates (August vs. September). While the relative abundance of major functional gene categories did not differ with depth, the abundance of individual functional genes for carbon cycling, nitrogen cycling, organic contaminant remediation, phosphorus cycling, sulfur cycling, virulence, and viruses differed between surface and bottom seawater samples. Aerobic oil degradation genes and taxa known to include oil-degrading bacteria were found at all three depths. These findings support previous observations that two different water masses contribute to a stratified water column in the summer open-water season of the Burger lease area, but indicate that potential function is fairly similar with depth despite differences in temperature, water chemistry, bacterial community structure, and individual functional gene alleles.

  20. Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

    SciTech Connect

    Maltz, Lauren

    2015-08-27

    By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure of the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β- lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes

  1. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus.

    PubMed

    Matsui, Takashi; Yamane, Junji; Mogi, Nobuyuki; Yamaguchi, Hiroto; Takemoto, Hiroshi; Yao, Min; Tanaka, Isao

    2012-09-01

    FtsZ is a key molecule in bacterial cell division. In the presence of GTP, it polymerizes into tubulin-like protofilaments by head-to-tail association. Protofilaments of FtsZ seem to adopt a straight or a curved conformation in relation to the bound nucleotide. However, although several bacterial and archaeal FtsZ structures have been determined, all of the structures reported previously are considered to have a curved conformation. In this study, structures of FtsZ from Staphylococcus aureus (SaFtsZ) were determined in apo, GDP-bound and inhibitor-complex forms and it was found that SaFtsZ undergoes marked conformational changes. The accumulated evidence suggests that the GDP-bound structure has the features of the straight form. The structural change between the curved and straight forms shows intriguing similarity to the eukaryotic cytoskeletal protein tubulin. Furthermore, the structure of the apo form showed an unexpectedly large conformational change in the core region. FtsZ has also been recognized as a novel target for antibacterial drugs. The structure of the complex with the inhibitor PC190723, which has potent and selective antistaphylococcal activity, indicated that the inhibitor binds at the cleft between the two subdomains.

  2. Changes in Bacterial and Eukaryotic Community Structure after Mass Lysis of Filamentous Cyanobacteria Associated with Viruses†

    PubMed Central

    van Hannen, Erik J.; Zwart, Gabriel; van Agterveld, Miranda P.; Gons, Herman J.; Ebert, Jeannine; Laanbroek, Hendrikus J.

    1999-01-01

    During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities. PMID:9925618

  3. Bacterial community structure and activity in different Cd-treated forest soils.

    PubMed

    Lazzaro, Anna; Hartmann, Martin; Blaser, Peter; Widmer, Franco; Schulin, Rainer; Frey, Beat

    2006-11-01

    In this study we compared indicators of Cd bioavailability (water extracts, Lakanen extracts, free ions) and ecotoxicity in forest soils with contrasting physico-chemical characteristics. Soil samples were treated with CdCl(2) solutions (0, 0.1, 1, 10 and 100 mM) and incubated for 30 days. Microbial activity indexes (acid phosphatase, beta-glucosidase, basal respiration) and changes in bacterial community structure using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting were investigated. The Cd concentrations measured ranged from 1% to 37% of the total additions in water extracts, to higher levels in Lakanen extracts. Effects of Cd were observed at bioavailable concentrations exceeding United Nations/European Economic Commission UN/ECE guidelines for total Cd in the soil solution. Basal respiration was the most affected index, while enzymatic activities showed variable responses to the Cd treatments. We also noticed that soils with pH higher than 6.7 and clay content higher than 50% showed inhibition of basal respiration but no marked shift in bacterial community structure. Soils with lower pH (pH <5.8) with less clay content (<50%) showed in addition strong changes in the bacterial community structure. Our results provide evidence for the importance of relating the effects of Cd on the soil communities to soil properties and to bioavailability.

  4. The Structure and Function of the Gram-Positive Bacterial RNA Degradosome.

    PubMed

    Cho, Kyu Hong

    2017-01-01

    The RNA degradosome is a highly structured protein complex responsible for bulk RNA decay in bacteria. The main components of the complex, ribonucleases, an RNA helicase, and glycolytic enzymes are well-conserved in bacteria. Some components of the degradosome are essential for growth and the disruption of degradosome formation causes slower growth, indicating that this complex is required for proper cellular function. The study of the Escherichia coli degradosome has been performed extensively for the last several decades and has revealed detailed information on its structure and function. On the contrary, the Gram-positive bacterial degradosome, which contains ribonucleases different from the E. coli one, has been studied only recently. Studies on the Gram-positive degradosome revealed that its major component RNase Y was necessary for the full virulence of medically important Gram-positive bacterial pathogens, suggesting that it could be a target of antimicrobial therapy. This review describes the structures and function of Gram-positive bacterial RNA degradosomes, especially those of a Gram-positive model organism Bacillus subtilis, and two important Gram-positive pathogens, Staphylococcus aureus and Streptococcus pyogenes.

  5. The Structure and Function of the Gram-Positive Bacterial RNA Degradosome

    PubMed Central

    Cho, Kyu Hong

    2017-01-01

    The RNA degradosome is a highly structured protein complex responsible for bulk RNA decay in bacteria. The main components of the complex, ribonucleases, an RNA helicase, and glycolytic enzymes are well-conserved in bacteria. Some components of the degradosome are essential for growth and the disruption of degradosome formation causes slower growth, indicating that this complex is required for proper cellular function. The study of the Escherichia coli degradosome has been performed extensively for the last several decades and has revealed detailed information on its structure and function. On the contrary, the Gram-positive bacterial degradosome, which contains ribonucleases different from the E. coli one, has been studied only recently. Studies on the Gram-positive degradosome revealed that its major component RNase Y was necessary for the full virulence of medically important Gram-positive bacterial pathogens, suggesting that it could be a target of antimicrobial therapy. This review describes the structures and function of Gram-positive bacterial RNA degradosomes, especially those of a Gram-positive model organism Bacillus subtilis, and two important Gram-positive pathogens, Staphylococcus aureus and Streptococcus pyogenes. PMID:28217125

  6. Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

    PubMed

    Gu, Huan; Hou, Shuyu; Yongyat, Chanokpon; De Tore, Suzanne; Ren, Dacheng

    2013-09-03

    Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

  7. Structural, Surface, in vitro Bacterial Adhesion and Biofilm Formation Analysis of Three Dental Restorative Composites

    PubMed Central

    Azam, Maria T.; Khan, Abdul S.; Muzzafar, Danish; Faryal, Rani; Siddiqi, Saadat A.; Ahmad, Riaz; Chauhdry, Aqif A.; Rehman, Ihtesham U.

    2015-01-01

    This study was conducted to investigate the relationship between dental materials and bacterial adhesion on the grounds of their chemical composition and physical properties. Three commercially available dental restorative materials (Filtek™Z350, Filtek™P90 and Spectrum®TPH®) were structurally analyzed and their wettability and surface roughness were evaluated by using Fourier Transform Infrared Spectroscopy, Contact Angle Measurement and Atomic Force Microscopy, respectively. These materials were molded into discs and tested with three bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia) for microbial attachment. The bacterial adhesion was observed at different time intervals, i.e., 0 h, 8 h, 24 h, 48 h and 72 h, along with Colony Forming Unit Count and Optical Density measurement of the media. It was found that all materials showed a degree of conversion with time intervals, i.e., 0 h, 8 h, 24 h, 48 h and 72 h, which led to the availability of functional groups (N–H and C–H) that might promote adhesion. The trend in difference in the extent of bacterial adhesion can be related to particle size, chemical composition and surface wettability of the dental materials.

  8. Shift of bacterial community structure in two Thai soil series affected by silver nanoparticles using ARISA.

    PubMed

    Chunjaturas, Wariya; Ferguson, John A; Rattanapichai, Wutthida; Sadowsky, Michael J; Sajjaphan, Kannika

    2014-07-01

    In this study we examined the influence of silver nanoparticles (SNP) on the bacterial community and microbial processes in two soils from Thailand, a Ayutthaya (Ay) and Kamphaengsaen soil series (Ks). Results of this analysis revealed that SNP did not affect to pH, electrical conductivity, cation exchange capacity, and organic matter in both the Ay and Ks series. Automated ribosomal intergenic spacer analysis (ARISA) analysis profiles showed that bacterial community decreased with increasing SNP concentration. Pearson's correlation coefficient and multidimensional scaling analyses indicated that the effects of SNP on the bacterial community structure depended more on soil types than SNP application rates and incubation periods. Additionally, the results showed that SNP application rates affected on amount of CO2 emissions, while SNP application rates had no effect on N mineralization in both soil types. This study is the first investigation of the effects of SNP on bacterial community using ARISA analysis. Our results might be useful to evaluate the risk associated with the applications of SNP for consumer products and agricultural practices.

  9. Structure and temporal dynamics of the bacterial communities associated to microhabitats of the coral Oculina patagonica.

    PubMed

    Rubio-Portillo, Esther; Santos, Fernando; Martínez-García, Manuel; de Los Ríos, Asunción; Ascaso, Carmen; Souza-Egipsy, Virginia; Ramos-Esplá, Alfonso A; Anton, Josefa

    2016-12-01

    Corals are known to contain a diverse microbiota that plays a paramount role in the physiology and health of holobiont. However, few studies have addressed the variability of bacterial communities within the coral host. In this study, bacterial community composition from the mucus, tissue and skeleton of the scleractinian coral Oculina patagonica were investigated seasonally at two locations in the Western Mediterranean Sea, to further understand how environmental conditions and the coral microbiome structure are related. We used denaturing gradient gel electrophoresis in combination with next-generation sequencing and electron microscopy to characterize the bacterial community. The bacterial communities were significantly different among coral compartments, and coral tissue displayed the greatest changes related to environmental conditions and coral health status. Species belonging to the Rhodobacteraceae and Vibrionaceae families form part of O. patagonica tissues core microbiome and may play significant roles in the nitrogen cycle. Furthermore, sequences related to the coral pathogens, Vibrio mediterranei and Vibrio coralliilyticus, were detected not only in bleached corals but also in healthy ones, even during cold months. This fact opens a new view onto unveiling the role of pathogens in the development of coral diseases in the future. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. Bacterial community structure in freshwater springs infested with the invasive plant species Hydrilla verticillata.

    PubMed

    Gordon-Bradley, N; Li, N; Williams, H N

    2015-01-01

    The phylogenetic composition and physiological profiles of bacterial communities in freshwater springs were evaluated during the blooming and non-blooming stages of the invasive plant species, Hydrilla verticillata. Community-level physiological profiles (CLPPs) and pyrosequencing of 16S rRNA gene amplicons were used to study potential Hydrilla mediated shifts in the physiological potential and phylogenetic composition of the bacterial community in infested systems. The results of CLPP revealed that the microbes in the Hydrilla invaded sites utilized less substrates during blooming periods than during nonblooming periods of the plant. Spearman's rank correlation analysis showed some relationships between the relative abundances of bacterial taxa and the Biolog substrate utilization pattern. The relative abundance of the identified taxa showed some striking differences based on the blooming status of Hydrilla and to a lesser extent on site variation. The relative abundance of Actinobacteria, Bacteriodetes, and Verrucomicrobia was generally higher during Hydrilla blooms, while Deltaproteobacteria was generally higher during non-blooming stages of Hydrilla. The detected genera also varied based on the blooming stages of the plant. Based on the findings, it appears that Hydrilla alters the phylogenetic composition and structure of the bacterial community during the blooming stage.

  11. Bacterial community structure in freshwater springs infested with the invasive plant species Hydrilla verticillata

    PubMed Central

    Gordon-Bradley, N.; Li, N.

    2015-01-01

    The phylogenetic composition and physiological profiles of bacterial communities in freshwater springs were evaluated during the blooming and non-blooming stages of the invasive plant species, Hydrilla verticillata. Community-level physiological profiles (CLPPs) and pyrosequencing of 16S rRNA gene amplicons were used to study potential Hydrilla mediated shifts in the physiological potential and phylogenetic composition of the bacterial community in infested systems. The results of CLPP revealed that the microbes in the Hydrilla invaded sites utilized less substrates during blooming periods than during nonblooming periods of the plant. Spearman’s rank correlation analysis showed some relationships between the relative abundances of bacterial taxa and the Biolog substrate utilization pattern. The relative abundance of the identified taxa showed some striking differences based on the blooming status of Hydrilla and to a lesser extent on site variation. The relative abundance of Actinobacteria, Bacteriodetes, and Verrucomicrobia was generally higher during Hydrilla blooms, while Deltaproteobacteria was generally higher during non-blooming stages of Hydrilla. The detected genera also varied based on the blooming stages of the plant. Based on the findings, it appears that Hydrilla alters the phylogenetic composition and structure of the bacterial community during the blooming stage. PMID:26207069

  12. Structural mechanism for bacterial oxidation of oceanic trimethylamine into trimethylamine N-oxide.

    PubMed

    Li, Chun-Yang; Chen, Xiu-Lan; Zhang, Dian; Wang, Peng; Sheng, Qi; Peng, Ming; Xie, Bin-Bin; Qin, Qi-Long; Li, Ping-Yi; Zhang, Xi-Ying; Su, Hai-Nan; Song, Xiao-Yan; Shi, Mei; Zhou, Bai-Cheng; Xun, Lu-Ying; Chen, Yin; Zhang, Yu-Zhong

    2017-03-01

    Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin-containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm (RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction, FAD is reduced and a C4a-hydroperoxyflavin intermediate forms. In the oxidative half-reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP(+) bends and interacts with D317, shutting off the entrance to create a protected micro-environment for catalysis and exposing C4a-hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP(+) undergoes a conformational change in the oxidative half-reaction of FMOs.

  13. Bacterial community structures associated with a natural spring phytoplankton bloom in the Yellow Sea, China

    NASA Astrophysics Data System (ADS)

    Liu, Min; Xiao, Tian; Sun, Jun; Wei, Hao; Wu, Ying; Zhao, Yuan; Zhang, Wuchang

    2013-12-01

    Bacterial community structures associated with a spring phytoplankton bloom were investigated using denaturing gradient gel electrophoresis (DGGE) and 16S rDNA clone libraries. Statistical and phylogenetic analyses applied on both molecular methods revealed differences in bacterial community composition between the bloom station and post-bloom station, as well as between two bloom stages (bloom- and decay-) at bloom station. At the class level, the bacterial community at the bloom station was dominated by Alphaproteobacteria, Flavobacteria and Gammaproteobacteria, whereas Alphaproteobacteria and Gammaproteobacteria were dominant at the post-bloom station. At order level, no obvious predominant subgroup was found at the post-bloom station. In contrast, predominant subgroups were observed in bloom samples and they changed over the course of bloom. Rhodobacterales (mainly Roseobacter) and Flavobacteriales (mainly Flavobacterium) were the predominant subgroups in the bloom period, whereas Roseobacter became the unique predominant subgroup in the decay-bloom period. Rhodobacterales and Flavobacteriales, which were dominant in the bloom-associated bacterial communities in the Yellow Sea, were also reported as dominant during bloom conditions in other ocean regions, suggesting that they play an important role in bloom events.

  14. Small RNAs in spermatogenesis.

    PubMed

    Yadav, Ram Prakash; Kotaja, Noora

    2014-01-25

    Spermatogenesis is characterized by meiotic divisions and major morphological changes to produce spermatozoa that are capable of independent movement and fertilization of an egg. Male germ cell differentiation is governed by orchestrated, phase-specific gene expression patterns that are tightly controlled at transcriptional and post-transcriptional level. Post-transcriptional regulation of protein-coding mRNAs becomes prominent during the late steps of spermatogenesis when the compacting sperm nucleus becomes transcriptionally inhibited. Small non-coding RNAs are important regulators of gene expression that mainly function post-transcriptionally to control the properties of their target mRNAs. Male germ cells express several classes of small RNAs, including Dicer-dependent microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), as well as Dicer-independent piwi-interacting RNAs (piRNAs). Increasing evidence supports the essential role of small RNA-mediated RNA regulation in normal spermatogenesis and male fertility.

  15. Complete structure of the bacterial flagellar hook reveals extensive set of stabilizing interactions

    PubMed Central

    Matsunami, Hideyuki; Barker, Clive S.; Yoon, Young-Ho; Wolf, Matthias; Samatey, Fadel A.

    2016-01-01

    The bacterial flagellar hook is a tubular helical structure made by the polymerization of multiple copies of a protein, FlgE. Here we report the structure of the hook from Campylobacter jejuni by cryo-electron microscopy at a resolution of 3.5 Å. On the basis of this structure, we show that the hook is stabilized by intricate inter-molecular interactions between FlgE molecules. Extra domains in FlgE, found only in Campylobacter and in related bacteria, bring more stability and robustness to the hook. Functional experiments suggest that Campylobacter requires an unusually strong hook to swim without its flagella being torn off. This structure reveals details of the quaternary organization of the hook that consists of 11 protofilaments. Previous study of the flagellar filament of Campylobacter by electron microscopy showed its quaternary structure made of seven protofilaments. Therefore, this study puts in evidence the difference between the quaternary structures of a bacterial filament and its hook. PMID:27811912

  16. Probing Induced Structural Changes in Biomimetic Bacterial Cell Membrane Interactions with Divalent Cations

    SciTech Connect

    Holt, Allison M; Standaert, Robert F; Jubb, Aaron M; Katsaras, John; Johs, Alexander

    2017-01-01

    Biological membranes, formed primarily by the self-assembly of complex mixtures of phospholipids, provide a structured scaffold for compartmentalization and structural processes in living cells. The specific physical properties of phospholipid species present in a given membrane play a key role in mediating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipid present in bacterial, yeast, and mammalian cell membranes, is exceptional. In addition to undergoing the standard lipid polymorphic transition between the gel and liquid-crystalline phase, it can also assume an unusual polymorphic state, the inverse hexagonal phase (HII). Divalent cations are among the factors that drive the formation of the HII phase, wherein the lipid molecules form stacked tubular structures by burying the hydrophilic head groups and exposing the hydrophobic tails to the bulk solvent. Most biological membranes contain a lipid species capable of forming the HII state suggesting that such lipid polymorphic structural states play an important role in structural biological processes such as membrane fusion. In this study, the interactions between Mg2+ and biomimetic bacterial cell membranes composed of PE and phosphatidylglycerol (PG) were probed using differential scanning calorimetry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectroscopy. The lipid phase transitions were examined at varying ratios of PE to PG and upon exposure to physiologically relevant concentrations of Mg2+. An understanding of these basic interactions enhances our understanding of membrane dynamics and how membrane-mediated structural changes may occur in vivo.

  17. Wheat and Rice Growth Stages and Fertilization Regimes Alter Soil Bacterial Community Structure, But Not Diversity.

    PubMed

    Wang, Jichen; Xue, Chao; Song, Yang; Wang, Lei; Huang, Qiwei; Shen, Qirong

    2016-01-01

    Maintaining soil fertility and the microbial communities that determine fertility is critical to sustainable agricultural strategies, and the use of different organic fertilizer (OF) regimes represents an important practice in attempts to preserve soil quality. However, little is known about the dynamic response of bacterial communities to fertilization regimes across crop growth stages. In this study, we examined microbial community structure and diversity across eight representative growth stages of wheat-rice rotation under four different fertilization treatments: no nitrogen fertilizer (NNF), chemical fertilizer (CF), organic-inorganic mixed fertilizer (OIMF), and OF. Quantitative PCR (QPCR) and high-throughput sequencing of bacterial 16S rRNA gene fragments revealed that growth stage as the best predictor of bacterial community abundance and structure. Additionally, bacterial community compositions differed between wheat and rice rotations. Relative to soils under wheat rotation, soils under rice rotation contained higher relative abundances (RA) of anaerobic and mesophilic microbes and lower RA of aerophilic microbes. With respect to fertilization regime, NNF plots had a higher abundance of nitrogen-fixing Cyanobacteria. OIMF had a lower abundance of ammonia-oxidizing Thaumarchaeota compared with CF. Application of chemical fertilizers (CF and OIMF treatments) significantly increased the abundance of some generally oligotrophic bacteria such those belonging to the Acidobacteria, while more copiotrophic of the phylum Proteobacteria increased with OF application. A high correlation coefficient was found when comparing RA of Acidobacteria based upon QPCR vs. sequence analysis, yet poor correlations were found for the α- and β- Proteobacteria, highlighting the caution required when interpreting these molecular data. In total, crop, fertilization scheme and plant developmental stage all influenced soil microbial community structure, but not total levels of alpha

  18. Wheat and Rice Growth Stages and Fertilization Regimes Alter Soil Bacterial Community Structure, But Not Diversity

    PubMed Central

    Wang, Jichen; Xue, Chao; Song, Yang; Wang, Lei; Huang, Qiwei; Shen, Qirong

    2016-01-01

    Maintaining soil fertility and the microbial communities that determine fertility is critical to sustainable agricultural strategies, and the use of different organic fertilizer (OF) regimes represents an important practice in attempts to preserve soil quality. However, little is known about the dynamic response of bacterial communities to fertilization regimes across crop growth stages. In this study, we examined microbial community structure and diversity across eight representative growth stages of wheat-rice rotation under four different fertilization treatments: no nitrogen fertilizer (NNF), chemical fertilizer (CF), organic–inorganic mixed fertilizer (OIMF), and OF. Quantitative PCR (QPCR) and high-throughput sequencing of bacterial 16S rRNA gene fragments revealed that growth stage as the best predictor of bacterial community abundance and structure. Additionally, bacterial community compositions differed between wheat and rice rotations. Relative to soils under wheat rotation, soils under rice rotation contained higher relative abundances (RA) of anaerobic and mesophilic microbes and lower RA of aerophilic microbes. With respect to fertilization regime, NNF plots had a higher abundance of nitrogen–fixing Cyanobacteria. OIMF had a lower abundance of ammonia-oxidizing Thaumarchaeota compared with CF. Application of chemical fertilizers (CF and OIMF treatments) significantly increased the abundance of some generally oligotrophic bacteria such those belonging to the Acidobacteria, while more copiotrophic of the phylum Proteobacteria increased with OF application. A high correlation coefficient was found when comparing RA of Acidobacteria based upon QPCR vs. sequence analysis, yet poor correlations were found for the α- and β- Proteobacteria, highlighting the caution required when interpreting these molecular data. In total, crop, fertilization scheme and plant developmental stage all influenced soil microbial community structure, but not total levels of

  19. Nanopods: a new bacterial structure and mechanism for deployment of outer membrane vesicles.

    PubMed

    Shetty, Ameesha; Chen, Shicheng; Tocheva, Elitza I; Jensen, Grant J; Hickey, William J

    2011-01-01

    Bacterial outer membrane vesicles (OMV) are packets of periplasmic material that, via the proteins and other molecules they contain, project metabolic function into the environment. While OMV production is widespread in proteobacteria, they have been extensively studied only in pathogens, which inhabit fully hydrated environments. However, many (arguably most) bacterial habitats, such as soil, are only partially hydrated. In the latter, water is characteristically distributed as films on soil particles that are, on average thinner, than are typical OMV (ca. ≤10 nm water film vs. 20 to >200 nm OMV;). We have identified a new bacterial surface structure, termed a "nanopod", that is a conduit for projecting OMV significant distances (e.g., ≥6 µm) from the cell. Electron cryotomography was used to determine nanopod three-dimensional structure, which revealed chains of vesicles within an undulating, tubular element. By using immunoelectron microscopy, proteomics, heterologous expression and mutagenesis, the tubes were determined to be an assembly of a surface layer protein (NpdA), and the interior structures identified as OMV. Specific metabolic function(s) for nanopods produced by Delftia sp. Cs1-4 are not yet known. However, a connection with phenanthrene degradation is a possibility since nanopod formation was induced by growth on phenanthrene. Orthologs of NpdA were identified in three other genera of the Comamonadaceae family, and all were experimentally verified to form nanopods. Nanopods are new bacterial organelles, and establish a new paradigm in the mechanisms by which bacteria effect long-distance interactions with their environment. Specifically, they create a pathway through which cells can effectively deploy OMV, and the biological activity these transmit, in a diffusion-independent manner. Nanopods would thus allow environmental bacteria to expand their metabolic sphere of influence in a manner previously unknown for these organisms.

  20. Structure of bacterial communities in soil following cover crop and organic fertilizer incorporation.

    PubMed

    Fernandez, Adria L; Sheaffer, Craig C; Wyse, Donald L; Staley, Christopher; Gould, Trevor J; Sadowsky, Michael J

    2016-11-01

    Incorporation of organic material into soils is an important element of organic farming practices that can affect the composition of the soil bacterial communities that carry out nutrient cycling and other functions crucial to crop health and growth. We conducted a field experiment to determine the effects of cover crops and fertilizers on bacterial community structure in agricultural soils under long-term organic management. Illumina sequencing of 16S rDNA revealed diverse communities comprising 45 bacterial phyla in corn rhizosphere and bulk field soil. Community structure was most affected by location and by the rhizosphere effect, followed by sampling time and amendment treatment. These effects were associated with soil physicochemical properties, including pH, moisture, organic matter, and nutrient levels. Treatment differences were apparent in bulk and rhizosphere soils at the time of peak corn growth in the season following cover crop and fertilizer application. Cover crop and fertilizer treatments tended to lower alpha diversity in early season samples. However, winter rye, oilseed radish, and buckwheat cover crop treatments increased alpha diversity in some later season samples compared to a no-amendment control. Fertilizer treatments and some cover crops decreased relative abundance of members of the ammonia-oxidizing family Nitrosomonadaceae. Pelleted poultry manure and Sustane® (a commercial fertilizer) decreased the relative abundance of Rhizobiales. Our data point to a need for future research exploring how (1) cover crops influence bacterial community structure and functions, (2) these effects differ with biomass composition and quantity, and (3) existing soil conditions and microbial community composition influence how soil microbial populations respond to agricultural management practices.

  1. miRNAs in brain development

    SciTech Connect

    Petri, Rebecca; Malmevik, Josephine; Fasching, Liana; Åkerblom, Malin; Jakobsson, Johan

    2014-02-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs have been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function.

  2. Natural Products at Work: Structural Insights into Inhibition of the Bacterial Membrane Protein MraY.

    PubMed

    Koppermann, Stefan; Ducho, Christian

    2016-09-19

    Natural(ly) fit: The X-ray crystal structure of the bacterial membrane protein MraY in complex with its natural product inhibitor muraymycin D2 is discussed. MraY catalyzes one of the membrane-associated steps in peptidoglycan biosynthesis and, therefore, represents a promising target for novel antibiotics. Structural insights derived from the protein-inhibitor complex might now pave the way for the development of new antimicrobial drugs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

    PubMed Central

    Kirby, A R; Gunning, A P; Morris, V J; Ridout, M J

    1995-01-01

    A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan. Images FIGURE 1 FIGURE 3 FIGURE 4 PMID:7711262

  4. Bacterial community structure on two alpine debris-covered glaciers and biogeography of Polaromonas phylotypes.

    PubMed

    Franzetti, Andrea; Tatangelo, Valeria; Gandolfi, Isabella; Bertolini, Valentina; Bestetti, Giuseppina; Diolaiuti, Guglielmina; D'Agata, Carlo; Mihalcea, Claudia; Smiraglia, Claudio; Ambrosini, Roberto

    2013-08-01

    High-elevation cold environments are considered ideal places to test hypotheses about mechanisms of bacterial colonization and succession, and about bacterial biogeography. Debris-covered glaciers (glaciers whose ablation area is mainly covered by a continuous layer of rock debris fallen from the surrounding mountains) have never been investigated in this respect so far. We used the Illumina technology to analyse the V5 and V6 hypervariable regions of the bacterial 16S rRNA gene amplified from 38 samples collected in July and September 2009 at different distances from the terminus on two debris-covered glaciers (Miage and Belvedere--Italian Alps). Heterotrophic taxa-dominated communities and bacterial community structure changed according to ice ablation rate, organic carbon content of the debris and distance from the glacier terminus. Bacterial communities therefore change during downwards debris transport, and organic carbon of these recently exposed substrates is probably provided more by allochthonous deposition of organic matter than by primary production by autotrophic organisms. We also investigated whether phylotypes of the genus Polaromonas, which is ubiquitous in cold environments, do present a biogeographical distribution by analysing the sequences retrieved in this study together with others available in the literature. We found that the genetic distance among phylotypes increased with geographic distance; however, more focused analyses using discrete distance classes revealed that both sequences collected at sites <100 km and at sites 9400-13,500 km to each other were more similar than those collected at other distance classes. Evidences of biogeographic distribution of Polaromonas phylotypes were therefore contrasting.

  5. Phaeocystis antarctica blooms strongly influence bacterial community structures in the Amundsen Sea polynya

    PubMed Central

    Delmont, Tom O.; Hammar, Katherine M.; Ducklow, Hugh W.; Yager, Patricia L.; Post, Anton F.

    2014-01-01

    Rising temperatures and changing winds drive the expansion of the highly productive polynyas (open water areas surrounded by sea ice) abutting the Antarctic continent. Phytoplankton blooms in polynyas are often dominated by the haptophyte Phaeocystis antarctica, and they generate the organic carbon that enters the resident microbial food web. Yet, little is known about how Phaeocystis blooms shape bacterial community structures and carbon fluxes in these systems. We identified the bacterial communities that accompanied a Phaeocystis bloom in the Amundsen Sea polynya during the austral summers of 2007–2008 and 2010–2011. These communities are distinct from those determined for the Antarctic Circumpolar Current (ACC) and off the Palmer Peninsula. Diversity patterns for most microbial taxa in the Amundsen Sea depended on location (e.g., waters abutting the pack ice near the shelf break and at the edge of the Dotson glacier) and depth, reflecting different niche adaptations within the confines of this isolated ecosystem. Inside the polynya, P. antarctica coexisted with the bacterial taxa Polaribacter sensu lato, a cryptic Oceanospirillum, SAR92 and Pelagibacter. These taxa were dominated by a single oligotype (genotypes partitioned by Shannon entropy analysis) and together contributed up to 73% of the bacterial community. Size fractionation of the bacterial community [<3 μm (free-living bacteria) vs. >3 μm (particle-associated bacteria)] identified several taxa (especially SAR92) that were preferentially associated with Phaeocystis colonies, indicative of a distinct role in Phaeocystis bloom ecology. In contrast, particle-associated bacteria at 250 m depth were enriched in Colwellia and members of the Cryomorphaceae suggesting that they play important roles in the decay of Phaeocystis blooms. PMID:25566197

  6. Impact of Arsenite on the Bacterial Community Structure and Diversity in Soil

    PubMed Central

    Dong, Dian-Tao; Yamamura, Shigeki; Amachi, Seigo

    2016-01-01

    The impact of arsenite (As[III]) on the bacterial community structure and diversity in soil was determined by incubating soil slurries with 50, 500, and 5,000 μM As(III). As(III) was oxidized to arsenate (As[V]), and the microbial contribution to As(III) oxidation was 70–100%. PCR-denaturing gradient gel electrophoresis revealed that soil bacterial diversity decreased in the presence of As(III). Bacteria closely related to the family Bacillaceae were predominant in slurry spiked with 5,000 μM As(III). The population size of culturable As(III)-resistant bacteria was 37-fold higher in this slurry than in unspiked slurry (p < 0.01), indicating that high levels of As(III) stimulate the emergence of As(III)-resistant bacteria. As(III)-resistant bacteria isolated from slurry spiked with 5,000 μM As(III) were mainly affiliated with the genus Bacillus; however, no strains showed As(III)-oxidizing capacity. An As(III)-oxidizing bacterial community analysis based on As(III) oxidase gene (aioA) sequences demonstrated that diversity was the lowest in slurry spiked with 5,000 μM As(III). The deduced AioA sequences affiliated with Alphaproteobacteria accounted for 91–93% of all sequences in this slurry, among which those closely related to Bosea spp. were predominant (48–86%). These results suggest that exposure to high levels of As(III) has a significant impact on the composition and diversity of the soil bacterial community, including the As(III)-oxidizing bacterial community. Certain As(III)-oxidizing bacteria with strong As(III) resistance may be enriched under high As(III) levels, while more sensitive As(III) oxidizers are eliminated under these conditions. PMID:26903368

  7. Bacterial community structure in treated sewage sludge with mesophilic and thermophilic anaerobic digestion.

    PubMed

    Stiborova, Hana; Wolfram, Jan; Demnerova, Katerina; Macek, Tomas; Uhlik, Ondrej

    2015-11-01

    Stabilized sewage sludge is applied to agricultural fields and farmland due to its high organic matter content. The aim of this study was to investigate the effects of two types of sludge stabilization, mesophilic anaerobic digestion (MAD) and thermophilic anaerobic digestion (TAD), on bacterial communities in sludge, including the presence of pathogenic microorganisms. Bacterial community structure and phylogenetic diversity were analyzed in four sewage sludge samples from the Czech Republic. Analysis of 16S ribosomal RNA (rRNA) genes showed that investigated sludge samples harbor diverse bacterial populations with only a few taxa present across all samples. Bacterial diversity was higher in sludge samples after MAD versus TAD treatment, and communities in MAD-treated sludge shared the highest genetic similarities. In all samples, the bacterial community was dominated by reads affiliated with Proteobacteria. The sludge after TAD treatment had considerably higher number of reads of thermotolerant/thermophilic taxa, such as the phyla Deinococcus-Thermus and Thermotogae or the genus Coprothermobacter. Only one operational taxonomic unit (OTU), which clustered with Rhodanobacter, was detected in all communities at a relative abundance >1 %. All of the communities were screened for the presence of 16S rRNA gene sequences of pathogenic bacteria using a database of 122 pathogenic species and ≥98 % identity threshold. The abundance of such sequences ranged between 0.23 and 1.57 % of the total community, with lower numbers present after the TAD treatment, indicating its higher hygienization efficiency. Sequences clustering with nontuberculous mycobacteria were present in all samples. Other detected sequences of pathogenic bacteria included Streptomyces somaliensis, Acinetobacter calcoaceticus, Alcaligenes faecalis, Gordonia spp., Legionella anisa, Bordetella bronchiseptica, Enterobacter aerogenes, Brucella melitensis, and Staphylococcus aureus.

  8. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.

    PubMed

    Bulgarelli, Davide; Rott, Matthias; Schlaeppi, Klaus; Ver Loren van Themaat, Emiel; Ahmadinejad, Nahal; Assenza, Federica; Rauf, Philipp; Huettel, Bruno; Reinhardt, Richard; Schmelzer, Elmon; Peplies, Joerg; Gloeckner, Frank Oliver; Amann, Rudolf; Eickhorst, Thilo; Schulze-Lefert, Paul

    2012-08-02

    The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found

  9. Bacterial community structure in two permafrost wetlands on the Tibetan Plateau and Sanjiang Plain, China.

    PubMed

    Yun, Juanli; Ju, Yiwen; Deng, Yongcui; Zhang, Hongxun

    2014-08-01

    Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10(10) 16S rRNA gene copies per gram of wet soil in both wetlands, with 10(8) pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.

  10. Evidence for selective bacterial community structuring in the freshwater sponge Ephydatia fluviatilis.

    PubMed

    Costa, Rodrigo; Keller-Costa, Tina; Gomes, Newton C M; da Rocha, Ulisses Nunes; van Overbeek, Leo; van Elsas, Jan Dirk

    2013-01-01

    To understand the functioning of sponges, knowledge of the structure of their associated microbial communities is necessary. However, our perception of sponge-associated microbiomes remains mainly restricted to marine ecosystems. Here, we report on the molecular diversity and composition of bacteria in the freshwater sponge Ephydatia fluviatilis inhabiting the artificial lake Vinkeveense Plassen, Utrecht, The Netherlands. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints revealed that the apparent diversities within the domain Bacteria and the phylum Actinobacteria were lower in E. fluviatilis than in bulk water. Enrichment of specific PCR-DGGE bands in E. fluviatilis was detected. Furthermore, sponge- and bulk water-derived bacterial clone libraries differed with respect to bacterial community composition at the phylum level. E. fluviatilis-derived sequences were affiliated with six recognized phyla, i.e., Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, Chlamydiae and Verrucomicrobia, in order of relative abundance; next to the uncultured candidate phylum TM7 and one deeply rooted bacterial lineage of undefined taxonomy (BLUT). Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in the freshwater clone library whereas sequences affiliated with Planctomycetes, Verrucomicrobia, Acidobacteria and Armatimonadetes were found at lower frequencies. Fine-tuned phylogenetic inference showed no or negligible overlaps between the E. fluviatilis and water-derived phylotypes within bacterial taxa such as Alphaproteobacteria, Bacteroidetes and Actinobacteria. We also ascertained the status of two alphaproteobacterial lineages as freshwater sponge-specific phylogenetic clusters, and report on high distinctiveness of other E. fluviatilis specific phylotypes, especially within the Bacteroidetes, Planctomycetes and Chlamydia taxa. This study supports the contention that the composition and

  11. Bacterial community structure on two alpine debris-covered glaciers and biogeography of Polaromonas phylotypes

    PubMed Central

    Franzetti, Andrea; Tatangelo, Valeria; Gandolfi, Isabella; Bertolini, Valentina; Bestetti, Giuseppina; Diolaiuti, Guglielmina; D'Agata, Carlo; Mihalcea, Claudia; Smiraglia, Claudio; Ambrosini, Roberto

    2013-01-01

    High-elevation cold environments are considered ideal places to test hypotheses about mechanisms of bacterial colonization and succession, and about bacterial biogeography. Debris-covered glaciers (glaciers whose ablation area is mainly covered by a continuous layer of rock debris fallen from the surrounding mountains) have never been investigated in this respect so far. We used the Illumina technology to analyse the V5 and V6 hypervariable regions of the bacterial 16S rRNA gene amplified from 38 samples collected in July and September 2009 at different distances from the terminus on two debris-covered glaciers (Miage and Belvedere—Italian Alps). Heterotrophic taxa-dominated communities and bacterial community structure changed according to ice ablation rate, organic carbon content of the debris and distance from the glacier terminus. Bacterial communities therefore change during downwards debris transport, and organic carbon of these recently exposed substrates is probably provided more by allochthonous deposition of organic matter than by primary production by autotrophic organisms. We also investigated whether phylotypes of the genus Polaromonas, which is ubiquitous in cold environments, do present a biogeographical distribution by analysing the sequences retrieved in this study together with others available in the literature. We found that the genetic distance among phylotypes increased with geographic distance; however, more focused analyses using discrete distance classes revealed that both sequences collected at sites <100 km and at sites 9400–13 500 km to each other were more similar than those collected at other distance classes. Evidences of biogeographic distribution of Polaromonas phylotypes were therefore contrasting. PMID:23535918

  12. [Changes of bacterial community structure on reusing domestic sewage of Daoxianghujing Hotel to landscape water].

    PubMed

    Zhu, Jing-nan; Wang, Xiao-dan; Zhai, Zhen-hua; Ma, Wen-lin; Li, Rong-qi; Wang, Xue-lian; Li, Yan-hong

    2010-05-01

    A 16S rDNA library was used to evaluate the bacterial diversity and identify dominant groups of bacteria in different treatment pools in the domestic sewage system of the Beijing Daoxianghujing Hotel. The results revealed that there were many types of bacteria in the hotel domestic sewage, and the bacterial Shannon-Weaver diversity index was 3.12. In addition, epsilon Proteobacteria was found to be the dominant group with the ratio of 32%. In addition, both the CFB phylum, Fusobacteria, gamma Proteobacteria and Firmicutes were also reached to 9%-15%. After treated with the reclaimed water station, the bacterial Shannon-Weaver diversity index was reduced to 2. 41 and beta Proteobacteria became the dominant group and occupied 73% of the total clones. However, following artificial wetland training, the bacterial Shannon-Weaver diversity index in the sample increased to 3.38, Actinobacteria arrived to 33% and became the most dominant group; Cyanobacteria reached to 26%, and was the second dominant group. But, the control sample comprised 38% Cyanobacteria, and mainly involved in Cyanobium, Synechoccus and Microcystis, with ratios of 47.1%, 17.6% and 8.8%, respectively. Some bacteria of Microcystis aenruginosa were also detected, which probably resulted in the light bloom finally. Therefore, the bacterial diversity and community structures changed in response to treatment of the hotel domestic sewage; there was no cyanobacteria bloom explosion in the treated water. This study will aid in investigation the changes of microbial ecology in different types of water and providing the useful information for enhancing the cyanobacteria blooms control from ecological angle.

  13. Diet is the primary determinant of bacterial community structure in the guts of higher termites.

    PubMed

    Mikaelyan, Aram; Dietrich, Carsten; Köhler, Tim; Poulsen, Michael; Sillam-Dussès, David; Brune, Andreas

    2015-10-01

    The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically 'lower termites' are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood-feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus-level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host-specific distribution of the bacterial lineages in termite guts are best explained by diet-related differences in the availability of microhabitats and functional niches.

  14. Effects of sieving, drying and rewetting upon soil bacterial community structure and respiration rates.

    PubMed

    Thomson, Bruce C; Ostle, Nick J; McNamara, Niall P; Whiteley, Andrew S; Griffiths, Robert I

    2010-10-01

    Soil microcosm studies often require some form of soil homogenisation, such as sieving, to provide a representative sample. Frequently, soils are also homogenised following drying and are then rewetted, yet little research has been done to understand how these methods impact upon microbial communities. Here we compared the molecular diversity and functional responses of intact cores from a Scottish grassland soil with homogenised samples prepared by drying, sieving and rewetting or freshly sieving wet soils. Results showed that there was no significant difference in total soil CO(2)-C efflux between the freshly sieved and intact core treatments, however, respiration was significantly higher in the dried and rewetted microcosms. Molecular fingerprinting (T-RFLP) of bacterial communities at two different time-points showed that both homogenisation methods significantly altered bacterial community structure with the largest differences being observed after drying and rewetting. Assessments of responsive taxa in each treatment showed that intact cores were dominated by Acidobacterial peaks whereas an increased relative abundance of Alphaproteobacterial terminal restriction fragments were apparent in both homogenised treatments. However, the shift in community structure was not as large in the freshly sieved soil. Our findings suggest that if soil homogenisation must be performed, then fresh sieving of wet soil is preferable to drying and rewetting in approximating the bacterial diversity and functioning of intact cores.

  15. Assessment of Ruminal Bacterial and Archaeal Community Structure in Yak (Bos grunniens)

    PubMed Central

    Zhou, Zhenming; Fang, Lei; Meng, Qingxiang; Li, Shengli; Chai, Shatuo; Liu, Shujie; Schonewille, Jan Thomas

    2017-01-01

    The aim of this study was to determine the microbial community composition in the rumen of yaks under different feeding regimes. Microbial communities were assessed by sequencing bacterial and archaeal 16S ribosomal RNA gene fragments obtained from yaks (Bos grunniens) from Qinghai-Tibetan Plateau, China. Samples were obtained from 14 animals allocated to either pasture grazing (Graze), a grazing and supplementary feeding regime (GSF), or an indoor feeding regime (Feed). The predominant bacterial phyla across feeding regimes were Bacteroidetes (51.06%) and Firmicutes (32.73%). At genus level, 25 genera were shared across all samples. The relative abundance of Prevotella in the graze and GSF regime group were significantly higher than that in the feed regime group. Meanwhile, the relative abundance of Ruminococcus was lower in the graze group than the feed and GSF regime groups. The most abundant archaeal phylum was Euryarchaeota, which accounted for 99.67% of the sequences. Ten genera were detected across feeding regimes, seven genera were shared by all samples, and the most abundant was genus Methanobrevibacter (91.60%). The relative abundance of the most detected genera were similar across feeding regime groups. Our results suggest that the ruminal bacterial community structure differs across yak feeding regimes while the archaeal community structures are largely similar. PMID:28223980

  16. Responses of bacterial community structure and denitrifying bacteria in biofilm to submerged macrophytes and nitrate

    PubMed Central

    Zhang, Songhe; Pang, Si; Wang, Peifang; Wang, Chao; Guo, Chuan; Addo, Felix Gyawu; Li, Yi

    2016-01-01

    Submerged macrophytes play important roles in constructed wetlands and natural water bodies, as these organisms remove nutrients and provide large surfaces for biofilms, which are beneficial for nitrogen removal, particularly from submerged macrophyte-dominated water columns. However, information on the responses of biofilms to submerged macrophytes and nitrogen molecules is limited. In the present study, bacterial community structure and denitrifiers were investigated in biofilms on the leaves of four submerged macrophytes and artificial plants exposed to two nitrate concentrations. The biofilm cells were evenly distributed on artificial plants but appeared in microcolonies on the surfaces of submerged macrophytes. Proteobacteria was the most abundant phylum in all samples, accounting for 27.3–64.8% of the high-quality bacterial reads, followed by Chloroflexi (3.7–25.4%), Firmicutes (3.0–20.1%), Acidobacteria (2.7–15.7%), Actinobacteria (2.2–8.7%), Bacteroidetes (0.5–9.7%), and Verrucomicrobia (2.4–5.2%). Cluster analysis showed that bacterial community structure can be significantly different on macrophytes versus from those on artificial plants. Redundancy analysis showed that electrical conductivity and nitrate concentration were positively correlated with Shannon index and operational taxonomic unit (OTU) richness (log10 transformed) but somewhat negatively correlated with microbial density. The relative abundances of five denitrifying genes were positively correlated with nitrate concentration and electrical conductivity but negatively correlated with dissolved oxygen. PMID:27782192

  17. The spatial structure of bacterial communities is influenced by historical environmental conditions.

    PubMed

    Andersson, Martin G I; Berga, Mercè; Lindström, Eva S; Langenheder, Silke

    2014-05-01

    The spatial structure of ecological communities, including that of bacteria, is often influenced by species sorting by contemporary environmental conditions. Moreover, historical processes, i.e., ecological and evolutionary events that have occurred at some point in the past, such as dispersal limitation, drift, priority effects, or selection by past environmental conditions, can be important, but are generally investigated much less. Here, we conducted a field study using 16 rock pools, where we specifically compared the importance of past vs. contemporary environmental conditions for bacterial community structure by correlating present differences in bacterial community composition among pools to environmental conditions measured on the same day, as well as to those measured 2, 4, 6, and 8 d earlier. The results prove that selection by past environmental conditions exists, since we were able to show that bacterial communities are, to a greater extent, an imprint of past compared to contemporary environmental conditions. We suggest that this is the result of a combination of different mechanisms, including priority effects that cause rapid adaptation to new environmental conditions of taxa that have been initially selected by past environmental conditions, and slower rates of turnover in community composition compared to environmental conditions.

  18. Comparative analysis of bacterial community structure in the rhizosphere of maize by high-throughput pyrosequencing

    PubMed Central

    Ye, Boping

    2017-01-01

    In this study, we designed a microcosm experiment to explore the composition of the bacterial community in the rhizosphere of maize and bulk soil by sequencing the V3-V4 region of the 16S rRNA gene on the Illumina system. 978–1239 OTUs (cut off level of 3%) were found in rhizosphere and bulk soil samples. Rhizosphere shared features with the bulk soil, such as predominance of Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes and TM7. At genus level, many of the dominant rhizosphere genera (Chitinophaga, Nitrospira, Flavobacterium, etc.) displayed different patterns of temporal changes in the rhizosphere as opposed to the bulk soil, showing rhizosphere has more impact on soil microorganisms. Besides, we found that significant growth-related dynamic changes in bacterial community structure were mainly associated with phylum Bacteroidetes, Proteobacteria and Actinobacteria (mainly genera Burkholderia, Flavisolibacter and Pseudomonas), indicating that different growth stages affected the bacterial community composition in maize soil. Furthermore, some unique genera in especial Plant-Growth Promoting Rhizobacteria (PGPR) such as Nonomuraea, Thiobacillus and Bradyrhizobium etc., which were beneficial for the plant growth appeared to be more abundant in the rhizosphere than bulk soil, indicating that the selectivity of root to rhizosphere microbial is an important mechanism leading to the differences in the bacteria community structure between rhizosphere and bulk soil. PMID:28542542

  19. The structure of resting bacterial populations in soil and subsoil permafrost.

    PubMed

    Soina, Vera S; Mulyukin, Andrei L; Demkina, Elena V; Vorobyova, Elena A; El-Registan, Galina I

    2004-01-01

    The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.

  20. The Structure of Resting Bacterial Populations in Soil and Subsoil Permafrost

    NASA Astrophysics Data System (ADS)

    Soina, Vera S.; Mulyukin, Andrei L.; Demkina, Elena V.; Vorobyova, Elena A.; El-Registan, Galina I.

    2004-09-01

    The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-sporeforming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.

  1. Comparative analysis of bacterial community structure in the rhizosphere of maize by high-throughput pyrosequencing.

    PubMed

    Yang, Yi; Wang, Na; Guo, Xinyan; Zhang, Yi; Ye, Boping

    2017-01-01

    In this study, we designed a microcosm experiment to explore the composition of the bacterial community in the rhizosphere of maize and bulk soil by sequencing the V3-V4 region of the 16S rRNA gene on the Illumina system. 978-1239 OTUs (cut off level of 3%) were found in rhizosphere and bulk soil samples. Rhizosphere shared features with the bulk soil, such as predominance of Acidobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes and TM7. At genus level, many of the dominant rhizosphere genera (Chitinophaga, Nitrospira, Flavobacterium, etc.) displayed different patterns of temporal changes in the rhizosphere as opposed to the bulk soil, showing rhizosphere has more impact on soil microorganisms. Besides, we found that significant growth-related dynamic changes in bacterial community structure were mainly associated with phylum Bacteroidetes, Proteobacteria and Actinobacteria (mainly genera Burkholderia, Flavisolibacter and Pseudomonas), indicating that different growth stages affected the bacterial community composition in maize soil. Furthermore, some unique genera in especial Plant-Growth Promoting Rhizobacteria (PGPR) such as Nonomuraea, Thiobacillus and Bradyrhizobium etc., which were beneficial for the plant growth appeared to be more abundant in the rhizosphere than bulk soil, indicating that the selectivity of root to rhizosphere microbial is an important mechanism leading to the differences in the bacteria community structure between rhizosphere and bulk soil.

  2. Responses of bacterial community structure and denitrifying bacteria in biofilm to submerged macrophytes and nitrate

    NASA Astrophysics Data System (ADS)

    Zhang, Songhe; Pang, Si; Wang, Peifang; Wang, Chao; Guo, Chuan; Addo, Felix Gyawu; Li, Yi

    2016-10-01

    Submerged macrophytes play important roles in constructed wetlands and natural water bodies, as these organisms remove nutrients and provide large surfaces for biofilms, which are beneficial for nitrogen removal, particularly from submerged macrophyte-dominated water columns. However, information on the responses of biofilms to submerged macrophytes and nitrogen molecules is limited. In the present study, bacterial community structure and denitrifiers were investigated in biofilms on the leaves of four submerged macrophytes and artificial plants exposed to two nitrate concentrations. The biofilm cells were evenly distributed on artificial plants but appeared in microcolonies on the surfaces of submerged macrophytes. Proteobacteria was the most abundant phylum in all samples, accounting for 27.3-64.8% of the high-quality bacterial reads, followed by Chloroflexi (3.7-25.4%), Firmicutes (3.0-20.1%), Acidobacteria (2.7-15.7%), Actinobacteria (2.2-8.7%), Bacteroidetes (0.5-9.7%), and Verrucomicrobia (2.4-5.2%). Cluster analysis showed that bacterial community structure can be significantly different on macrophytes versus from those on artificial plants. Redundancy analysis showed that electrical conductivity and nitrate concentration were positively correlated with Shannon index and operational taxonomic unit (OTU) richness (log10 transformed) but somewhat negatively correlated with microbial density. The relative abundances of five denitrifying genes were positively correlated with nitrate concentration and electrical conductivity but negatively correlated with dissolved oxygen.

  3. Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

    PubMed Central

    Crow, Allister; Race, Paul R.; Jubelin, Grégory; Varela Chavez, Carolina; Escoubas, Jean-Michel; Oswald, Eric; Banfield, Mark J.

    2009-01-01

    A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins. PMID:19440549

  4. Crystal structures of Cif from bacterial pathogens Photorhabdus luminescens and Burkholderia pseudomallei.

    PubMed

    Crow, Allister; Race, Paul R; Jubelin, Grégory; Varela Chavez, Carolina; Escoubas, Jean-Michel; Oswald, Eric; Banfield, Mark J

    2009-01-01

    A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a gamma-proteobacterium) and human pathogen Burkholderia pseudomallei (a beta-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the 'tail-domain' (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.

  5. Crystal structure of SmcL, a bacterial neutral sphingomyelinase C from Listeria.

    PubMed

    Openshaw, Amy E A; Race, Paul R; Monzó, Hector J; Vázquez-Boland, José-Antonio; Banfield, Mark J

    2005-10-14

    Sphingomyelinases C are enzymes that catalyze the hydrolysis of sphingomyelin in biological membranes to ceramide and phosphorylcholine. Various pathogenic bacteria produce secreted neutral sphingomyelinases C that act as membrane-damaging virulence factors. Mammalian neutral sphingomyelinases C, which display sequence homology to the bacterial enzymes, are involved in sphingolipid metabolism and signaling. This article describes the first structure to be determined for a member of the neutral sphingomyelinase C family, SmcL, from the intracellular bacterial pathogen Listeria ivanovii. The structure has been refined to 1.9-A resolution with phases derived by single isomorphous replacement with anomalous scattering techniques from a single iridium derivative. SmcL adopts a DNase I-like fold, and is the first member of this protein superfamily to have its structure determined that acts as a phospholipase. The structure reveals several unique features that adapt the protein to its phospholipid substrate. These include large hydrophobic beta-hairpin and hydrophobic loops surrounding the active site that may bind and penetrate the lipid bilayer to position sphingomyelin in a catalytically competent position. The structure also provides insight into the proposed general base/acid catalytic mechanism, in which His-325 and His-185 play key roles.