RESUMO
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Citosol/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Salmonella/microbiologia , Salmonella enterica/fisiologia , Virulência , Proteínas de Bactérias/genética , Citosol/microbiologia , Ilhas Genômicas , Células HeLa , Humanos , RNA-Seq , Infecções por Salmonella/metabolismoRESUMO
The phenomenon of contact-dependent growth inhibition (CDI) and the genes required for CDI (cdiBAI) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAIEC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system (cdiBAIEC93-2) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI+ bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Plasmídeos/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Proteínas de Membrana/genética , Interações Microbianas , Plasmídeos/metabolismoRESUMO
The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secYS281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secYS281F target cells and absent in secYS281F ΔppiD or secYS281F ΔyfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secYG313W ) renders ΔppiD or ΔyfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins.IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Canais de Translocação SEC/genética , Inibição de Contato , Mutação , Transporte Proteico , Canais de Translocação SEC/fisiologiaRESUMO
Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by 'accessory' or 'moron' loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.
Assuntos
Bacteriófagos/fisiologia , Lisogenia , Transcriptoma , Bacteriófagos/genética , Prófagos/genética , Prófagos/fisiologia , Salmonella/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Salmonella typhimurium/genética , Sistemas Toxina-Antitoxina/genética , Animais , Proteínas de Bactérias/metabolismo , Loci Gênicos/genética , Camundongos , Mutação , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Células RAW 264.7 , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Seleção GenéticaRESUMO
Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.
Assuntos
Infecções por Salmonella/genética , Salmonella typhimurium/genética , Animais , Gastroenterite/microbiologia , Perfilação da Expressão Gênica/métodos , Variação Genética/genética , Humanos , Macrófagos , Camundongos , Infecções por Salmonella/microbiologia , VirulênciaRESUMO
Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.
Assuntos
Nucleotídeos de Citosina/metabolismo , Farmacorresistência Bacteriana/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Nucleotídeos de Citosina/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genótipo , Proteínas de Membrana/genéticaRESUMO
Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only â¼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.
Assuntos
Evolução Molecular , Genoma Bacteriano/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , DNA Bacteriano/genética , Epidemias , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Virulência/genética , Fatores de Virulência/genéticaRESUMO
In the past 30 years, Salmonella bloodstream infections have become a significant health problem in sub-Saharan Africa and are responsible for the deaths of an estimated 390,000 people each year. The disease is predominantly caused by a recently described sequence type of Salmonella Typhimurium: ST313, which has a distinctive set of prophage sequences. We have thoroughly characterized the ST313-associated prophages both genetically and experimentally. ST313 representative strain D23580 contains five full-length prophages: BTP1, Gifsy-2D23580, ST64BD23580, Gifsy-1D23580, and BTP5. We show that common S. Typhimurium prophages Gifsy-2, Gifsy-1, and ST64B are inactivated in ST313 by mutations. Prophage BTP1 was found to be a functional novel phage, and the first isolate of the proposed new species "Salmonella virus BTP1", belonging to the P22virus genus. Surprisingly, â¼109 BTP1 virus particles per ml were detected in the supernatant of non-induced, stationary-phase cultures of strain D23580, representing the highest spontaneously induced phage titer so far reported for a bacterial prophage. High spontaneous induction is shown to be an intrinsic property of prophage BTP1, and indicates the phage-mediated lysis of around 0.2% of the lysogenic population. The fact that BTP1 is highly conserved in ST313 poses interesting questions about the potential fitness costs and benefits of novel prophages in epidemic S. Typhimurium ST313.
RESUMO
Streptococcus pneumoniae (pneumococcus) is the primary cause of bacterial meningitis. Pneumococcal bacteria penetrates the blood-brain barrier (BBB), but the bacterial factors that enable this process are not known. Here, we determined that expression of pneumococcal pilus-1, which includes the pilus adhesin RrgA, promotes bacterial penetration through the BBB in a mouse model. S. pneumoniae that colonized the respiratory epithelium and grew in the bloodstream were chains of variable lengths; however, the pneumococci that entered the brain were division-competent, spherical, single cocci that expressed adhesive RrgA-containing pili. The cell division protein DivIVA, which is required for an ovoid shape, was localized at the poles and septum of pneumococcal chains of ovoid, nonseparated bacteria, but was absent in spherical, single cocci. In the bloodstream, a small percentage of pneumococci appeared as piliated, RrgA-expressing, DivIVA-negative single cocci, suggesting that only a minority of S. pneumoniae are poised to cross the BBB. Together, our data indicate that small bacterial cell size, which is signified by the absence of DivIVA, and the presence of an adhesive RrgA-containing pilus-1 mediate pneumococcal passage from the bloodstream through the BBB into the brain to cause lethal meningitis.
Assuntos
Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Meningite Pneumocócica/microbiologia , Streptococcus pneumoniae , Fatores de Virulência/metabolismo , Adesinas Bacterianas , Animais , Aderência Bacteriana , Barreira Hematoencefálica , Encéfalo/microbiologia , Parede Celular , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
RNase E is an essential bacterial endoribonuclease with a central role in processing tRNAs and rRNA, and turning over mRNAs. Previous studies in strains carrying mutations in the rne structural gene have shown that tRNA processing is likely to be an essential function of RNase E but have not determined whether mRNA turnover is also an essential function. To address this we selected extragenic suppressors of temperature-sensitive mutations in rne that cause a large increase in mRNA half-life at the non-permissive temperature. Fifteen suppressors were mapped to three different loci: relBE (toxin-antitoxin system); vacB (RNase R); and rpsA (ribosomal protein S1). Each suppressor class has the potential to interact with mRNA and each restores wild-type levels of mRNA turnover but does not reverse the minor defects in tRNA and rRNA processing. RelE toxin is especially interesting because its only known activity is to cleave mRNAs in the ribosomal A-site. The relBE suppressor mutations increase transcription of relE, and controlled overexpression of RelE alone was sufficient to suppress the rne ts phenotype. Suppression increased turnover of some major mRNAs (tufA, ompA) but not all mRNAs. We propose that turnover of some mRNAs is one of the essential functions of RNase E.
Assuntos
Endorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Supressão Genética , Proteínas da Membrana Bacteriana Externa/genética , Exorribonucleases/metabolismo , Fenótipo , RNA Ribossômico/metabolismo , Salmonella enterica/genética , TemperaturaRESUMO
Salmonellaâ Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP) (1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP) (1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP) (1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP) (1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP) (1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP) (1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP) (1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor.
Assuntos
Glicosídeo Hidrolases/metabolismo , Lipopolissacarídeos/química , Antígenos O/química , Prófagos/enzimologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Glicosídeo Hidrolases/genética , Humanos , Lipopolissacarídeos/metabolismo , Lisogenia , Dados de Sequência Molecular , Antígenos O/metabolismo , Prófagos/genética , Prófagos/fisiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Alinhamento de Sequência , Proteínas Virais/genética , VirulênciaRESUMO
Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.
Assuntos
Proteínas de Bactérias/metabolismo , Guanosina Tetrafosfato/metabolismo , Ligases/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Pirofosfatases/metabolismo , Proteínas de Bactérias/genética , Cromatografia em Camada Fina , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Genótipo , Ligases/genética , Mutação de Sentido Incorreto , Fator Tu de Elongação de Peptídeos/genética , Fenótipo , Pirofosfatases/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência de Prolina/genética , RNA de Transferência de Prolina/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismoRESUMO
Bacterial transcriptional networks consist of hundreds of transcription factors and thousands of promoters. However, the true complexity of transcription in a bacterial pathogen and the effect of the environments encountered during infection remain to be established. We present a simplified approach for global promoter identification in bacteria using RNA-seq-based transcriptomic analyses of 22 distinct infection-relevant environmental conditions. Individual RNA samples were combined to identify most of the 3,838 Salmonella enterica serovar Typhimurium promoters in just two RNA-seq runs. Individual in vitro conditions stimulated characteristic transcriptional signatures, and the suite of 22 conditions induced transcription of 86% of all S. Typhimurium genes. We highlight the environmental conditions that induce the Salmonella pathogenicity islands and present a small RNA expression landscape of 280 sRNAs. This publicly available compendium of environmentally controlled expression of every transcriptional feature of S. Typhimurium constitutes a useful resource for the bacterial research community.
Assuntos
Redes Reguladoras de Genes , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
The availability of thousands of genome sequences of bacterial pathogens poses a particular challenge because each genome contains hundreds of genes of unknown function (FUN). How can we easily discover which FUN genes encode important virulence factors? One solution is to combine two different functional genomic approaches. First, transcriptomics identifies bacterial FUN genes that show differential expression during the process of mammalian infection. Second, global mutagenesis identifies individual FUN genes that the pathogen requires to cause disease. The intersection of these datasets can reveal a small set of candidate genes most likely to encode novel virulence attributes. We demonstrate this approach with the Salmonella infection model, and propose that a similar strategy could be used for other bacterial pathogens.
Assuntos
Genes Bacterianos , Genômica/métodos , Técnicas Microbiológicas/métodos , Salmonella/genética , Salmonella/fisiologia , Análise Mutacional de DNA , Perfilação da Expressão Gênica/métodosRESUMO
RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66âCys, Ile207âSer, Ile207âAsn, and Ala327âPro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Endorribonucleases/química , Endorribonucleases/genética , Mutação , Salmonella typhimurium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Endorribonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Salmonella typhimurium/química , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , TemperaturaRESUMO
Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n= 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n= 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.