RESUMO
Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.
Assuntos
Guanosina Tetrafosfato , Transdução de Sinais/fisiologia , Animais , Bactérias/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/metabolismo , Guanosina Tetrafosfato/fisiologia , Pirofosfatases/metabolismo , Pirofosfatases/fisiologia , Sistemas do Segundo MensageiroRESUMO
Riboswitches are noncoding portions of certain mRNAs that bind metabolite, coenzyme, signaling molecule, or inorganic ion ligands and regulate gene expression. Most known riboswitches sense derivatives of RNA monomers. This bias in ligand chemical composition is consistent with the hypothesis that widespread riboswitch classes first emerged during the RNA World, which is proposed to have existed before proteins were present. Here we report the discovery and biochemical validation of a natural riboswitch class that selectively binds guanosine tetraphosphate (ppGpp), a widespread signaling molecule and bacterial "alarmone" derived from the ribonucleotide GTP. Riboswitches for ppGpp are predicted to regulate genes involved in branched-chain amino acid biosynthesis and transport, as well as other gene classes that previously had not been implicated to be part of its signaling network. This newfound riboswitch-alarmone partnership supports the hypothesis that prominent RNA World signaling pathways have been retained by modern cells to control key biological processes.
Assuntos
Guanosina Tetrafosfato/fisiologia , Riboswitch/fisiologia , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Ligantes , Conformação de Ácido Nucleico , RNA Bacteriano , Ribonucleotídeos/metabolismo , Riboswitch/genética , Transdução de SinaisRESUMO
The cyanobacterium Synechococcus elongatus relies upon photosynthesis to drive metabolism and growth. During darkness, Synechococcus stops growing, derives energy from its glycogen stores, and greatly decreases rates of macromolecular synthesis via unknown mechanisms. Here, we show that the stringent response, a stress response pathway whose genes are conserved across bacteria and plant plastids, contributes to this dark adaptation. Levels of the stringent response alarmone guanosine 3'-diphosphate 5'-diphosphate (ppGpp) rise after a shift from light to dark, indicating that darkness triggers the same response in cyanobacteria as starvation in heterotrophic bacteria. High levels of ppGpp are sufficient to stop growth and dramatically alter many aspects of cellular physiology, including levels of photosynthetic pigments and polyphosphate, DNA content, and the rate of translation. Cells unable to synthesize ppGpp display pronounced growth defects after exposure to darkness. The stringent response regulates expression of a number of genes in Synechococcus, including ribosomal hibernation promoting factor (hpf), which causes ribosomes to dimerize in the dark and may contribute to decreased translation. Although the metabolism of Synechococcus differentiates it from other model bacterial systems, the logic of the stringent response remains remarkably conserved, while at the same time having adapted to the unique stresses of the photosynthetic lifestyle.
Assuntos
Adaptação Fisiológica , Synechococcus/fisiologia , Aclimatação , Replicação do DNA , Escuridão , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/análise , Guanosina Tetrafosfato/fisiologia , FotossínteseRESUMO
The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases--RsgA, RbgA, Era, HflX, and ObgE--as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Tetrafosfato/fisiologia , Biogênese de Organelas , Ribossomos/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Enterococcus faecalis/metabolismo , Escherichia coli , Biblioteca Gênica , Guanosina Trifosfato/biossíntese , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Fases de Leitura Aberta , Ligação Proteica , Ribossomos/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
In bacteria a second messenger, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), synthesized upon nutrient starvation, controls many gene expressions and enzyme activities, which is necessary for growth under changeable environments. Recent studies have shown that ppGpp synthase and hydrolase are also conserved in eukaryotes, although their functions are not well understood. We recently showed that ppGpp-overaccumulation in Arabidopsis chloroplasts results in robust growth under nutrient-limited conditions, demonstrating that the bacterial-like stringent response at least functions in plastids. To test if ppGpp also functions in the cytosol, we constructed the transgenic Arabidopsis expressing Bacillus subtilis ppGpp synthase gene yjbM. Upon induction of the gene, the mutant synthesizes â¼10-20-fold higher levels of ppGpp, and its fresh weight was reduced to Ë80% that of the wild type. These results indicate that cytosolic ppGpp negatively regulates plant growth and development.
Assuntos
Arabidopsis/genética , Guanosina Tetrafosfato/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Citosol/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Plantas Geneticamente ModificadasRESUMO
The stringent response, mediated by second messenger (p)ppGpp, results in swift and massive transcriptional reprogramming under nutrient limited conditions. In this study, the role of (p)ppGpp on virulence of Pseudomonas syringae pv. syringaeâ B728a (PssB728a) was investigated. The virulence of the relA/spoT (ppGpp(0) ) double mutant was completely impaired on bean, and bacterial growth was significantly reduced, suggesting that (p)ppGpp is required for full virulence of P. syringae. Expression of T3SS and other virulence genes was reduced in ppGpp(0) mutants. In addition, ppGpp deficiency resulted in loss of swarming motility, reduction of pyoverdine production, increased sensitivity to oxidative stress and antibiotic tolerance, as well as reduced ability to utilize γ-amino butyric acid. Increased levels of ppGpp resulted in reduced cell size of PssB728a when grown in a minimal medium and on plant surfaces, while most ppGpp(0) mutant cells were not viable on plant surfaces 24 h after spray inoculation, suggesting that ppGpp-mediated stringent response temporarily limits cell growth, and might control cell survival on plants by limiting their growth. These results demonstrated that ppGpp-mediated stringent response plays a central role in P. syringae virulence and survival and indicated that ppGpp serves as a global signal for regulating various virulence traits in PssB728a.
Assuntos
Proteínas de Bactérias/genética , Guanosina Pentafosfato/fisiologia , Guanosina Tetrafosfato/fisiologia , Doenças das Plantas/microbiologia , Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Farmacorresistência Bacteriana , Guanosina Pentafosfato/genética , Guanosina Tetrafosfato/genética , Oligopeptídeos/biossíntese , Estresse Oxidativo/genética , Folhas de Planta/microbiologia , Pseudomonas syringae/genética , Sistemas do Segundo Mensageiro/genética , Virulência , Fatores de Virulência/genéticaRESUMO
During active growth of Escherichia coli, majority of the transcriptional activity is carried out by the housekeeping sigma factor (sigma(70)), whose association with core RNAP is generally favoured because of its higher intracellular level and higher affinity to core RNAP. In order to facilitate transcription by alternative sigma factors during nutrient starvation, the bacterial cell uses multiple strategies by which the transcriptional ability of sigma(70) is diminished in a reversible manner. The facilitators of shifting the balance in favour of alternative sigma factors happen to be as diverse as a small molecule (p)ppGpp (represents ppGpp or pppGpp), proteins (DksA, Rsd) and a species of RNA (6S RNA). Although 6S RNA and (p)ppGpp were known in literature for a long time, their role in transcriptional switching has been understood only in recent years. With the elucidation of function of DksA, a new dimension has been added to the phenomenon of stringent response. As the final outcome of actions of (p)ppGpp, DksA, 6S RNA and Rsd is similar, there is a need to analyse these mechanisms in a collective manner. We review the recent trends in understanding the regulation of sigma(70) by (p)ppGpp, DksA, Rsd and 6S RNA and present a case for evolving a unified model of RNAP redistribution during starvation by modulation of sigma(70) activity in E. coli.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Guanosina Tetrafosfato/fisiologia , RNA Bacteriano/fisiologia , RNA não Traduzido , Proteínas Repressoras/fisiologia , Fator sigma/genéticaRESUMO
In most bacteria, nutrient limitations provoke the stringent control by the rapid synthesis of the alarmones pppGpp and ppGpp. The most prominent and highly conserved reaction is the repression of rRNA synthesis. Additionally, (p)ppGpp synthesis is also linked to many other physiological changes involving gene activation/repression but also protein translation, enzyme activation and replication. Whereas much of the basic research was performed with Escherichia coli there is now growing evidence that in gram-positive bacteria there are fundamental differences in (p)ppGpp synthesis, regulation and molecular function. Here we will focus on basic differences between firmicutes and proteobacteria, particularly E. coli.
Assuntos
Bactérias Gram-Positivas/metabolismo , Guanosina Pentafosfato/fisiologia , Guanosina Tetrafosfato/fisiologia , Adaptação Fisiológica , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/genética , Ligases/química , Ligases/genética , Ligases/metabolismo , Proteobactérias/química , Proteobactérias/genética , Proteobactérias/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/genéticaRESUMO
Group II introns are hypothesized to share common ancestry with both nuclear spliceosomal introns and retrotransposons, which collectively occupy the majority of genome space in higher eukaryotes. These phylogenetically diverse introns are mobile retroelements that move through an RNA intermediate. Disruption of Escherichia coli genes encoding enzymes that catalyze synthesis of global regulators cAMP and ppGpp inhibits group II intron retromobility. These small molecules program genetic transitions between nutrient excess and starvation. Accordingly, we demonstrated that glucose depletion of wild-type cells and cAMP supplementation of mutants stimulated retromobility. Likewise, amino acid starvation, which induces the alarmone ppGpp, activated retromobility. In both cases, retrotransposition to ectopic sites was favored over retrohoming. Interestingly, these stimulatory effects are mediated at the level of the DNA target, rather than of expression of the retroelement. Thereby, during metabolic stress, cAMP and ppGpp control group II intron movement in concert with the cell's global genetic circuitry, stimulating genetic diversity.
Assuntos
AMP Cíclico/fisiologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Tetrafosfato/fisiologia , Íntrons/genética , Retroelementos/fisiologia , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Aminoácidos/metabolismo , Cromossomos Bacterianos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética , Pirofosfatases/genética , Pirofosfatases/metabolismo , RNA Bacteriano/metabolismo , Retroelementos/genéticaRESUMO
The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp(0)) and/or DksA (DeltadksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp(0) DeltadksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/fisiologia , Transcrição Gênica/genética , Western Blotting , Quimiotaxia/genética , Proteínas de Escherichia coli/genética , Flagelos/genética , Guanosina Tetrafosfato/genética , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although in bacterial cells all genes are transcribed by RNA polymerase, there are 2 additional enzymes capable of catalyzing RNA synthesis: poly(A) polymerase I, which adds poly(A) residues to transcripts, and primase, which produces primers for DNA replication. Mechanisms of actions of these 3 RNA-synthesizing enzymes were investigated for many years, and schemes of their regulations have been proposed and generally accepted. Nevertheless, recent discoveries indicated that apart from well-understood mechanisms, there are additional regulatory processes, beyond the established schemes, which allow bacterial cells to respond to changing environmental and physiological conditions. These newly discovered mechanisms, which are discussed in this review, include: (i) specific regulation of gene expression by RNA polyadenylation, (ii) control of DNA replication by interactions of the starvation alarmones, guanosine pentaphosphate and guanosine tetraphosphate, (p)ppGpp, with DnaG primase, (iii) a role for the DksA protein in ppGpp-mediated regulation of transcription, (iv) allosteric modulation of the RNA polymerase catalytic reaction by specific inhibitors of transcription, rifamycins, (v) stimulation of transcription initiation by proteins binding downstream of the promoter sequences, and (vi) promoter-dependent control of transcription antitermination efficiency.
Assuntos
Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/biossíntese , Guanosina Tetrafosfato/fisiologia , Transcrição GênicaRESUMO
Bacillus subtilis and Streptomyces spp. provide tractable experimental systems for studying cellular responses to adverse environmental conditions. During conditions of extreme nutrient limitation, these prokaryotes exhibit a wide range of adaptations, including the production and secretion of antibiotics and enzymes and the formation of aerial mycelium and spores. In response to these conditions, all bacteria, but not eukaryotic microorganisms, exhibit a "stringent response," during which the unusual guanosine tetraphosphate, ppGpp, accumulates intracellularly. This is accompanied by a marked reduction in the GTP pool, due to ppGpp inhibition of IMP-dehydrogenase, and immediate repression of rRNA synthesis, due to the binding of ppGpp to RNA polymerase. This review summarizes our studies on the bacterial stringent response and its use in applied microbiology. We found that morphological differentiation results from a decrease in the pool of GTP, whereas physiological differentiation (antibiotic production) results from a more direct function of ppGpp. That is, we found that the Streptomyces GTP-binding protein Obg functions by sensing intracellular GTP levels and that certain mutations in the RNA polymerase beta-subunit circumvent dependence on ppGpp in antibiotic production. X-ray crystallographic analysis provided a structural basis for the ppGpp regulation of transcription. On the basis of these findings, we have developed the novel concept of "ribosome engineering," focusing on activation of dormant genes to elicit cellular function fully. Ribosome engineering can be applied to strain improvement, screening of novel metabolites, plant breeding, cell-free translation systems, and the treatment of tuberculosis.
Assuntos
Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Engenharia de Proteínas/métodos , Ribossomos/genética , Antibacterianos/biossíntese , Bacillus subtilis/genética , RNA Polimerases Dirigidas por DNA/genética , Guanosina Tetrafosfato/metabolismo , Guanosina Tetrafosfato/fisiologia , Streptomyces/genéticaRESUMO
The recent discovery that the protein DksA acts as a coregulator of genes controlled by ppGpp led us to investigate the similarities and differences between the relaxed phenotype of a ppGpp-deficient mutant and the phenotype of a strain lacking DksA. We demonstrate that the absence of DksA and ppGpp has similar effects on many of the observed phenotypes but that DksA and ppGpp also have independent and sometimes opposing roles in the cell. Specifically, we show that overexpression of DksA can compensate for the loss of ppGpp with respect to transcription of the promoters P(uspA), P(livJ), and P(rrnBP1) as well as amino acid auxotrophy, cell-cell aggregation, motility, filamentation, and stationary phase morphology, suggesting that DksA can function without ppGpp in regulating gene expression. In addition, ppGpp and DksA have opposing effects on adhesion. In the course of our analysis, we also discovered new features of the relaxed mutant, namely, defects in cell-cell aggregation and motility.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Guanosina Tetrafosfato/metabolismo , Aminoácidos/metabolismo , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Western Blotting , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/fisiologia , Microscopia Eletrônica de Varredura , Modelos Biológicos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
During infection of mammalian hosts, facultative intracellular pathogens have to adjust rapidly to different environmental conditions encountered during passage through the gastrointestinal tract and following uptake into epithelial cells and macrophages. Successful establishment within the host therefore requires the coordinated expression of a large number of virulence genes necessary for the adaptation between the extracellular and intracellular phases of infection. In this study we show that the bacterial signal molecule, ppGpp, plays a major role in mediating the environmental signals involved in the regulation of both the extracellular and intracellular virulence gene programs. Under oxygen limiting conditions, we observed a strong ppGpp dependence for invasion gene expression, the result of severe reductions in expression of the Salmonella pathogenicity island (SPI) 1 transcriptional regulator genes hilA, C, and D and invF. Overexpression of the non-SPI1-encoded regulator RtsA restored hilA expression in the absence of ppGpp. SPI2-encoded genes, required for intracellular proliferation in macrophages, were activated in the wild type strain under aerobic, late log phase growth conditions. The expression of SPI2 genes was also shown to be ppGpp-dependent under these conditions. The results from this study suggest a mechanism for the alternate regulation of the opposing extracellular and intracellular virulence gene programs and indicate a remarkable specificity for ppGpp in the regulation of genes involved in virulence compared with the rest of the genome. This is the first demonstration that this highly conserved regulatory system is involved in bacterial virulence gene expression on a global scale.
Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Guanosina Tetrafosfato/fisiologia , Líquido Intracelular/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Transdução de Sinais/genética , Fatores de Virulência/genética , Líquido Intracelular/fisiologia , Salmonella typhimurium/fisiologia , Fatores de Virulência/biossíntese , Fatores de Virulência/fisiologiaAssuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/fisiologia , Regiões Promotoras Genéticas , Fator sigma/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Fator sigma/genética , Transcrição GênicaRESUMO
The sigma subunit of procaryotic RNA polymerases is responsible for specific promoter recognition and transcription initiation. In addition to the major sigma factor, sigma 70, in Escherichia coli, which directs most of the transcription in the cell, bacteria possess multiple, alternative sigma factors that direct RNA polymerase to distinct sets of promoters in response to environmental signals. By activating an alternative sigma factor, gene expression can be rapidly reprogrammed to meet the needs of the cell as the environment changes. Sigma factors are subject to multiple levels of regulation that control their levels and activities. The alternative sigma factor sigmaE in Escherichia coli is induced in response to extracytoplasmic stress. Here we demonstrate that sigmaE can also respond to signals other than extracytoplasmic stress. sigmaE activity increases in a growth phase-dependent manner as a culture enters stationary phase. The signaling pathway that activates sigmaE during entry into stationary phase is dependent upon the alarmone guanosine 3',5'-bispyrophosphate (ppGpp) and is distinct from the pathway that signals extracytoplasmic stress. ppGpp is the first cytoplasmic factor shown to control sigmaE activity, demonstrating that sigmaE can respond to internal signals as well as signals originating in the cell envelope. ppGpp is a general signal of starvation stress and is also required for activation of the sigmaS and sigma 54 alternative sigma factors upon entry into stationary phase, suggesting that this is a key mechanism by which alternative sigma factors can be activated in concert to provide a coordinated response to nutritional stress.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/fisiologia , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/metabolismo , Regulação para CimaRESUMO
In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp-deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp(0) suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp(0) strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp-stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/fisiologia , Guanosina Tetrafosfato/fisiologia , Integrases/genética , Aglutinação , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , DNA Nucleotidiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Guanosina Pentafosfato/genética , Guanosina Pentafosfato/farmacologia , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/farmacologia , Mutação , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator sigma/metabolismo , Supressão Genética , Transcrição Gênica/efeitos dos fármacos , Doenças Urológicas/microbiologia , Leveduras/citologiaRESUMO
Starvation-induced development of Myxococcus xanthus is an excellent model for biofilm formation because it involves cell-cell signaling to coordinate formation of multicellular mounds, gene expression, and cellular differentiation into spores. The role of sigma(D), an alternative sigma factor important for viability in stationary phase and for stress responses, was investigated during development by measuring signal production, gene expression, and sporulation of a sigD null mutant alone and upon codevelopment with wild-type cells or signaling mutants. The sigD mutant responded to starvation by inducing (p)ppGpp synthesis normally but was impaired for production of A-signal, an early cell density signal, and for production of the morphogenetic C-signal. Induction of early developmental genes was greatly reduced, and expression of those that depend on A-signal was not restored by codevelopment with wild-type cells, indicating that sigma(D) is needed for cellular responses to A-signal. Despite these early developmental defects, the sigD mutant responded to C-signal supplied by codeveloping wild-type cells by inducing a subset of late developmental genes. sigma(D) RNA polymerase is dispensable for transcription of this subset, but a distinct regulatory class, which includes genes essential for sporulation, requires sigma(D) RNA polymerase or a gene under its control, cell autonomously. The level of sigD transcript in a relA mutant during growth is much lower than in wild-type cells, suggesting that (p)ppGpp positively regulates sigD transcription in growing cells. The sigD transcript level drops in wild-type cells after 20 min of starvation and remains low after 40 min but rises in a relA mutant after 40 min, suggesting that (p)ppGpp negatively regulates sigD transcription early in development. We conclude that sigma(D) synthesized during growth occupies a position near the top of a regulatory hierarchy governing M. xanthus development, analogous to sigma factors that control biofilm formation of other bacteria.
Assuntos
Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus/fisiologia , Fator sigma/fisiologia , Transdução de Sinais , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Guanosina Tetrafosfato/fisiologia , Myxococcus xanthus/genética , Myxococcus xanthus/crescimento & desenvolvimento , Óperon , Fosfotransferases/metabolismo , Fator sigma/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Fatores de TempoRESUMO
Guanosine 3',5'-bispyrophosphate (ppGpp), also known as "magic spot," has been shown to bind prokaryotic RNA polymerase to down-regulate ribosome production and increase transcription of amino acid biosynthesis genes during the stringent response to amino acid starvation. Because many environmental growth perturbations cause ppGpp to accumulate, we hypothesize ppGpp to have an overarching role in regulating the genetic program that coordinates transitions between logarithmic growth (feast) and growth arrest (famine). We used the classic glucose-lactose diauxie as an experimental system to investigate the temporal changes in transcription that accompany growth arrest and recovery in wild-type Escherichia coli and in mutants that lack RelA (ppGpp synthetase) and other global regulators, i.e., RpoS and Crp. In particular, diauxie was delayed in the relA mutant and was accompanied by a 15% decrease in the number of carbon sources used and a 3-fold overall decrease in the induction of RpoS and Crp regulon genes. Thus the data significantly expand the previously known role of ppGpp and support a model wherein the ppGpp-dependent redistribution of RNA polymerase across the genome is the driving force behind control of the stringent response, general stress response, and starvation-induced carbon scavenging. Our conceptual model of diauxie describes these global control circuits as dynamic, interconnected, and dependent upon ppGpp for the efficient temporal coordination of gene expression that programs the cell for transitions between feast and famine.