RESUMO
Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.
Assuntos
Bacillus subtilis , Regulação Bacteriana da Expressão Gênica , Clivagem do RNA , Estabilidade de RNA , RNA Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimologia , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Conformação de Ácido Nucleico , Sequência de BasesRESUMO
Mycobacterium tuberculosis, the bacterium responsible for human tuberculosis, has a genome encoding a remarkably high number of toxin-antitoxin systems of largely unknown function. We have recently shown that the M. tuberculosis genome encodes four of a widespread, MenAT family of nucleotidyltransferase toxin-antitoxin systems. In this study we characterize MenAT1, using tRNA sequencing to demonstrate MenT1 tRNA modification activity. MenT1 activity is blocked by MenA1, a short protein antitoxin unrelated to the MenA3 kinase. X-ray crystallographic analysis shows blockage of the conserved MenT fold by asymmetric binding of MenA1 across two MenT1 protomers, forming a heterotrimeric toxin-antitoxin complex. Finally, we also demonstrate tRNA modification by toxin MenT4, indicating conserved activity across the MenT family. Our study highlights variation in tRNA target preferences by MenT toxins, selective use of nucleotide substrates, and diverse modes of MenA antitoxin activity.
Assuntos
Antitoxinas , Mycobacterium tuberculosis , Toxinas Biológicas , Humanos , Antitoxinas/genética , Nucleotidiltransferases , Nucleotídeos , RNA de Transferência/genéticaRESUMO
Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo. mRNA cleavage by the toxin occurs after the second nucleotide of the ribosomal A-site codon during translation, with a strong preference for CCA codons in vivo. Finally, we report the cryo-EM structure of the ribosome-bound TAC toxin in the presence of native M. tuberculosis cspA mRNA, revealing the specific mechanism by which the TAC toxin interacts with the ribosome and the tRNA in the P-site to cleave its mRNA target.
Assuntos
Antitoxinas , Mycobacterium tuberculosis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Chaperonas Moleculares/genética , Mycobacterium tuberculosis/genética , RNA Mensageiro/genética , RibossomosRESUMO
Plasmids need to ensure their transmission to both daughter-cells when their host divides, but should at the same time avoid overtaxing their hosts by directing excessive host-resources toward production of plasmid factors. Naturally occurring plasmids have therefore evolved regulatory mechanisms to restrict their copy-number in response to the volume of the cytoplasm. In many plasmid families, copy-number control is mediated by a small plasmid-specified RNA, which is continuously produced and rapidly degraded, to ensure that its concentration is proportional to the current plasmid copy-number. We show here that pSA564 from the RepA_N-family is regulated by a small antisense RNA (RNA1), which, when over-expressed in trans, blocks plasmid replication and cures the bacterial host. The 5' untranslated region (5'UTR) of the plasmid replication initiation gene (repA) potentially forms two mutually exclusive secondary structures, ON and OFF, where the latter both sequesters the repA ribosome binding site and acts as a rho-independent transcriptional terminator. Duplex formation between RNA1 and the 5'UTR shifts the equilibrium to favor the putative OFF-structure, enabling a single small RNA to down-regulate repA expression at both transcriptional and translational levels. We further examine which sequence elements on the antisense RNA and on its 5'UTR target are needed for this regulation. Finally, we identify the host-encoded exoribonucleases RNase J1 and J2 as the enzymes responsible for rapidly degrading the replication-inhibiting section of RNA1. This region accumulates and blocks RepA expression in the absence of either RNase J1 or J2, which are therefore essential host factors for pSA564 replication in Staphylococcus aureus.
RESUMO
The identification of fecal contamination in coastal marine ecosystems is one of the main requirements for evaluation of potential risks to human health. The objective of this study was to investigate the occurrence and distribution of fecal indicators and pathogenic bacteria in seawaters and mussels collected monthly during a period of 1 year from four different sites in Northeastern Algeria (sites S1 to S4), through biochemical and molecular analyses. Our research is the first to use molecular analysis to unambiguously identify the potentially pathogenic bacteria present in Algerian Perna perna mussels. The obtained results revealed that the levels of fecal indicator bacteria (FIB) from both P. perna and seawater samples largely exceeded the permissible limits at S2 and S3. This is mainly related to their location close to industrial and coastal activity zones, which contain a mixture of urban, agricultural, and industrial pollutants. Besides, P. perna collected from all sites were severalfold more contaminated by FIB than seawater samples, primarily during the warm season of the study period. Biochemical and molecular analyses showed that isolated bacteria from both seawater and mussels were mainly potentially pathogenic species such as E. coli, Salmonella spp., Staphylococcus spp., Klebsiella spp., Pseudomonas spp., and Proteus spp.
Assuntos
Perna (Organismo) , Poluentes Químicos da Água , Animais , Bactérias/genética , Ecossistema , Monitoramento Ambiental , Escherichia coli , Humanos , Água do Mar , Poluentes Químicos da Água/análiseRESUMO
A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.
Assuntos
Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Proteínas de Escherichia coli/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Antibacterianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Humanos , Óperon/genética , RNA Mensageiro/genética , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Especificidade por Substrato , Transcriptoma/genéticaRESUMO
Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Regulon/genética , Staphylococcus aureus/metabolismo , Proteínas de Transporte de Cátions/genética , Mutação com Ganho de Função , Homeostase , Staphylococcus aureus/genéticaRESUMO
Opportunistic pathogens have to be ready to change life-style whenever the occasion arises, and therefore need to keep tight control over the expression of their virulence factors. Doubly so for commensal bacteria, such as Staphylococcus aureus, which should avoid harming their hosts when they are in a state of peaceful co-existence. S. aureus carries very few sigma factors to help define the transcriptional programs, but instead uses a plethora of small RNA molecules and RNA-RNA interactions to regulate gene expression post-transcriptionally. The endoribonucleases RNase III and RNase Y contribute to this regulatory diversity, and provide a link to RNA-decay and intra-cellular spatiotemporal control of expression. In this review we describe some of these post-transcriptional mechanisms as well as some of the novel transcriptomic approaches that have been used to find and to study them.
Assuntos
RNA Bacteriano/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Endorribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Bacteriano/química , Ribonuclease III/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , SimbioseRESUMO
Knowing the exact 5'-end of a specific RNA molecule has long been of great interest, since this information can reveal transcription start sites, which subsequently pinpoints promoter regions and potential binding sites for transcriptional regulators. Mapping of the 5'-end can also identify endoribonucleolytic cleavage sites, for the maturation of ribosomal RNA or events which initiate degradation of all or parts of the RNA molecule. If the 5' nucleotide of an RNA is triphosphorylated, then the nucleotide represents the original, unaltered 5'-end of the RNA. Similarly, if an endoribonuclease cleaves the RNA with a mechanism that leaves a monophosphate on the 5'-end downstream cleavage product, then it is useful to search exclusively for monophosphorylated 5'-ends to identify the cleavage sites of the endoribonuclease. This chapter details three variations of the EMOTE (exact mapping of transcriptome ends) protocol, which can identify RNA 5'-ends on a transcriptome-wide scale, and simultaneously provide information about the phosphorylation state of the detected ends. EMOTE, TSS-EMOTE (transcription start specific EMOTE), and nEMOTE (nonphosphorylated EMOTE) reveals mono-, tri-, and nonphosphorylated 5'-ends, respectively, and the protocols can be performed individually or in parallel on the same RNA sample.
Assuntos
Sítio de Iniciação de Transcrição/fisiologia , Transcriptoma/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Fosforilação , Regiões Promotoras Genéticas/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
Correct balance between bacterial RNA degradation and synthesis is essential for controlling expression level of all RNAs. The RNA polymerase, which performs the RNA synthesis, is highly conserved across the bacterial domain. However, this is surprisingly not the case for the RNA degradation machinery, which is composed of different subunits and performs different enzymatic reactions, depending on the organism. In Escherichia coli, the RNA decay is performed by the degradosome complex, which forms around the membrane-associated endoribonuclease RNase E, and is stable enough to be purified without falling apart. In contrast, many Firmicutes, for example, Bacillus subtilis, Staphylococcus aureus, and Streptococcus pneumoniae, do not encode an RNase E homolog, but instead have the endoribonuclease RNase Y and the exo- and endo-ribonuclease RNase J complex. A wide range of experiments have been performed, mainly with B. subtilis and S. aureus, to determine which interactions exist between the various RNA decay enzymes in the Firmicutes, with the goal of understanding how RNA degradation (and thus gene expression homeostasis and regulation) is organized in these organisms. The in vivo and in vitro data is diverse, and does not always concur. This overview gathers the data on interactions between Firmicute RNA degradation factors, to highlight the similarities and differences between experimental data from different experiments and from different organisms. WIREs RNA 2018, 9:e1460. doi: 10.1002/wrna.1460 This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability.
Assuntos
Firmicutes/genética , Estabilidade de RNA/genética , Firmicutes/metabolismo , Ribonucleases/química , Ribonucleases/genética , Ribonucleases/metabolismo , RibossomosRESUMO
RNA decay and RNA maturation are important steps in the regulation of bacterial gene expression. RNase J, which is present in about half of bacterial species, has been shown to possess both endo- and 5' to 3' exo-ribonuclease activities. The exonucleolytic activity is clearly involved in the degradation of mRNA and in the maturation of at least the 5' end of 16S rRNA in the 2 Firmicutes Staphylococcus aureus and Bacillus subtilis. The endoribonuclease activity of RNase J from several species has been shown to be weak in vitro and 3-D structural data of different RNase J orthologs have not provided a clear explanation for the molecular basis of this activity. Here, we show that S. aureus RNase J1 is a manganese dependent homodimeric enzyme with strong 5' to 3' exo-ribonuclease as well as endo-ribonuclease activity. In addition, we demonstrated that SauJ1 can efficiently degrade 5' triphosphorylated RNA. Our results highlight RNase J1 as an important player in RNA turnover in S. aureus.
Assuntos
Manganês/metabolismo , Ribonucleases/metabolismo , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Fosforilação , Estrutura Quaternária de Proteína , Ribonucleases/química , Ribonucleases/genética , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of α-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5'-GANTC-3' sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different α-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.
Assuntos
Caulobacter crescentus/genética , Ciclo Celular/genética , Metilação de DNA/genética , Transcrição Gênica , Divisão Celular/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Regulação Bacteriana da Expressão Gênica , Genoma Microbiano , Metiltransferases/genética , Fosfatos/metabolismo , Regiões Promotoras Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Inanição/genética , Inanição/metabolismoRESUMO
Internal bacterial concentration of Mg2+, the most abundant divalent cation in living cells, is estimated to be in the single millimolar range. However, many bacteria will thrive in media with only micromolars of Mg2+, by using a range of intensely studied and highly efficient import mechanisms, as well as in media with very high magnesium concentration, presumably mediated by currently unknown export mechanisms. Staphylococcus aureus has a particularly high Mg2+ tolerance for a pathogen, growing unimpaired in up to 770 mM Mg2+, and we here identify SA0657, a key factor in this tolerance. The predicted domain structure of SA0657 is shared with a large number of proteins in bacteria, archaea and even eukarya, for example CorB from Salmonella and the human CNNM protein family. One of the shared domains, a CBS pair potentially involved in Mg2+ sensing, contains the conserved Glycine326 which we establish to be a key residue for SA0657 function. In light of our findings, we propose the name MpfA, Magnesium Protection Factor A, for SA0657.
RESUMO
BACKGROUND: Bacteria rely on efficient gene regulatory mechanisms to switch between genetic programs when they are facing new environments. Although this regulation can occur at many different levels, one of the key steps is the initiation of transcription. Identification of the first nucleotide transcribed by the RNA polymerase is therefore essential to understand the underlying regulatory processes, since this provides insight on promoter strength and binding sites for transcriptional regulators, and additionally reveals the exact 5' untranslated region of the transcripts, which often contains elements that regulate translation. RESULTS: Here we present data from a novel TSS-EMOTE assay (Transcription Start Specific Exact Mapping Of Transcriptome Ends) to precisely map the transcription initiation sites of four entire transcriptomes. TSS-EMOTE is a variation of the dRNA-seq method, which has been combined with the EMOTE protocol, in order to increase detection of longer transcripts and limit biases introduced by PCR amplification of the Illumina sequencing library. Using TSS-EMOTE, 2018 promoters were detected in the opportunistic pathogen Staphylococcus aureus, and detailed consensus sequences could be obtained for the RNA polymerase recognition elements (e.g. sigma factor binding sites). The data also revealed a 94 nt median length of the 5' untranslated region in S. aureus, giving important insights for future work on translational regulation. Additionally, the transcriptomes of three other opportunistic pathogens, Staphylococcus epidermidis, Acinetobacter baumannii and Enterobacter aerogenes, were examined, and the identified promoter locations were then used to generate a map of the operon structure for each of the four organisms. CONCLUSIONS: Mapping transcription start sites, and subsequent correlation with the genomic sequence, provides a multitude of important information about the regulation of gene expression, both at the transcriptional and translational level, by defining 5' untranslated regions and sigma-factor binding sites. We have here mapped transcription start sites in four important pathogens using TSS-EMOTE, a method that works with both long and 3'-phosphorylated RNA molecules, and which incorporates Unique Molecular Identifiers (UMIs) to allow unbiased quantification.
Assuntos
Bactérias/genética , Mapeamento Cromossômico , Genômica/métodos , Sítio de Iniciação de Transcrição , Transcrição Gênica , Bactérias/patogenicidade , Sequência de Bases , Análise por Conglomerados , Sequência Consenso , Perfilação da Expressão Gênica , Genes Bacterianos , Sequenciamento de Nucleotídeos em Larga Escala , Óperon , Matrizes de Pontuação de Posição Específica , Regiões Promotoras Genéticas , Transcriptoma , Fatores de Virulência/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1005577.].
RESUMO
Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/genética , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Serina Endopeptidases/metabolismo , Sistemas Toxina-Antitoxina , Adaptação FisiológicaRESUMO
Recently a number of seminal studies have revealed that both sequence and spatio-temporal factors govern RNA decay in bacteria, which is crucial for regulation of gene expression. Ribonucleases have been described that not only exhibit sequence preferences, but also are sub-cellularly localised. Furthermore, the RNA itself is distributed in an organised manner and does not diffuse freely or randomly within the bacterial cells. Thus, even within the sub-micrometer distances of the bacterial intra-cellular space, the positions of the enzymes and their substrates are kept in check. Adding to this complexity is the secondary structure and sequence specificity that many, perhaps all, ribonucleases exhibit, including those that are responsible for "general" RNA degradation. In this review, the implications of these novel findings are discussed and specific examples from Staphylococcus aureus are analysed.
Assuntos
Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Membrana Celular/metabolismo , Transporte Proteico , Transporte de RNA , Ribonucleases/metabolismo , Especificidade por SubstratoRESUMO
Bacteria depend on efficient RNA turnover, both during homeostasis and when rapidly altering gene expression in response to changes. Nevertheless, remarkably few details are known about the rate-limiting steps in targeting and decay of RNA. The membrane-anchored endoribonuclease RNase Y is a virulence factor in Gram-positive pathogens. We have obtained a global picture of Staphylococcus aureus RNase Y sequence specificity using RNA-seq and the novel transcriptome-wide EMOTE method. Ninety-nine endoribonucleolytic sites produced in vivo were precisely mapped, notably inside six out of seven genes whose half-lives increase the most in an RNase Y deletion mutant, and additionally in three separate transcripts encoding degradation ribonucleases, including RNase Y itself, suggesting a regulatory network. We show that RNase Y is required to initiate the major degradation pathway of about a hundred transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.
Assuntos
Estabilidade de RNA/genética , Ribonucleases/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Regulação Bacteriana da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , Ribonucleases/biossíntese , Deleção de Sequência , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Transcriptoma/genéticaRESUMO
Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes.
Assuntos
Endorribonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Staphylococcus aureus/metabolismo , Estrutura Terciária de Proteína , Staphylococcus aureus/enzimologiaRESUMO
RNA helicases of the DEAD-box and DEAH-box families are important players in many processes involving RNA molecules. These proteins can modify RNA secondary structures or intermolecular RNA interactions and modulate RNA-protein complexes. In bacteria, they are known to be involved in ribosome biogenesis, RNA turnover and translation initiation. They thereby play an important role in the adaptation of bacteria to changing environments and to respond to stress conditions.