Effect of Antibiotics on Sporulation Caused by the Stringent Response in Bacillus subtilis

Ochi, Kozo; Freese, Ernst

doi:10.1099/00221287-129-12-3709

Cited by 15 publications

(14 citation statements)

References 10 publications

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“…6C). Antibiotics that block protein synthesis by binding to the ribosome are known to interfere with ribosome-dependent (p)ppGpp synthesis at a relatively low concentration (40). And as expected, in vitro pppGpp synthesis by ribosomes from the wild-type T. thermophilus HB8 was severely inhibited by addition of thiostrepton or tetracycline (10 g/ml [each]) (Fig.…”

Section: Resultsmentioning

confidence: 93%

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium,Thermus thermophilus

et al. 2006

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Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel ؉ ; wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA Ser aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

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Section: Resultsmentioning

confidence: 93%

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium,Thermus thermophilus

et al. 2006

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“…4). Thus, it is reasonable to conclude that the inability to sufficiently reduce the level of GTP in the relA strain is a major cause for the inability of this strain to initiate genetic competence and sporulation (16)(17)(18). The observed intracellular concentrations of GTP during the transition phase (t0 to t1) were estimated to be 1.5 to 2.5 mM (for the stringent strain) and 3.5 to 4.5 mM (for the relaxed strain) by the method of Ochi (15).…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid depletion in B. subtilis gives rise to the transient increase of (p)ppGpp, accompanied by the reduction of intracellular GTP, eventually leading to the induction of spore formation in relA ϩ (stringent) cells but not in relA (relaxed) mutant cells (11,12,(16)(17)(18). In the course of studying the ribosomal function of B. subtilis, we have recently found that genetic competence in a certain aspartate-auxotrophic B. subtilis strain is affected significantly by the relA mutation.…”

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confidence: 99%

“…The stringent response depends on the transient increase of hyperphosphorylated guanosine nucleotides [(p)ppGpp], which are synthesized from GDP or GTP by the relA gene product (called stringent factor [ppGpp synthetase]) in response to binding of uncharged tRNA to the ribosomal A site (reviewed by Cashel et al [1]). Amino acid depletion in B. subtilis gives rise to the transient increase of (p)ppGpp, accompanied by the reduction of intracellular GTP, eventually leading to the induction of spore formation in relA ϩ (stringent) cells but not in relA (relaxed) mutant cells (11,12,(16)(17)(18). In the course of studying the ribosomal function of B. subtilis, we have recently found that genetic competence in a certain aspartate-auxotrophic B. subtilis strain is affected significantly by the relA mutation.…”

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RelA Protein Is Involved in Induction of Genetic Competence in CertainBacillus subtilisStrains by Moderating the Level of Intracellular GTP

2002

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We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain of Bacillus subtilis is significantly lower than that of the isogenic stringent (relA ؉ ) strain. Transcriptional fusion analysis utilizing a lacZ reporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain. We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography. The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added at t0 (the transition point between exponential to stationary growth phase). Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development. Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting the codY gene, which is known to negatively control comK expression. Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation.When Bacillus subtilis cells encounter adverse nutrient conditions, cells start to approach the stationary growth phase by initiating several development processes that contribute to its survival. Of these processes, genetic competence and sporulation have been studied in great detail. Genetic competence is a physiological state that enables cells to incorporate exogenous DNA, which is then incorporated into the endogenous DNA by crossing over. Competence development is tightly controlled by a complex regulatory network involving kinases, phosphatases, and transcriptional regulators (4). The most critical regulator is a transcriptional factor named ComK, which has been shown to be necessary for inducing the late competence genes such as comG (27,28). It has been demonstrated that the cellular level of ComK is precisely controlled by several regulatory proteins at the transcriptional and posttranscriptional levels (2,5,6,25,26). During the exponential growth phase, ComK activity is inhibited by a complex of two proteins, MecA and ClpC (10, 26). The ComS protein, which is encoded by the srfA operon and is synthesized in response to quorum-sensing oligopeptide pheromones (13, 23), releases cells from ComK inhibition by dissociating the ternary complex with MecA and ClpC (25). In addition, Serror and Sonenshein (21) demonstrated that CodY, which is a global regulator for stationary-phase genes, directly binds to the srfA and comK promoter region. Thus, it is highly likely that CodY acts as a negative regulator for competence genes.One of the most important adaptation s...

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“…This effect is accompanied by an increase in the intracellular concentrations of ppGpp (guanosine 5'-diphosphate 3' -diphosphate) and pppGpp (guanosine 5'-triphosphate 3' -diphosphate), compounds believed to be responsible for the stringent response.10,ll) Sporulation is also known to be initiated directly by inhibitors of GMP synthesis, such as decoyinine (a nucleoside antibiotic) or mycophenolate, which results in a decrease of the intracellular GTP pool. 5,9) Very recently, it has been reported that B. subtilis sporulation results from a decrease of GTP (caused by the 905 stringent response), whereas antibiotic synthesis results from a different effect of the stringent response. 12 ) I report here that decoyinine also restores sporulation (differentiation) of B. subtilis deficient in an intracellular serine protease, while it does not initiate antibiotic synthesis (secondary metabolism).…”

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Sporulation and Antibiotic Production byBacillus subtilisMutants Deficient in Intracellular Proteases

Ocm¹

1985

Agricultural and Biological Chemistry

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Effect of Antibiotics on Sporulation Caused by the Stringent Response in Bacillus subtilis

Cited by 15 publications

References 10 publications

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium,Thermus thermophilus

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium,Thermus thermophilus

RelA Protein Is Involved in Induction of Genetic Competence in CertainBacillus subtilisStrains by Moderating the Level of Intracellular GTP

Sporulation and Antibiotic Production byBacillus subtilisMutants Deficient in Intracellular Proteases

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