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IMPORTANCE: Shigella species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. Shigella virulence phenotypes are controlled by the transcriptional regulator, VirB. We show that VirB belongs to a fast-evolving, plasmid-borne clade of the ParB superfamily, which has diverged from versions with a distinct cellular role-DNA partitioning. We report that, like classic members of the ParB family, VirB binds a highly unusual ligand, CTP. Mutants predicted to be defective in CTP binding are compromised in a variety of virulence attributes controlled by VirB, likely because these mutants cannot engage DNA. This study (i) reveals that VirB binds CTP, (ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, (iii) provides new insight into VirB-CTP-DNA interactions, and (iv) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many bacteria.
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Proteínas de Ligação a DNA , Shigella , Virulência/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Ligantes , Shigella flexneri , Shigella/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
In September 2022, an international summer course entitled 'The new microbiology' took place in Greece, on the island of Spetses. The organizers aimed to highlight the spectacular advances and the renaissance occurring in Microbiology, driven by developments in genomics, proteomics, imaging techniques, and bioinformatics. Combinations of these advances allow for single cell analyses, rapid and relatively inexpensive metagenomic and transcriptomic data analyses and comparisons, visualization of previously unsuspected mechanisms, and large-scale studies. A 'New Microbiology' is emerging which allows studies that address the critical roles of microbes in health and disease, in humans, animals, and the environment. The concept of one health is now transforming microbiology. The goal of the course was to discuss all these topics with members of the new generation of microbiologists all of whom were highly motivated and fully receptive.
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Undergraduate students often struggle to understand the basics of bacterial gene regulation, a key concept in microbiology. They find it hard to visualize the architecture of a bacterial operon or how the gene, RNA, and protein components interact with each other to regulate the operon. To better visualize the molecular interactions, students engaged in a role-playing exercise on bacterial gene regulation in the classroom. Before beginning the activity, they received a shortened, traditional lecture on the architecture and function of the lac operon under "on" and "off" conditions. Students chose one or more placards detailing a molecular role (such as promoter, repressor, RNA polymerase, gene X, gene Y, etc.). Upon receiving instructor prompts, they assembled in linear order to mimic correct genomic locations of genes and regulatory elements on the operon. When given a prompt for "operon on" or "operon off" condition, students identified all the necessary components (roles) for that condition, assembled in the correct order, and then moved through the assembled operon to mimic what happens inside the cell under that condition. Students were tested before and after the activity using a set of eight multiple-choice questions. Students showed significant gains in their ability to answer these questions correctly immediately after the activity. More importantly, the improved understanding was also reflected in a high median score on summative assessments given a few weeks after the completion of the activity. This activity can also be readily adapted to online or a hybrid mode of teaching to benefit larger student populations.
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Genetically identical bacterial cells commonly display different phenotypes. This phenotypic heterogeneity is well known for stress responses, where it is often explained as bet hedging against unpredictable environmental threats. Here, we explore phenotypic heterogeneity in a major stress response of Escherichia coli and find it has a fundamentally different basis. We characterize the response of cells exposed to hydrogen peroxide (H2O2) stress in a microfluidic device under constant growth conditions. A machine-learning model reveals that phenotypic heterogeneity arises from a precise and rapid feedback between each cell and its immediate environment. Moreover, we find that the heterogeneity rests upon cell-cell interaction, whereby cells shield each other from H2O2 via their individual stress responses. Our work shows how phenotypic heterogeneity in bacterial stress responses can emerge from short-range cell-cell interactions and result in a collective phenotype that protects a large proportion of the population.
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Peróxido de Hidrogênio , Estresse Oxidativo , Peróxido de Hidrogênio/toxicidade , Fenótipo , Comunicação Celular , Oxirredução , Bactérias/genéticaRESUMO
Riboswitches are a class of RNA motifs in the untranslated regions of bacterial messenger RNAs (mRNAs) that can adopt different conformations to regulate gene expression. The binding of specific small molecule or ion ligands, or other RNAs, influences the conformation the riboswitch adopts. Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) offers an approach for probing this structural isomerization, or conformational switching, at the level of single mRNA molecules. SiM-KARTS utilizes fluorescently labeled, short, sequence-complementary DNA or RNA oligonucleotide probes that transiently access a specific RNA conformation over another. Binding and dissociation to a surface-immobilized target RNA of arbitrary length are monitored by Total Internal Reflection Fluorescence Microscopy (TIRFM) and quantitatively analyzed, via spike train and burst detection, to elucidate the rate constants of isomerization, revealing mechanistic insights into riboswitching.
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Riboswitch , DNA Complementar , Cinética , Ligantes , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos , Sondas RNA , RNA Bacteriano/metabolismoRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for thousands of deaths every year in the United States. P. aeruginosa virulence factor production is mediated by quorum sensing, a mechanism of bacterial cell-cell communication that relies on the production and detection of signal molecules called autoinducers. In P. aeruginosa, the transcription factor receptor RhlR is activated by a RhlI-synthesized autoinducer. We recently showed that RhlR-dependent transcription is enhanced by a physical interaction with the enzyme PqsE via increased affinity of RhlR for promoter DNA. However, the molecular basis for complex formation and how complex formation enhanced RhlR transcriptional activity remained unclear. Here, we report the structure of ligand-bound RhlR in complex with PqsE. Additionally, we determined the structure of the complex bound with DNA, revealing the mechanism by which RhlR-mediated transcription is enhanced by PqsE, thereby establishing the molecular basis for RhlR-dependent virulence factor production in P. aeruginosa.
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Pseudomonas aeruginosa , Percepção de Quorum , Percepção de Quorum/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The serine protease autotransporters of the Enterobacteriaceae (SPATEs) are a large family of virulence factors commonly found in enteric bacteria. These secreted virulence factors have diverse functions during bacterial infection, including adhesion, aggregation and cell toxicity. One such SPATE, the Pic mucinase (protein involved in colonisation) cleaves mucin, allowing enteric bacterial cells to utilise mucin as a carbon source and to penetrate the gut mucus lining, thereby increasing mucosal colonisation. The pic gene is widely distributed within the Enterobacteriaceae, being found in human pathogens, such as enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC) and Shigella flexneri 2a. As the pic promoter regions from EAEC strain 042 and UPEC strain CFT073 differ, we have investigated the regulation of each promoter. Here, using in vivo and in vitro techniques, we show that both promoters are activated by the global transcription factor, CRP (cyclic AMP receptor protein), but the architectures of the EAEC and the UPEC pic promoter differ. Expression from both pic promoters is repressed by the nucleoid-associated factor, Fis, and maximal promoter activity occurs when cells are grown in minimal medium. As CRP activates transcription in conditions of nutrient depletion, whilst Fis levels are maximal in nutrient-rich environments, the regulation of the EAEC and UPEC pic promoters is consistent with Pic's nutritional role in scavenging mucin as a suitable carbon source during colonisation and infection.
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Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Serina Endopeptidases , Escherichia coli Uropatogênica , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mucinas/metabolismo , Regiões Promotoras Genéticas , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis, a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.
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There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements.
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Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/genética , Inativação Gênica , Heterocromatina/genética , Fator Proteico 1 do Hospedeiro/genética , Prófagos/genética , Bacillus subtilis , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/virologia , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismoRESUMO
Primary σ70 factors are key conserved bacterial regulatory proteins that interact with regulatory DNA to control gene expression. It is, however, poorly understood whether σ70 sequence diversity in different bacteria reflects functional differences. Here, we employ comparative and functional genomics to explore the sequence and function relationship of primary σ70. Using multiplex automated genome engineering and deep sequencing (MAGE-seq), we generate a saturation mutagenesis library and high-resolution fitness map of E. coli σ70 in domains 2-4. Mapping natural σ70 sequence diversity to the E. coli σ70 fitness landscape reveals significant predicted fitness deficits across σ70 orthologs. Interestingly, these predicted deficits are larger than observed fitness changes for 15 σ70 orthologs introduced into E. coli. Finally, we use a multiplexed transcriptional reporter assay and RNA sequencing (RNA-seq) to explore functional differences of several σ70 orthologs. This work provides an in-depth analysis of σ70 sequence and function to improve efforts to understand the evolution and engineering potential of this global regulator.
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Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Mapeamento Cromossômico , Dissecação , Escherichia coli/genética , Genômica/métodos , Regiões Promotoras Genéticas/genéticaRESUMO
Sulfate Transport Anti-Sigma antagonist domains (Pfam01740) are found in all branches of life, from eubacteria to mammals, as a conserved fold encoded by highly divergent amino acid sequences. These domains are present as part of larger SLC26/SulP anion transporters, where the STAS domain is associated with transmembrane anchoring of the larger multidomain protein. Here, we focus on STAS Domain only Proteins (SDoPs) in eubacteria, initially described as part of the Bacillus subtilis Regulation of Sigma B (RSB) regulatory system. Since their description in B. subtilis, SDoPs have been described to be involved in the regulation of sigma factors, through partner-switching mechanisms in various bacteria such as: Mycobacterium. tuberculosis, Listeria. monocytogenes, Vibrio. fischeri, Bordetella bronchiseptica, among others. In addition to playing a canonical role in partner-switching with an anti-sigma factor to affect the availability of a sigma factor, several eubacterial SDoPs show additional regulatory roles compared to the original RSB system of B. subtilis. This is of great interest as these proteins are highly conserved, and often involved in altering gene expression in response to changes in environmental conditions. For many of the bacteria we will examine in this review, the ability to sense environmental changes and alter gene expression accordingly is critical for survival and colonization of susceptible hosts.
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Proteínas de Transporte de Ânions , Genes Bacterianos , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Imidazóis , Estrutura Terciária de Proteína , Fator sigma/genéticaRESUMO
Pseudomonas syringae can rapidly deploy specialized functions to deal with abiotic and biotic stresses. Host niches pose specific sets of environmental challenges driven, in part, by immune defenses. Bacteria use a "just-in-time" strategy of gene regulation, meaning that they only produce the functions necessary for survival as needed. Extracytoplasmic function (ECF) sigma factors transduce a specific set of environmental signals and change gene expression patterns by altering RNA polymerase promoter specificity, to adjust bacterial physiology, structure, or behavior, singly or in combination, to improve chances of survival. The broadly conserved ECF sigma factor AlgU affects virulence in both animal and plant pathogens. Pseudomonas syringae AlgU controls expression of more than 800 genes, some of which contribute to suppression of plant immunity and bacterial fitness in plants. This review discusses AlgU activation mechanisms, functions controlled by AlgU, and how these functions contribute to P. syringae survival in plants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.
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Pseudomonas syringae , Fator sigma , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas syringae/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico , VirulênciaRESUMO
Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children's Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, blaCTX-M and blaTEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.
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Antibacterianos/farmacologia , Diarreia/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biofilmes , Pré-Escolar , Egito , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Genes Bacterianos , Genoma Bacteriano , Humanos , Lactente , Virulência , Fatores de Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by more than 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.
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Sistemas CRISPR-Cas/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/efeitos dos fármacos , Evolução Molecular Direcionada , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Genes Reporter/efeitos dos fármacos , Genes Reporter/genética , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , Alinhamento de Sequência , Software , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd ) for the SvWhiD-σHrdB4 complex as â¼0.7 µm, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8-10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.
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Proteínas de Bactérias , Proteínas de Ligação a DNA , Fator sigma , Streptomyces , Fatores de Transcrição , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Fator sigma/química , Fator sigma/metabolismo , Streptomyces/química , Streptomyces/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5'-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.
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In this paper, we propose a computational strategy for performing genome-wide analyses of intergenic sequences in bacterial genomes. Following similar directions of a previous paper, where a method for genome-wide analysis of eucaryotic Intergenic sequences was proposed, here we developed a tool for implementing similar concepts in bacteria genomes. This allows us to (i) classify intergenic sequences into clusters, characterized by specific global structural features and (ii) draw possible relations with their functional features.
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DNA Intergênico/genética , Regulação Bacteriana da Expressão Gênica , Genômica/métodos , Análise de Sequência de DNA/métodos , Software , Análise por Conglomerados , DNA Intergênico/química , Genoma BacterianoRESUMO
A wide variety of mechanisms that control gene expression in bacteria are based on conditional transcription termination. Generally, in these mechanisms, a transcription terminator is located between a promoter and a downstream gene(s), and the efficiency of the terminator is controlled by a regulatory effector that can be a metabolite, protein, or RNA. The most common type of regulation involving conditional termination is transcription attenuation, in which the primary regulatory target is an essential element of a single terminator. The terminator can be either intrinsic or Rho dependent, with each presenting unique regulatory targets. Transcription attenuation mechanisms can be divided into five classes based primarily on the manner in which transcription termination is rendered conditional. This review summarizes each class of control mechanisms from a historical perspective, describes important examples in a physiological context and the current state of knowledge, highlights major advances, and discusses expectations of future discoveries.
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Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Bactérias/metabolismo , Escherichia coli/genética , Genes Bacterianos , RNA Bacteriano/genética , Proteínas de Ligação a RNA , RiboswitchRESUMO
Riboswitches are dynamic RNA motifs that are mostly embedded in the 5'-untranslated regions of bacterial mRNAs, where they regulate gene expression transcriptionally or translationally by undergoing conformational changes upon binding of a small metabolite or ion. Due to the small size of typical ligands, relatively little free energy is available from ligand binding to overcome the often high energetic barrier of reshaping RNA structure. Instead, most riboswitches appear to take advantage of the directional and hierarchical folding of RNA by employing the ligand as a structural 'linchpin' to adjust the kinetic partitioning between alternate folds. In this model, even small, local structural and kinetic effects of ligand binding can cascade into global RNA conformational changes affecting gene expression. Single-molecule (SM) microscopy tools are uniquely suited to study such kinetically controlled RNA folding since they avoid the ensemble averaging of bulk techniques that loses sight of unsynchronized, transient, and/or multi-state kinetic behavior. This review summarizes how SM methods have begun to unravel riboswitch-mediated gene regulation.
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Regulação Bacteriana da Expressão Gênica/genética , Dobramento de RNA/genética , Riboswitch/genética , Imagem Individual de Molécula/métodos , Bactérias/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Cinética , Microscopia de Fluorescência/métodos , Pinças ÓpticasRESUMO
CsrBs are bacterial highly conserved and multiple-copy noncoding small RNAs (sRNAs) that play major roles in cell physiology and virulence. In the Vibrio genus, they are known to be regulated by the two-component system VarS/VarA. They modulate the well-characterized quorum sensing pathway controlling virulence and luminescence in Vibrio cholerae and Vibrio harveyi, respectively. Remarkably, Vibrio tasmaniensis LGP32, an oyster pathogen that belongs to the Splendidus clade, was found to have four copies of csrB, named csrB1-4, compared to two to three copies in other Vibrio species. Here, we show that the extra csrB4 copy results from a csrB3 gene duplication, a characteristic of the Splendidus clade. Interestingly, csrB genes are regulated in different ways in V. tasmaniensis, with csrB1 expression being independent of the VarS/VarA system. We found that a complex regulatory network involving CsrBs, quorum sensing, and the stationary-phase sigma factor σS redundantly but differentially controls the production of two secreted metalloproteases, Vsm and PrtV, the former being a major determinant of the V. tasmaniensis extracellular product toxicity. In particular, we identified a novel VarS/VarA-dependent but CsrB-independent pathway that controls positively both Vsm production and PrtV production as well as rpoS expression. Altogether, our data show that a csrB gene duplication event in V. tasmaniensis supported the evolution of the regulatory network controlling the expression of major toxic secreted metalloproteases, thereby increasing redundancy and enabling the integration of additional input signals.IMPORTANCE The conserved CsrB sRNAs are an example of sibling sRNAs, i.e., sRNAs which are present in multiple copies in genomes. This report illustrates how new copies arise through gene duplication events and highlights two evolutionary advantages of having such multiple copies: differential regulation of the multiple copies allows integration of different input signals into the regulatory network of which they are parts, and the high redundancy that they provide confers a strong robustness to the system.