RESUMO
Pathogenic bacteria employ complex systems to cope with metal ion shortage conditions and propagate in the host. IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum Staphylococcus aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here, we report that IsrR regulates the tricarboxylic acid (TCA) cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop, underscoring the tight control required to prevent aconitase expression. Beyond its canonical enzymatic role, aconitase becomes an RNA-binding protein with regulatory activity in iron-deprived conditions, a feature that is conserved in S. aureus. Aconitase not only negatively regulates its own expression, but also impacts the enzymes involved in both its substrate supply and product utilization. This moonlighting activity concurrently upregulates pyruvate carboxylase expression, allowing it to compensate for the TCA cycle deficiency associated with iron scarcity. These results highlight the cascade of complex posttranscriptional regulations controlling S. aureus central metabolism in response to iron deficiency.
Assuntos
Aconitato Hidratase , Proteínas de Bactérias , Ciclo do Ácido Cítrico , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus , Aconitato Hidratase/metabolismo , Aconitato Hidratase/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/enzimologia , Ciclo do Ácido Cítrico/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ferro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Pequeno RNA não Traduzido/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Deficiências de FerroRESUMO
In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS-silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5' rapid amplification of complementary DNA ends sequencing (5' RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD's ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Salmonella , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Salmonella/genética , Salmonella/patogenicidade , Análise de Célula Única , Transcrição Gênica , Virulência/genéticaRESUMO
Staphylococcus aureus, a human opportunist pathogen, adjusts its metabolism to cope with iron deprivation within the host. We investigated the potential role of small non-coding RNAs (sRNAs) in dictating this process. A single sRNA, named here IsrR, emerged from a competition assay with tagged-mutant libraries as being required during iron starvation. IsrR is iron-repressed and predicted to target mRNAs expressing iron-containing enzymes. Among them, we demonstrated that IsrR down-regulates the translation of mRNAs of enzymes that catalyze anaerobic nitrate respiration. The IsrR sequence reveals three single-stranded C-rich regions (CRRs). Mutational and structural analysis indicated a differential contribution of these CRRs according to targets. We also report that IsrR is required for full lethality of S. aureus in a mouse septicemia model, underscoring its role as a major contributor to the iron-sparing response for bacterial survival during infection. IsrR is conserved among staphylococci, but it is not ortholog to the proteobacterial sRNA RyhB, nor to other characterized sRNAs down-regulating mRNAs of iron-containing enzymes. Remarkably, these distinct sRNAs regulate common targets, illustrating that RNA-based regulation provides optimal evolutionary solutions to improve bacterial fitness when iron is scarce.
Assuntos
RNA Bacteriano , Pequeno RNA não Traduzido , Animais , Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Ferro/metabolismo , Camundongos , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Staphylococcus/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.
Assuntos
RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Animais , Feminino , Regulação Bacteriana da Expressão Gênica , Hemólise , Camundongos , RNA Bacteriano/química , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Virulência/genéticaRESUMO
Bacterial small RNAs (sRNAs) contribute to a variety of regulatory mechanisms that modulate a wide range of pathways, including metabolism, virulence, and antibiotic resistance. We investigated the involvement of sRNAs in rifampicin resistance in the opportunistic pathogen Staphylococcus aureus. Using a competition assay with an sRNA mutant library, we identified 6S RNA as being required for protection against low concentrations of rifampicin, an RNA polymerase (RNAP) inhibitor. This effect applied to rifabutin and fidaxomicin, two other RNAP-targeting antibiotics. 6S RNA is highly conserved in bacteria, and its absence in two other major pathogens, Salmonella enterica and Clostridioides difficile, also impaired susceptibility to RNAP inhibitors. In S. aureus, 6S RNA is produced from an autonomous gene and accumulates in stationary phase. In contrast to what was reported for Escherichia coli, S. aureus 6S RNA does not appear to play a critical role in the transition from exponential to stationary phase but affects σB-regulated expression in prolonged stationary phase. Nevertheless, its protective effect against rifampicin is independent of alternative sigma factor σB activity. Our results suggest that 6S RNA helps maintain RNAP-σA integrity in S. aureus, which could in turn help bacteria withstand low concentrations of RNAP inhibitors.
Assuntos
Rifampina , Staphylococcus aureus , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA não Traduzido , Rifampina/farmacologia , Fator sigma/genética , Fator sigma/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Transcrição GênicaRESUMO
Transcription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity: (i) constant and independent of RNA levels, (ii) increased once RNA has accumulated, and (iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes and can serve as a blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control.
Assuntos
Adaptação Fisiológica , RNA Bacteriano/genética , RNA/genética , Iniciação da Transcrição Genética , Escherichia coli , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Bacteriano/metabolismoRESUMO
Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viability even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.
Assuntos
Genoma Bacteriano/genética , Fator Rho/genética , Terminação da Transcrição Genética , Transcrição Gênica , Bacteriófago T4/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cromossomos/genética , DNA Helicases/genética , Replicação do DNA/genética , DNA Antissenso/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes de RNAr/genética , Genoma Bacteriano/efeitos dos fármacos , Fator Rho/antagonistas & inibidores , Ribonuclease H/genética , Proteínas Virais/genéticaRESUMO
RsaE is a regulatory RNA highly conserved amongst Firmicutes that lowers the amount of mRNAs associated with the TCA cycle and folate metabolism. A search for new RsaE targets in Staphylococcus aureus revealed that in addition to previously described substrates, RsaE down-regulates several genes associated with arginine catabolism. In particular, RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. The faster growth rate of ΔrsaE compared to its parental strain in media containing amino acids as sole carbon source points to an underlying role for RsaE in amino acid catabolism. Collectively, the data support a model in which RsaE acts as a global regulator of functions associated with metabolic adaptation.
Assuntos
Arginina/metabolismo , RNA Bacteriano/fisiologia , Sequências Reguladoras de Ácido Ribonucleico , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Sequência Conservada , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Organismos Geneticamente Modificados , Sequências Reguladoras de Ácido Ribonucleico/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.
Assuntos
Aminoácidos de Cadeia Ramificada/biossíntese , Regulação Bacteriana da Expressão Gênica , Óperon , Transcrição Gênica , Vibrio alginolyticus/genética , Acetolactato Sintase/metabolismo , Organismos Aquáticos , Escherichia coli/genética , Isoleucina/biossíntese , Leucina/biossíntese , Sequências Reguladoras de Ácido Nucleico , Valina/biossínteseRESUMO
Bacteria optimize their fitness in response to a changing environment by tight regulation of gene expression. Regulation can be controlled at both transcriptional and post-transcriptional levels via key players such as sigma factors, regulatory proteins and regulatory RNAs. The identification of phenotypes associated with gene deletions is the established method for finding gene functions but may require testing many conditions for each studied mutant. As regulatory RNAs often contribute to fine-tuning gene expression, phenotypes associated with their inactivation are often weak and difficult to detect. Nevertheless, minor phenotypes conferring modest advantages, may allow bacteria to emerge after some generations under selective pressure. A strategy employing DNA barcodes can be used to perform competition experiments between mutants and to monitor fitness associated with mutations in different growth conditions. We combined this strategy with deep sequencing to study regulatory RNAs in Staphylococcus aureus, a major opportunistic pathogen.
Assuntos
Bioensaio , Regulação Bacteriana da Expressão Gênica , Interações Microbianas/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Staphylococcus aureus/genética , Código de Barras de DNA Taxonômico , Escherichia coli/genética , Escherichia coli/metabolismo , Aptidão Genética , Mutação , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Fator sigma/genética , Fator sigma/metabolismo , Staphylococcus aureus/metabolismo , Transcrição Gênica , Transformação BacterianaRESUMO
Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5' RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.
Assuntos
Regiões 5' não Traduzidas/genética , Mapeamento Cromossômico/métodos , Enterococcus faecalis/genética , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , Sitios de Sequências Rotuladas , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Desnaturação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Processamento Pós-Transcricional do RNA , Sítio de Iniciação de Transcrição , TranscriptomaRESUMO
BACKGROUND: Streptococcus agalactiae, or Group B Streptococcus, is a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. In an effort to reconstruct the transcriptional networks involved in S. agalactiae physiology and pathogenesis, we performed an extensive and robust characterization of its transcriptome through a combination of differential RNA-sequencing in eight different growth conditions or genetic backgrounds and strand-specific RNA-sequencing. RESULTS: Our study identified 1,210 transcription start sites (TSSs) and 655 transcript ends as well as 39 riboswitches and cis-regulatory regions, 39 cis-antisense non-coding RNAs and 47 small RNAs potentially acting in trans. Among these putative regulatory RNAs, ten were differentially expressed in response to an acid stress and two riboswitches sensed directly or indirectly the pH modification. Strikingly, 15% of the TSSs identified were associated with the incorporation of pseudo-templated nucleotides, showing that reiterative transcription is a pervasive process in S. agalactiae. In particular, 40% of the TSSs upstream genes involved in nucleotide metabolism show reiterative transcription potentially regulating gene expression, as exemplified for pyrG and thyA encoding the CTP synthase and the thymidylate synthase respectively. CONCLUSIONS: This comprehensive map of the transcriptome at the single nucleotide resolution led to the discovery of new regulatory mechanisms in S. agalactiae. It also provides the basis for in depth analyses of transcriptional networks in S. agalactiae and of the regulatory role of reiterative transcription following variations of intra-cellular nucleotide pools.
Assuntos
Nucleotídeos/análise , RNA Mensageiro/análise , Streptococcus agalactiae/genética , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Genes Bacterianos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Bacteriano/análise , Análise de Sequência de RNA/métodos , Streptococcus agalactiae/crescimento & desenvolvimentoRESUMO
YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , NADH Desidrogenase/deficiência , Proteínas Ribossômicas/deficiência , Aerobiose , Proteínas de Escherichia coli , Fermentação , Perfilação da Expressão Gênica , Humanos , Análise do Fluxo Metabólico , Análise em Microsséries , Modelos Biológicos , Dados de Sequência Molecular , Transtornos Parkinsonianos/patologia , Análise de Sequência de DNARESUMO
Most bacterial regulatory RNAs exert their function through base-pairing with target RNAs. Computational prediction of targets is a busy research field that offers biologists a variety of web sites and software. However, it is difficult for a non-expert to evaluate how reliable those programs are. Here, we provide a simple benchmark for bacterial sRNA target prediction based on trusted E. coli sRNA/target pairs. We use this benchmark to assess the most recent RNA target predictors as well as earlier programs for RNA-RNA hybrid prediction. Moreover, we consider how the definition of mRNA boundaries can impact overall predictions. Recent algorithms that exploit both conservation of targets and accessibility information offer improved accuracy over previous software. However, even with the best predictors, the number of true biological targets with low scores and non-targets with high scores remains puzzling.
Assuntos
Biologia Computacional/métodos , Escherichia coli/genética , RNA Bacteriano/genética , Pareamento de Bases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regiões não Traduzidas/genéticaRESUMO
Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio "sRNA core."
Assuntos
Ostreidae/microbiologia , RNA Bacteriano/genética , Vibrio/genética , Vibrio/patogenicidade , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Evolução Molecular , Gammaproteobacteria/genética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Especificidade da Espécie , Vibrio/classificação , Vibrio/metabolismoRESUMO
Staphylococcus aureus is one of the main etiological agents of mastitis in ruminants. In the present retrospective study, we evaluated the potential interest of a previously described automated multiple loci Variable Number of Tandem Repeats (VNTR) Assay (MLVA) comprising 16 loci as a first line tool to investigate the population structure of S. aureus from mastitis. We determined the genetic diversity of S. aureus strains from cases of clinical and subclinical mastitis in dairy cattle (n = 118, of which 16 were methicillin-resistant), sheep (n = 18) and goats (n = 16). The 152 strains could be subdivided into 115 MLVA genotypes (including 14 genotypes for the ovine strains and 15 genotypes for the caprine strains). This corresponds to a discriminatory index (D) value of 0.9936. Comparison with published MLVA data obtained using the same protocol applied to strains from diverse human and animal origins revealed a low number (8.5%) of human-related MLVA genotypes among the present collection. Eighteen percent of the S. aureus mastitis collection belonged to clonal complexes apparently not associated with other pathological conditions. Some of them displayed a relatively low level of diversity in agreement with a restricted ecological niche. These findings provide arguments suggesting that specific S. aureus lineages particularly adapted to ruminant mammary glands have emerged and that MLVA is a convenient tool to provide a broad overview of the population, owing to the availability via internet of databases compiling published MLVA genotypes.
Assuntos
Mastite/veterinária , Repetições Minissatélites , Tipagem de Sequências Multilocus/veterinária , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Alelos , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Evolução Molecular , Feminino , Doenças das Cabras/microbiologia , Cabras , Mastite/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Estudos Retrospectivos , Ovinos , Doenças dos Ovinos/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismoRESUMO
In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to "unroll" disrupting the H-NS bridges and releasing H-NS.
Assuntos
Cromatina , Proteínas de Ligação a DNA , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Inativação Gênica , Regulação Bacteriana da Expressão Gênica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Transcrição GênicaRESUMO
YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet-undefined function in the oxidative-stress response. YajL protects cells against oxidative-stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. Compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA repair proteins, RNA chaperones, and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative-stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/biossíntese , Estresse Oxidativo/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ferro/metabolismo , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Mutação , Proteínas Oncogênicas/genética , Oxirredução , Transtornos Parkinsonianos/genética , Proteína Desglicase DJ-1RESUMO
Staphylococcus aureus is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and RNAs. The aptitude of bacteria to use RNAs to rapidly modify gene expression, including virulence factors in response to stress or environmental changes, and to survive in a host is an evolving concept. Here, we focus on the recently inventoried S. aureus regulatory RNAs, with emphasis on those with identified functions, two of which are directly involved in pathogenicity.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Interações Hospedeiro-Patógeno/genética , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Variação Genética , RNA Bacteriano/metabolismo , RNA Interferente Pequeno/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismoRESUMO
The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure.