RESUMO
Diet affects nearly every aspect of animal life such as development, metabolism, behavior, and aging, both directly by supplying nutrients and indirectly through gut microbiota. C. elegans feeds on bacteria, and like other animals, different bacterial diets induce distinct dietary responses in the worm. However, the lack of certain critical tools hampers the use of worms as a model for dietary signaling. Here, we genetically engineered the bacterial strain OP50, the standard laboratory diet for C. elegans, making it compatible for dsRNA production and delivery. Using this RNAi-compatible OP50 strain and the other bacterial strain HT115, we feed worms different diets while delivering RNAi to interrogate the genetic basis underlying diet-dependent differential modulation of development, metabolism, behavior, and aging. We show by RNAi that neuroendocrine and mTOR pathways are involved in mediating differential dietary responses. This genetic tool greatly facilitates the use of C. elegans as a model for dietary signaling.
Assuntos
Bactérias/genética , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/fisiologia , Dieta , Modelos Animais , Envelhecimento/fisiologia , Animais , Genes Bacterianos , Engenharia Genética , Interferência de RNARESUMO
Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production.
Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Síndrome Hemolítico-Urêmica/metabolismo , Nefropatias/metabolismo , Ativação Viral/fisiologia , Animais , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/virologia , Feminino , Síndrome Hemolítico-Urêmica/mortalidade , Síndrome Hemolítico-Urêmica/virologia , Nefropatias/mortalidade , Nefropatias/virologia , Longevidade , Masculino , Camundongos , Toxina Shiga II/metabolismo , Organismos Livres de Patógenos Específicos , Taxa de Sobrevida , UrináliseRESUMO
Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/metabolismo , RNA Ribossômico 16S/biossíntese , Ribonuclease III/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Deleção de Genes , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/genética , Subunidades Ribossômicas Menores/metabolismo , Temperatura , Fatores de Transcrição/genética , Fatores de Elongação da TranscriçãoRESUMO
Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.
Assuntos
Escherichia coli/genética , 5-Metilcitosina/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Biotecnologia , Mapeamento Cromossômico , Metilação de DNA/genética , Enzimas de Restrição-Modificação do DNA/deficiência , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Amplificação de Genes , Deleção de Genes , Genoma Bacteriano , Análise de Sequência de DNA/métodos , Compostos de Espiro , Especificidade por SubstratoRESUMO
Bacteria in their struggle for survival have evolved or acquired defences against attacking phage. However, phage often contribute to this defence through mechanisms in which a prophage protects the bacterial population from attack by another, often unrelated, phage. The 933W prophage, which carries Shiga toxin genes that enhance pathogenicity of enterohaemorrhagic Escherichia coli strain O157:H7, also carries the stk gene encoding a eukaryotic-like tyrosine kinase that excludes (aborts) infection by phage HK97. This exclusion requires the kinase activity of Stk. Little, if any, protein tyrosine phosphorylation can be detected in a 933W lysogen prior to infection with HK97, while extensive Stk-mediated tyrosine phosphorylation is evident following infection. This includes autophosphorylation that stabilizes Stk protein from degradation. Although increased levels of Stk are found following HK97 infection, these higher levels are not necessary or sufficient for exclusion or protein phosphorylation. An HK97 open reading frame, orf41, is necessary for exclusion and Stk kinase activity. We hypothesize that interaction with gp41 stimulates Stk kinase activity. Exclusion of HK97 appears to be specific since other phages tested, λ, φ80, H-19B, λ-P22dis and T4rII, were not excluded. Infection of the 933W lysogen with a non-excluded phage fails to induce Stk-determined phosphorylation.
Assuntos
Escherichia coli O157/virologia , Prófagos/enzimologia , Prófagos/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Interferência Viral , Ativação Viral , Sequência de Aminoácidos , Modelos Biológicos , Dados de Sequência MolecularRESUMO
The fate of lysogens following prophage induction has assumed added significance with the finding that in many pathogens virulence genes are carried on prophages and, in some, the production and/or release of the virulence factor is under control of the phage lytic regulatory program. We outline a method for identifying and characterizing from a total lysogen population, the subpopulation in which the prophage is induced. The prophage is genetically altered so that on induction it does not go through the lytic pathway, but does express a resolvase that acts at a reporter cassette located at another site on the bacterial chromosome to irreversibly change the resistance of the bacterium from tetracycline to chloramphenicol. Thus, induced derivatives survive and are easily identified even if they make up a small fraction of the population.
Assuntos
Lisogenia/genética , Prófagos/genética , Bacteriófagos/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Regulação Viral da Expressão Gênica , Modelos Biológicos , Reação em Cadeia da Polimerase , Recombinases/genética , Proteínas Virais/genéticaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes hemorrhagic colitis and acute renal failure. We used a germ-free mouse model to investigate the role of host factors, Shiga toxin 2 (Stx2), and bacterial strain in disease due to EHEC. Germ-free male and female Swiss-Webster mice that were 3 days to 12 weeks old were orally inoculated with 1 of 10 EHEC strains or derivatives of two of these strains with Stx2 deleted. All inoculated mice became infected regardless of the inoculum dose. All bacterial strains colonized the intestines, reaching levels of 10(9) to 10(12) CFU/g of feces by 4 days after inoculation. Seven of the 10 wild-type strains caused disease. However, the two Stx2 deletion mutants, unlike the Stx2(+) parental strains, did not cause disease. The clinical signs of disease in mice included lethargy, dehydration, polyuria, polydypsia, and death. Postmortem examination of affected mice revealed dehydration and luminal cecal fluid accumulation. Histologic examination revealed close adherence of bacteria to the intestinal epithelium in the ileum and cecum but not in the colon. Other lesions included progressive renal tubular necrosis, glomerular fibrin thrombosis, and red blood cell sludging. The severity of disease varied according to the bacterial strain and age, but not sex, of the host. This study demonstrated that EHEC colonizes germ-free mice in large numbers, adheres to the intestinal epithelium, and causes luminal cecal fluid accumulation and progressive renal failure. The disease in mice was Stx2 and bacterial strain dependent. This animal model should be a useful tool for studying the pathogenesis of renal disease secondary to EHEC infection.
Assuntos
Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/patogenicidade , Toxina Shiga II/metabolismo , Animais , Contagem de Colônia Microbiana , Escherichia coli Êntero-Hemorrágica/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/fisiopatologia , Escherichia coli O157/patogenicidade , Feminino , Vida Livre de Germes , Intestinos/microbiologia , Nefropatias/microbiologia , Nefropatias/fisiopatologia , Masculino , Camundongos , Mutação , Insuficiência Renal/microbiologia , Insuficiência Renal/fisiopatologia , Índice de Gravidade de Doença , Toxina Shiga II/genética , VirulênciaRESUMO
The N protein of phage lambda acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the lambda early P(R) promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P(R), at positions -14 and -45 (yielding P(R-GA)), reduces effectiveness of N modification, while an additional mutation at position -30 (yielding P(R-GCA)) suppresses this effect. In vivo, the level of P(R-GA)-directed transcription was twice as great as the wild-type level, while transcription directed by P(R-GCA) was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P(R-GA) in vitro was roughly one-third the rate for wild-type P(R). We ascribe this apparent discrepancy to an effect of the mutations in P(R-GCA) on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.
Assuntos
Bacteriófago lambda/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/metabolismo , Fusão Gênica Artificial , Bacteriófago lambda/genética , Sequência de Bases , Regulação Viral da Expressão Gênica , Genes Reporter/genética , Genes Reporter/fisiologia , Dados de Sequência Molecular , Mutação Puntual , RNA Viral/biossíntese , Supressão Genética , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
OBJECTIVE: The purpose of the study was to determine if bedside ultrasound (US) and perception of wound foreign bodies (FBs) are useful screening tools for detecting wound FBs in children. METHODS: Prospective consecutive sample of children aged 18 years or younger presenting to a pediatric emergency department with wounds considered by the pediatric emergency department attending physician to be at risk for FBs was enrolled. Patients were asked if they had FB sensation in their wound(s). A bedside US of each wound was performed by the pediatric emergency department attending physician. A radiograph of each wound was obtained and interpreted by a radiologist blinded to US results and patient perception. Wound FBs were defined by the removal of a FB. The utilities of US and US with FB perception were compared with radiography for screening for wound FBs. Differences in performance characteristics among the 3 modalities were assessed using Fisher exact test. RESULTS: One hundred thirty-one wounds were studied in 105 patients. FBs were identified in 12 wounds (9.2%). A subanalysis was performed on patients able to answer questions regarding their perception of wound FBs. There were no significant differences in the test performance characteristics of bedside US alone compared with radiography for detecting wound FBs. Except for specificity, there were no significant differences in the test performance characteristics of bedside US combined with perception compared with radiography for detecting wound FBs. CONCLUSIONS: Bedside US is comparable to the performance of radiography interpreted by an attending pediatric radiologist. Bedside US alone or combined with patient perception may be an adequate initial screening tool for detecting wound FBs.
Assuntos
Corpos Estranhos/diagnóstico por imagem , Sistemas Automatizados de Assistência Junto ao Leito , Criança , Feminino , Humanos , Masculino , Pediatria , Estudos Prospectivos , Radiografia , Radiologia , Lesões dos Tecidos Moles/diagnóstico por imagem , Ultrassonografia , Ferimentos Penetrantes/diagnóstico por imagemRESUMO
In many pathogenic bacteria, genes that encode virulence factors are located in the genomes of prophages. Clearly bacteriophages are important vectors for disseminating virulence genes, but, in addition, do phage regulatory circuits contribute to expression of these genes? Phages of the lambda family that have genes encoding Shiga toxin are found in certain pathogenic Escherichia coli (known as Shiga toxin producing E. coli) and the filamentous phage CTXphi, that carries genes encoding cholera toxin (CTX), is found in Vibrio cholerae. Both the lambda and CTXphi phages have repressor systems that maintain their respective prophages in a quiescent state, and in both types of prophages this repressed state is abolished when the host cell SOS response is activated. In the lambda type of prophages, only binding of the phage-encoded repressor is involved in repression and this repressor ultimately controls Shiga toxin production and/or release. In the CTXphi prophage, binding of LexA, the bacterial regulator of SOS, in addition to binding of the repressor is involved in repression; the repressor has only limited control over CTX production and has no influence on its release.
Assuntos
Bacteriófagos/genética , Toxina da Cólera/metabolismo , Escherichia coli/patogenicidade , Regulação Viral da Expressão Gênica , Toxina Shiga/metabolismo , Vibrio cholerae/patogenicidade , Bacteriófagos/metabolismo , Toxina da Cólera/genética , Colífagos/genética , Colífagos/metabolismo , Escherichia coli/virologia , Humanos , Toxina Shiga/genética , Vibrio cholerae/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , VirulênciaRESUMO
The genes encoding Shiga toxin (Stx), the major virulence factor of Shiga toxin-producing Escherichia coli, are carried in the genomes of bacteriophages that belong to the lambdoid family of phages. Previous studies demonstrated that induction of prophages encoding stx significantly enhances the production and/or release of Stx from the bacterium. Therefore, factors that regulate the switch between lysogeny and lytic growth, e.g., repressor, operator sites, and associated phage promoters, play important roles in regulating the production and/or release of Stx. We report the results of genetic and biochemical studies characterizing these elements of the Stx-encoding bacteriophage 933W. Like lambda, 933W has three operator repeats in the right operator region (OR), but unlike lambda and all other studied lambdoid phages, which have three operator repeats in the left operator region (OL), 933W only has two operator repeats in OL. As was observed with lambda, the 933W OR and OL regions regulate transcription from the early PR and PL promoters, respectively. A lysogen carrying a 933W derivative encoding a noncleavable repressor fails to produce Stx, unlike a lysogen carrying a 933W derivative encoding a cleavable repressor. This finding provides direct evidence that measurable expression of the stx genes encoded by a 933W prophage requires induction of that prophage with the concomitant initiation of phage gene expression.
Assuntos
Bacteriófago lambda/genética , Regulação Viral da Expressão Gênica , Regiões Operadoras Genéticas/genética , Regiões Promotoras Genéticas/genética , Toxina Shiga II/metabolismo , Bacteriófago lambda/patogenicidade , Sequência de Bases , Escherichia coli/virologia , Dados de Sequência Molecular , Mutação , Prófagos/genética , Prófagos/patogenicidade , Toxina Shiga II/genética , Virulência , Ativação ViralRESUMO
The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.
Assuntos
Colífagos/genética , Fases de Leitura Aberta , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Toxina Shiga/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/químicaRESUMO
Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.
Assuntos
Bacteriófago lambda/genética , Colífagos/genética , Escherichia coli/metabolismo , Lisogenia , Toxinas Shiga/genética , Animais , Bacteriófago lambda/fisiologia , Colífagos/fisiologia , Colífagos/ultraestrutura , Escherichia coli/genética , Escherichia coli/virologia , Regulação Viral da Expressão Gênica , Toxinas Shiga/biossíntese , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
Transfer-messenger RNA (tmRNA, or SsrA), found in all eubacteria, has both transfer and messenger RNA activity. Relieving ribosome stalling by a process called trans-translation, tmRNAala enters the ribosome and adds its aminoacylated alanine to the nascent polypeptide. The original mRNA is released and tmRNA becomes the template for translation of a 10-amino-acid tag that signals for proteolytic degradation. Although essential in a few bacterial species, tmRNA is nonessential in Escherichia coli and many other bacteria. Proteins known to be associated with tmRNA include SmpB, ribosomal protein S1, RNase R, and phosphoribosyl pyrophosphate. SmpB, having no other known function, is essential for tmRNA activity. trans-translation operates within ribosomes stalled both at the end of truncated mRNAs and at rare codons and some natural termination sites. Both the release of stalled ribosomes and the subsequent degradation of tagged proteins are important consequences of trans-translation.
Assuntos
Biossíntese de Proteínas/fisiologia , RNA Bacteriano/metabolismo , Ribossomos/metabolismo , Códon/metabolismo , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas/genéticaAssuntos
Bacteriófago lambda/genética , Bioquímica/métodos , Transcrição Gênica , Sequência de Bases , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Toxina Shiga/química , beta-Galactosidase/metabolismoRESUMO
The NusA transcription elongation protein, which binds RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains. An essential function in E. coli, NusA is also one of the host factors required for action of the N transcription antitermination protein of lambda. Tandem KH domains have been identified downstream of the S1 domain. We changed the first Gly to Asp of the GXXG motif, a tetrapeptide diagnostic of KH domains, of both NusA KH domains. The change in the first, G253D, has a large effect, while the change in the second, G319D, has a small effect on NusA action. The changes in both KH domains interfere with NusA binding to RNA. A change of a highly conserved Arg in the S1 domain, R199A, has previously been reported to interfere with RNA binding while exerting a small effect on NusA action. However, a nusA allele with both the R199A and G319D changes encodes a functionally inactive NusA protein. These studies provide direct evidence that the both KH as well as the S1 RNA binding domains are important for NusA action in support of bacterial viability as well as transcription antitermination mediated by the lambda N protein.
Assuntos
Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Estrutura Terciária de Proteína/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da TranscriçãoRESUMO
Transcription antitermination by the bacteriophage lambda N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófago lambda/metabolismo , Proteínas de Escherichia coli , Fatores de Alongamento de Peptídeos/metabolismo , Regiões Terminadoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Virais Reguladoras e Acessórias/metabolismo , Escherichia coli/virologiaRESUMO
The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the lambda bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage phi 361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.
Assuntos
Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Escherichia coli/metabolismo , Escherichia coli/virologia , Toxina Shiga I/biossíntese , Toxina Shiga I/genética , Bacteriófago lambda/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Dosagem de Genes , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais/genética , Genoma Viral , Ferro/metabolismo , Ferro/farmacologia , Mitomicina/farmacologia , Regiões Promotoras Genéticas/genética , Provírus/efeitos dos fármacos , Provírus/genética , Provírus/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Toxina Shiga I/metabolismo , Regiões Terminadoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica , Proteínas Virais/genética , Virulência/genética , Ativação Viral , Replicação ViralRESUMO
The unique transfer-messenger RNA (tmRNA) molecule has been identified in all bacterial species examined, suggesting that its action confers an important survival advantage to bacteria. Acting both as a tRNA and an mRNA, in a process known as trans-translation, tmRNA adds a short peptide tag to undesirable proteins. Trans-translation plays at least two physiological roles: removing ribosomes stalled upon mRNA, and targeting the resulting truncated proteins for degradation by proteases. The first of these roles is required for all known activities of tmRNA, whereas the second may be dispensed with in most cases with little biological effect. However, tmRNA-targeted proteolysis may be important for fine-tuning expression of certain genes by altering the concentration of regulatory proteins. Here, we review recent literature that addresses the biological functions of tmRNA.