RESUMO
The alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen. IMPORTANCE Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , GTP Pirofosfoquinase/metabolismo , Guanosina Tetrafosfato/biossíntese , Doenças das Plantas/microbiologia , Pirofosfatases/metabolismo , Xanthomonas campestris/crescimento & desenvolvimento , Xanthomonas campestris/patogenicidade , Proteínas de Bactérias/genética , GTP Pirofosfoquinase/genética , Pirofosfatases/genética , Raphanus/microbiologia , Virulência , Xanthomonas campestris/enzimologia , Xanthomonas campestris/genéticaRESUMO
Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.
Assuntos
Antibacterianos/farmacologia , Cólera/dietoterapia , Isotiocianatos/farmacologia , Mariposas/microbiologia , Sulfóxidos/farmacologia , Vibrio cholerae/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , DNA/biossíntese , Modelos Animais de Doenças , Guanosina Tetrafosfato/biossíntese , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA/biossíntese , Células Vero , Vibrio cholerae/patogenicidade , Virulência/efeitos dos fármacos , Fatores de Virulência/biossínteseRESUMO
The nucleotide derivatives (p)ppGpp, comprising ppGpp and pppGpp, are important signalling molecules that control various facets of gene regulation and protein synthesis in Escherichia coli. Their synthesis is catalysed by RelA (in response to amino acid limitation) and by SpoT (in response to the limitation of carbon source or fatty acids). SpoT is also a hydrolase for degradation of both ppGpp and pppGpp, while GppA catalyses the conversion of pppGpp to ppGpp. Here we provide evidence to show that pppGpp exerts heightened toxicity compared to that by ppGpp. Thus, gppA spoT double mutants exhibited lethality under conditions in which the single mutants were viable. The extent of RelA-catalysed (p)ppGpp accumulation in the gppA spoT strain was substantially greater than that in its isogenic gppA+ derivative. The data is interpreted in terms of a model in which toxicity of pppGpp in the gppA spoT mutants is mediated by its activation of RelA so as to result in a vicious cycle of (p)ppGpp synthesis.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Guanosina Pentafosfato/análise , Guanosina Pentafosfato/biossíntese , Guanosina Tetrafosfato/análise , Guanosina Tetrafosfato/biossíntese , Pirofosfatases/metabolismo , Mutação , Fenótipo , PlasmídeosRESUMO
Glyphosate, the most widely used herbicide in both agricultural and urban areas is toxic for plants and for many bacterial species. The mechanism of action of glyphosate is through the inhibition of the EPSP synthase, a key enzyme in the biosynthetic pathway of aromatic amino acids. Here we show that glyphosate induces the stringent response in Escherichia coli. Bacteria treated with glyphosate stop growing and accumulate ppGpp. Both growth arrest and ppGpp accumulation are restored to normal levels upon addition of aromatic amino acids. Glyphosate-induced ppGpp accumulation is dependent on the presence of the (p)ppGpp synthetase RelA. However, unlike other cases of amino acid starvation, pppGpp could not be discerned. In a gppA background both ppGpp and pppGpp accumulated when exposed to glyphosate. Conversely, the wild-type strain and gppA mutant treated with serine hydroxamate accumulated high levels of both ppGpp and pppGpp. Altogether, the data indicate that glyphosate induces amino acid starvation resulting in a moderate accumulation of ppGpp and a reversible stringent response.
Assuntos
Escherichia coli/efeitos dos fármacos , Glicina/análogos & derivados , Guanosina Tetrafosfato/biossíntese , Aminoácidos Aromáticos/farmacologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Glicina/farmacologia , Ligases/genética , Ligases/metabolismo , Mutação , Serina/análogos & derivados , Serina/farmacologia , GlifosatoRESUMO
RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNAâ RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.
Assuntos
Escherichia coli K12/enzimologia , Guanosina Tetrafosfato/biossíntese , Ligases/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/enzimologia , Aminoácidos/deficiência , Sítios de Ligação , Escherichia coli K12/genética , Ligases/genética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , RNA de Transferência/genética , Ribossomos/genéticaRESUMO
The stringent response is a conserved bacterial stress response mechanism that allows bacteria to respond to nutritional challenges. It is mediated by the alarmones pppGpp and ppGpp, nucleotides that are synthesized and hydrolyzed by members of the RSH superfamily. Whilst there are key differences in the binding targets for (p)ppGpp between Gram-negative and Gram-positive bacterial species, the transient accumulation of (p)ppGpp caused by nutritional stresses results in a global change in gene expression in all species. The RSH superfamily of enzymes is ubiquitous throughout the bacterial kingdom, and can be split into three main groups: the long-RSH enzymes; the small alarmone synthetases (SAS); and the small alarmone hydrolases (SAH). Despite the prevalence of these enzymes, there are important differences in the way in which they are regulated on a transcriptional and post-translational level. Here we provide an overview of the diverse regulatory mechanisms that are involved in governing this crucial signalling network. Understanding how the RSH superfamily members are regulated gives insights into the varied important biological roles for this signalling pathway across the bacteria.
Assuntos
Fenômenos Fisiológicos Bacterianos , Guanosina Pentafosfato/biossíntese , Guanosina Tetrafosfato/biossíntese , Transdução de Sinais , Estresse Fisiológico/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Especificidade por SubstratoRESUMO
Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia coli strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non-dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non-dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that persisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Perfilação da Expressão Gênica , Ofloxacino/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/biossíntese , Anaerobiose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Dimetil Sulfóxido/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Guanosina Tetrafosfato/biossíntese , L-Lactato Desidrogenase/biossíntese , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Nitritos/metabolismoRESUMO
Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.
Assuntos
Regulação Alostérica/fisiologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/biossíntese , Guanosina Tetrafosfato/biossíntese , Ligases/fisiologia , Proteínas de Bactérias/química , Catálise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Clonagem Molecular , Cristalização , Escherichia coli , Ligases/metabolismo , Espectrometria de Massas , MutagêneseRESUMO
UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.
Assuntos
Enterococcus faecalis/enzimologia , Enterococcus faecalis/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/biossíntese , Ligases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biossíntese , Desoxiguanosina/química , Dipeptídeos/biossíntese , Dipeptídeos/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/genética , Magnésio , Estrutura Molecular , Estresse Fisiológico , Especificidade por SubstratoRESUMO
Two genes encoding RelA/SpoT homologs, PpRSH2a and PpRSH2b, which are involved in the synthesis of bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) for the stringent response, were isolated from the moss, Physcomitrella patens. A complementary analysis of PpRSH2a and PpRSH2b in Escherichia coli showed that these genes had ppGpp biosynthetic activity. The recombinant PpRSH2a and PpRSH2b were also shown to synthesize ppGpp in vitro. Both proteins were localized to the chloroplasts of P. patens. Expression of the PpRSH genes was induced upon treatment with abscisic acid or abiotic stresses, such as dehydration and UV irradiation. Overexpression of PpRSH2a and PpRSH2b caused suppression of the growth in response to 1% (w/v) of glucose. The present study suggests the existence of a mechanism to regulate the growth of P. patens, which is governed by plant RSH in chloroplasts.
Assuntos
Bryopsida/genética , Cloroplastos/genética , Guanosina Tetrafosfato/biossíntese , Ligases/genética , Proteínas de Plantas/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Bryopsida/efeitos dos fármacos , Bryopsida/crescimento & desenvolvimento , Bryopsida/efeitos da radiação , Cloroplastos/efeitos dos fármacos , Cloroplastos/enzimologia , Cloroplastos/efeitos da radiação , Dessecação , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ligases/metabolismo , Dados de Sequência Molecular , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estresse Fisiológico , Raios UltravioletaRESUMO
Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.
Assuntos
Proteínas de Bactérias/genética , Ligases/genética , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Guanosina Tetrafosfato/biossíntese , Hidrólise , Ligases/química , Ligases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In addition, expression of E. coli GppA, a pppGpp 5'-gamma phosphate hydrolase that converts pppGpp to ppGpp, is manipulated to fine tune differential accumulation of ppGpp and pppGpp. In vivo and in vitro experiments show that pppGpp is less potent than ppGpp with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter transcription inhibition, threonine operon promoter activation and RpoS induction. To provide further insights into regulation by (p)ppGpp, we have also determined crystal structures of E. coli RNA polymerase-σ(70) holoenzyme with ppGpp and pppGpp. We find that both nucleotides bind to a site at the interface between ß' and ω subunits.
Assuntos
Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Arabinose/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Guanosina Pentafosfato/biossíntese , Guanosina Pentafosfato/química , Guanosina Tetrafosfato/biossíntese , Guanosina Tetrafosfato/química , Hidrolases/metabolismo , Ligases/metabolismo , Óperon , Regiões Promotoras Genéticas , RNA Bacteriano/biossíntese , RNA Ribossômico/genética , Fator sigma/química , Fator sigma/metabolismo , Especificidade por SubstratoRESUMO
Antibiotic-producing Streptomyces are complex bacteria that remodel global transcription patterns and their nucleoids during development. Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp. Owing to its N-terminal domain, the protein can efficiently bind and condense DNA in vitro. Loss of function of this DNA-binding protein results in changes in both DNA condensation during development and the ability to adjust DNA supercoiling in response to osmotic stress. Initial analysis of the DksA-like activity of DdbA indicates that overexpression of the protein suppresses a conditional deficiency in antibiotic production of relA mutants that are unable to synthesise ppGpp, just as DksA overexpression in Escherichia coli can suppress ppGpp(0) phenotypes. The null mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor. Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.
Assuntos
DNA Fúngico/química , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Streptomyces coelicolor/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Guanosina Tetrafosfato/biossíntese , Histonas/química , Ligases/genética , Conformação de Ácido Nucleico , Pressão Osmótica , Estrutura Terciária de Proteína , Esporos Fúngicos/fisiologia , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/fisiologia , Estresse Fisiológico/genética , Transcrição GênicaRESUMO
During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.
Assuntos
Escherichia coli/enzimologia , Guanosina Tetrafosfato/biossíntese , Ligases/metabolismo , Regulação Alostérica , Ativação Enzimática , Escherichia coli/metabolismo , Ribossomos/metabolismoRESUMO
OBJECTIVE: Nucleotide guanosine-3', 5'-(bis) pyrophosphate (ppGpp) synthesized by (ppGpp) synthesase RelA or bifunctional ppGpp synthase/degradase RelA/SpoT, mediates bacterial stringent response to various stressful conditions. Here we characterized the slr1325 (syn-rsh) gene encoding a RelA/SpoT homolog (Syn-RSH) of the cyanobacterium Synechocystis sp. PCC6803. METHODS: We performed phenotypic complement test using Escherichia coli strain with(p) ppGpp-synthesis defect to determine Syn-RSH function(s), and employed chromatographic analysis of 32P-labeled cellular mononucleotides to detect the accumulation of ppGpp in Escherichia coli strains expressing Syn-RSH and in Synechocystis sp. PCC6803. RESULTS: Syn-RSH expression in E. coli relA/spoT double mutant was able to restore the cell growth arrest; Chromatographic analysis of 32P-labeled cellular mononucleotides revealed that Syn-RSH expression resulted in the synthesis of ppGpp in E. coli strain with relA and spoT mutant mutation. Additionally, Synechocystis cells accumulated a low level of ppGpp under laboratory growth conditions. CONCLUSION: Syn-RSH possesses ppGpp synthase/degradase activities, and ppGpp is required for Synechocystis cell viability under normal growth conditions.
Assuntos
Genes Bacterianos , Ligases/genética , Synechocystis/genética , Escherichia coli/genética , Guanosina Tetrafosfato/biossínteseRESUMO
The hyperphosphorylated guanine ribonucleotide ppGpp mediates the stringent response in bacteria. Biochemical and genetic studies of this response in Escherichia coli have shown that the biosynthesis of ppGpp is catalysed by two homologous enzymes, RelA and SpoT. RelA is activated in response to amino acid starvation, and SpoT responds to abiotic physical stress beside nutritional stress. All free-living bacteria, including Gram-positive firmicutes, contain RelA-SpoT homologues (RSH). Further, novel ppGpp biosynthetic enzymes, designated small alarmone synthetases (SASs), were recently identified in a subset of bacteria, including the Gram-positive organism Bacillus subtilis, and were shown to consist only of a ppGpp synthetase domain. Studies suggest that these SAS proteins contribute to ppGpp signalling in response to stressful conditions in a manner distinct from that of RelA-SpoT enzymes. SAS proteins currently appear to always occur in addition to RSH enzymes in various combinations but never alone. RSHs have also been identified in chloroplasts, organelles of photosynthetic eukaryotes that originated from endosymbiotic photosynthetic bacteria. These chloroplast RSHs are exclusively encoded in nuclear DNA and targeted into chloroplasts. The findings suggest that ppGpp may regulate chloroplast functions similar to those regulated in bacteria, including transcription and translation. In addition, a novel ppGpp synthetase that is regulated by Ca²âº as a result of the presence of two EF-hand motifs at its COOH terminus was recently identified in chloroplasts of land plants. This finding indicates the existence of a direct connection between eukaryotic Ca²âº signalling and prokaryotic ppGpp signalling in chloroplasts. The new observations with regard to ppGpp signalling in land plants suggest that such signalling contributes to the regulation of a wider range of cellular functions than previously anticipated.
Assuntos
Bactérias/metabolismo , Cloroplastos/metabolismo , Guanosina Tetrafosfato/metabolismo , Plantas/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Bactérias/genética , Cloroplastos/enzimologia , Cloroplastos/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/biossíntese , Guanosina Tetrafosfato/genética , Ligases/metabolismo , Plantas/enzimologia , Plantas/genética , Pirofosfatases/genética , Pirofosfatases/metabolismo , Transdução de SinaisRESUMO
Bacteria have developed sophisticated signal transduction pathways to sense and respond to environmental stresses. These pathways include intracellular regulators that elicit adaptive changes in the physiology of the cell. Extensive work, mostly performed in Escherichia coli, showed that the modified nucleotide ppGpp plays a key regulatory role by co-ordinating the cellular responses to adverse environmental conditions. In this issue of Molecular Microbiology, Traxler et al. define two sets of ppGpp-dependent genes that are expressed at different times after induction of ppGpp synthesis. Their results suggest that quantitative differences in the ppGpp intracellular concentration determine the precise pattern of gene expression during adaptation process: low levels of ppGpp suffice to activate the Lrp regulon, which, by activating the synthesis of some amino acid pathways, can generate a negative feedback loop while high levels activate RpoS and a feed-forward amplification of the general stress response. These dose-dependent effects on gene expression open new perspectives on the complex regulatory pathways mediated by ppGpp during environmental adaptation.
Assuntos
Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/biossíntese , Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Proteína Reguladora de Resposta a Leucina/metabolismo , Regulon , Fator sigma/metabolismo , Estresse FisiológicoRESUMO
Bacteria comprehensively reorganize their global gene expression when faced with starvation. The alarmone ppGpp facilitates this massive response by co-ordinating the downregulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation requires the activities of multiple regulators, yet the regulatory network downstream of ppGpp remains poorly defined. Transcription profiling during isoleucine depletion, which leads to gradual starvation (over > 100 min), allowed us to identify genes that required ppGpp, Lrp and RpoS for their induction and to deduce the regulon response times. Although the Lrp and RpoS regulons required ppGpp for their activation, they were not induced simultaneously. The data suggest that metabolic genes, i.e. those of the Lrp regulon, require only a low level of ppGpp for their induction. In contrast, the RpoS regulon was induced only when high levels of ppGpp accumulated. We tested several predictions of a model that explains how bacteria allocate transcriptional resources between metabolism and stress response by discretely tuning two regulatory circuits to different levels of ppGpp. The emergent regulatory structure insures that stress survival circuits are only triggered if homeostatic metabolic networks fail to compensate for environmental deficiencies.
Assuntos
Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/biossíntese , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Isoleucina/metabolismo , Proteína Reguladora de Resposta a Leucina/metabolismo , Redes e Vias Metabólicas , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Regulon , Fator sigma/metabolismo , Estresse FisiológicoRESUMO
Pseudomonas sp. MIS38 produces an effective biosurfactant named arthrofactin, which is a cyclic lipopeptide synthesized by a mega complex composed of three nonribosomal peptide synthetases. In order to gain insight into the control mechanism of arthrofactin production, a Tn5 mutant library was constructed and screened for arthrofactin-deficient mutants. Along with a number of mutations that occurred in the arthrofactin synthetase operon, three other mutants harbored distinct Tn5 insertions in the genes encoding SyrF-like protein (arfF), heat shock protein (htpG), and (p)ppGpp synthetase/hydrolase (spoT). Epistasis analyses revealed that spoT functions early in the arthrofactin production pathway. We also found that spoT affects MIS38 swarming, biofilm formation, and the cell morphology.
Assuntos
Genes Bacterianos/genética , Lipopeptídeos/biossíntese , Peptídeos Cíclicos/biossíntese , Pseudomonas/genética , Pseudomonas/metabolismo , Elementos de DNA Transponíveis/genética , Epistasia Genética/genética , Guanosina Tetrafosfato/biossíntese , Lipopeptídeos/deficiência , Mutagênese , Mutação , Peptídeos Cíclicos/deficiênciaRESUMO
Deficient antibiotic production in an afsB mutant, BH5, of Streptomyces coelicolor A3(2) was recently shown to be due to a mutation (G243D) in region 1.2 of the primary sigma factor sigma(HrdB). Here we show that intracellular ppGpp levels during growth, as well as after amino acid depletion, in the mutant BH5 are lower than those of the afsB(+) parent strain. The introduction of certain rifampicin resistance (rif) mutations, which bypassed the requirement of ppGpp for transcription of pathway-specific regulatory genes, actII-ORF4 and redD, for actinorhodin and undecylprodigiosin, respectively, completely restored antibiotic production by BH5. Antibiotic production was restored also by introduction of a new class of thiostrepton-resistance (tsp) mutations, which provoked aberrant accumulation of intracellular ppGpp. Abolition of ppGpp synthesis in the afsB tsp mutant Tsp33 again abolished antibiotic production. These results indicate that intracellular ppGpp level is finely tuned for successful triggering of antibiotic production in the wild-type strain, and that this fine tuning was absent from the afsB mutant BH5, resulting in a failure to initiate antibiotic production in this strain.