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

Benoist C, Guérin C, Noirot P, Dervyn E - PLoS ONE (2015)

Bottom Line: Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways.This link was confirmed by (p)ppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains.Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 Micalis, 78350, Jouy-en-Josas, France.

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 (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 involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc 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 Δsmc under non permissive conditions. We showed that production of (p)ppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

No MeSH data available.


Related in: MedlinePlus

Restoration of Δsmc viability by inducible expression of relA.Δsmc cells carrying different alleles of the E. coli relA genes either as an inactive form (relAi) either as a truncated form (relAt) under the transcriptional control of the Phyperspank promoter were grown in permissive condition (LB at 23°C) with or without IPTG 1mM. Cells were then diluted, spread on both LB plates and LB plates containing IPTG and incubated at permissive (23°C) and non permissive temperature (37°C) for 48h or on coumermycinA1 (1μg/ml) at 23°C. For each condition the ratio of cells growing in non permissive condition versus the number of cells grown in permissive condition (panels A and B) was calculated. The results are the means and the standard deviation measured on at least five different clones. Cells grown at 37°C were also analyzed by microscopic observation after DAPI treatment in order to calculate the ratio of anucleate cells (panel C) and to illustrate the condensation phenotype of the DNA in the WT strain (panel D1), Δsmc (panel D2), ΔsmcPhyperspank::relAt (panel D3), ΔsmcPhyperspank::relAt with 1mM of IPTG (panel D4), ΔsmcPhyperspank::relAi (panel D5), ΔsmcPhyperspank::relAi with 1mM of IPTG (panel D6).
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pone.0142308.g007: Restoration of Δsmc viability by inducible expression of relA.Δsmc cells carrying different alleles of the E. coli relA genes either as an inactive form (relAi) either as a truncated form (relAt) under the transcriptional control of the Phyperspank promoter were grown in permissive condition (LB at 23°C) with or without IPTG 1mM. Cells were then diluted, spread on both LB plates and LB plates containing IPTG and incubated at permissive (23°C) and non permissive temperature (37°C) for 48h or on coumermycinA1 (1μg/ml) at 23°C. For each condition the ratio of cells growing in non permissive condition versus the number of cells grown in permissive condition (panels A and B) was calculated. The results are the means and the standard deviation measured on at least five different clones. Cells grown at 37°C were also analyzed by microscopic observation after DAPI treatment in order to calculate the ratio of anucleate cells (panel C) and to illustrate the condensation phenotype of the DNA in the WT strain (panel D1), Δsmc (panel D2), ΔsmcPhyperspank::relAt (panel D3), ΔsmcPhyperspank::relAt with 1mM of IPTG (panel D4), ΔsmcPhyperspank::relAi (panel D5), ΔsmcPhyperspank::relAi with 1mM of IPTG (panel D6).

Mentions: RHX induces the stringent response by mimicking an amino acid starvation, and could have pleiotropic effects unrelated to (p)ppGpp-mediated response. To test whether the viability of Δsmc under non permissive conditions could be restored by accumulation of (p)ppGpp independently of amino acid starvation, we used alleles of the E. coli relA gene: one producing a constitutively active (p)ppGpp synthase (relAt) and the other producing a catalytic site mutant inactivated for (p)ppGpp production (relAi)[50] (gift from L. Jannière, ISSB, Evry, France). The relAt and relAi genes were placed under the control of the IPTG-inducible PHyperSpank(pHS) promoter and integrated at the amyE locus in the B. subtilis chromosome. We observed that induction by IPTG of the relAt gene resulted in ppGpp accumulation in the cell (ratio (p)ppGpp/GTP increased from 0.45 to 1.14) whereas a similar induction of the relAi gene did not ((p)ppGpp/GTP ratios of 0.42 and 0.47 with and without IPTG, respectively).We conclude that the E. coli RelAt is functional for ppGpp synthesis in B. subtilis. The strains carrying the relAt and relAi genes under the control of the IPTG-inducible promoter were grown under permissive conditions (LB at 23°C), diluted 200 times in either LB or LB supplemented with IPTG, incubated for 24h at 23°C and plated on LB with or without IPTG under the various conditions used to test the suppressor phenotypes. Specifically, plating efficiencies relative to the permissive condition (LB at 23°C) were measured for nalidixic acid 2μg/ml, coumermycin A1 0.5μg/ml, and non permissive conditions (LB at 37°C) (Fig 7, S3 Fig). Strikingly, the expression of the constitutively active EcRelAt conferred full viability under non permissive conditions, whereas the inactive EcRelAi did not (Fig 7A). The surviving colonies did not acquire additional chromosomal mutations because they were unable to form colonies when streaked at 37°C without IPTG. Therefore, (p)ppGpp synthesis by EcRelAt suppressed Δsmc defects in the absence of amino acid starvation.


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

Benoist C, Guérin C, Noirot P, Dervyn E - PLoS ONE (2015)

Restoration of Δsmc viability by inducible expression of relA.Δsmc cells carrying different alleles of the E. coli relA genes either as an inactive form (relAi) either as a truncated form (relAt) under the transcriptional control of the Phyperspank promoter were grown in permissive condition (LB at 23°C) with or without IPTG 1mM. Cells were then diluted, spread on both LB plates and LB plates containing IPTG and incubated at permissive (23°C) and non permissive temperature (37°C) for 48h or on coumermycinA1 (1μg/ml) at 23°C. For each condition the ratio of cells growing in non permissive condition versus the number of cells grown in permissive condition (panels A and B) was calculated. The results are the means and the standard deviation measured on at least five different clones. Cells grown at 37°C were also analyzed by microscopic observation after DAPI treatment in order to calculate the ratio of anucleate cells (panel C) and to illustrate the condensation phenotype of the DNA in the WT strain (panel D1), Δsmc (panel D2), ΔsmcPhyperspank::relAt (panel D3), ΔsmcPhyperspank::relAt with 1mM of IPTG (panel D4), ΔsmcPhyperspank::relAi (panel D5), ΔsmcPhyperspank::relAi with 1mM of IPTG (panel D6).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4634966&req=5

pone.0142308.g007: Restoration of Δsmc viability by inducible expression of relA.Δsmc cells carrying different alleles of the E. coli relA genes either as an inactive form (relAi) either as a truncated form (relAt) under the transcriptional control of the Phyperspank promoter were grown in permissive condition (LB at 23°C) with or without IPTG 1mM. Cells were then diluted, spread on both LB plates and LB plates containing IPTG and incubated at permissive (23°C) and non permissive temperature (37°C) for 48h or on coumermycinA1 (1μg/ml) at 23°C. For each condition the ratio of cells growing in non permissive condition versus the number of cells grown in permissive condition (panels A and B) was calculated. The results are the means and the standard deviation measured on at least five different clones. Cells grown at 37°C were also analyzed by microscopic observation after DAPI treatment in order to calculate the ratio of anucleate cells (panel C) and to illustrate the condensation phenotype of the DNA in the WT strain (panel D1), Δsmc (panel D2), ΔsmcPhyperspank::relAt (panel D3), ΔsmcPhyperspank::relAt with 1mM of IPTG (panel D4), ΔsmcPhyperspank::relAi (panel D5), ΔsmcPhyperspank::relAi with 1mM of IPTG (panel D6).
Mentions: RHX induces the stringent response by mimicking an amino acid starvation, and could have pleiotropic effects unrelated to (p)ppGpp-mediated response. To test whether the viability of Δsmc under non permissive conditions could be restored by accumulation of (p)ppGpp independently of amino acid starvation, we used alleles of the E. coli relA gene: one producing a constitutively active (p)ppGpp synthase (relAt) and the other producing a catalytic site mutant inactivated for (p)ppGpp production (relAi)[50] (gift from L. Jannière, ISSB, Evry, France). The relAt and relAi genes were placed under the control of the IPTG-inducible PHyperSpank(pHS) promoter and integrated at the amyE locus in the B. subtilis chromosome. We observed that induction by IPTG of the relAt gene resulted in ppGpp accumulation in the cell (ratio (p)ppGpp/GTP increased from 0.45 to 1.14) whereas a similar induction of the relAi gene did not ((p)ppGpp/GTP ratios of 0.42 and 0.47 with and without IPTG, respectively).We conclude that the E. coli RelAt is functional for ppGpp synthesis in B. subtilis. The strains carrying the relAt and relAi genes under the control of the IPTG-inducible promoter were grown under permissive conditions (LB at 23°C), diluted 200 times in either LB or LB supplemented with IPTG, incubated for 24h at 23°C and plated on LB with or without IPTG under the various conditions used to test the suppressor phenotypes. Specifically, plating efficiencies relative to the permissive condition (LB at 23°C) were measured for nalidixic acid 2μg/ml, coumermycin A1 0.5μg/ml, and non permissive conditions (LB at 37°C) (Fig 7, S3 Fig). Strikingly, the expression of the constitutively active EcRelAt conferred full viability under non permissive conditions, whereas the inactive EcRelAi did not (Fig 7A). The surviving colonies did not acquire additional chromosomal mutations because they were unable to form colonies when streaked at 37°C without IPTG. Therefore, (p)ppGpp synthesis by EcRelAt suppressed Δsmc defects in the absence of amino acid starvation.

Bottom Line: Three suppressor mutations were mapped in genes involved in tRNA aminoacylation and maturation pathways.This link was confirmed by (p)ppGpp quantification which indicated a constitutive induction of the stringent response in multiple suppressor strains.Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 Micalis, 78350, Jouy-en-Josas, France.

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 (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 involved in tRNA aminoacylation and maturation pathways. A transcriptomic survey of isolated suppressor mutations pointed to a potential link between suppression of Δsmc 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 Δsmc under non permissive conditions. We showed that production of (p)ppGpp alone was sufficient to suppress the thermosensitivity exhibited by the Δsmc mutant. Our findings shed new light on the coordination between chromosome dynamics mediated by SMC-ScpAB and other cellular processes during rapid bacterial growth.

No MeSH data available.


Related in: MedlinePlus