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Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

ABSTRACT: Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (?smc) to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of ?smc. Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of ?smc and induction of the stringent response. This link was confirmed by (p)ppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains. Furthermore, sublethal concentrations of arginine hydroxamate (RHX), a potent inducer of stringent response, restored growth of ?smc under non permissive conditions. We showed that production of (p)ppGpp alone was sufficient to suppress the thermosensitivity exhibited by the ?smc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

SUBMITTER: Benoist C

PROVIDER: S-EPMC4634966 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

Benoist Camille C Guérin Cyprien C Noirot Philippe P Dervyn Etienne E

PloS one 20151105 11

Bacillus subtilis mutants lacking the SMC-ScpAB complex are severely impaired for chromosome condensation and partitioning, DNA repair, and cells are not viable under standard laboratory conditions. We isolated suppressor mutations that restored the capacity of a smc deletion mutant (Δsmc) to grow under standard conditions. These suppressor mutations reduced chromosome segregation defects and abrogated hypersensitivity to gyrase inhibitors of Δsmc. Three suppressor mutations were mapped in genes ...[more]

PMID: 26539825

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Project description:BackgroundPlipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.ResultsThe 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.ConclusionsThis study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Dataset Information

Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

Publications

Constitutive Stringent Response Restores Viability of Bacillus subtilis Lacking Structural Maintenance of Chromosome Protein.

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