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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

The (p)ppGpp synthase RelA is required for viability of SMC  mutant strains.A strain background was constructed in which the two minor (p)ppGpp synthases were inactivated (ΔywaC ΔyjbM) and expression of only remaining (p)ppGpp synthase activity was placed under control of the IPTG-inducible promoter (PSpac::relA). This strain formed isolated colonies on LB plates without and with IPTG 1mM at permissive temperature (23°C). However, in absence of SMC (Δsmc) in this background, IPTG was absolutely required to form isolated colonies. Note that only residual growth in the mass of the streak is observed without IPTG.
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pone.0142308.g005: The (p)ppGpp synthase RelA is required for viability of SMC mutant strains.A strain background was constructed in which the two minor (p)ppGpp synthases were inactivated (ΔywaC ΔyjbM) and expression of only remaining (p)ppGpp synthase activity was placed under control of the IPTG-inducible promoter (PSpac::relA). This strain formed isolated colonies on LB plates without and with IPTG 1mM at permissive temperature (23°C). However, in absence of SMC (Δsmc) in this background, IPTG was absolutely required to form isolated colonies. Note that only residual growth in the mass of the streak is observed without IPTG.

Mentions: The combination of the four deletions was attempted by transforming the triple mutant Δsmc ΔyjbM ΔywaC with chromosomal DNA from ΔrelA ΔyjbM ΔywaC strain and selecting for the antibiotic resistance marker associated with ΔrelA under permissive conditions (LB at 23°C). A total of 30 transformants were obtained from four independent experiments, and their chromosome integrity at the 4 loci was verified (see Materials and Methods). All transformants except one had recovered the wild type smc locus. The transformant which carried the ΔrelA Δsmc ΔyjbM ΔywaC deletions exhibited a plating efficiency reduced nearly 10-fold relative to Δsmc under permissive conditions (S4 Table), suggesting that (p)ppGpp synthesis is important for the fitness of the Δsmc mutant. However, since we cannot rule out that the quadruple deletion mutant has acquired compensatory mutation(s), we cannot conclude from these results that (p)ppGpp synthesis is essential for Δsmc viability. Thus, we placed the relA gene under control of the inducible pSpac promoter in the ΔyjbM ΔywaC background (see Materials and Methods). The resulting strain did not require IPTG for growth (Fig 5). The Δsmc allele was introduced in the presence of IPTG. The resulting strain Δsmc ΔyjbM ΔywaC pSpac::relA grew similarly to the Δsmc and Δsmc ΔyjbM ΔywaC strains under permissive conditions (LB + IPTG 1mM at 23°C) (Fig 5). In sharp contrast, when relA expression was not induced, the Δsmc ΔyjbM ΔywaC pSpac::relA strain could not form isolated colonies. Our results strongly support that an active (p)ppGpp synthase is required for the viability of the Δsmc.


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)

The (p)ppGpp synthase RelA is required for viability of SMC  mutant strains.A strain background was constructed in which the two minor (p)ppGpp synthases were inactivated (ΔywaC ΔyjbM) and expression of only remaining (p)ppGpp synthase activity was placed under control of the IPTG-inducible promoter (PSpac::relA). This strain formed isolated colonies on LB plates without and with IPTG 1mM at permissive temperature (23°C). However, in absence of SMC (Δsmc) in this background, IPTG was absolutely required to form isolated colonies. Note that only residual growth in the mass of the streak is observed without IPTG.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4634966&req=5

pone.0142308.g005: The (p)ppGpp synthase RelA is required for viability of SMC mutant strains.A strain background was constructed in which the two minor (p)ppGpp synthases were inactivated (ΔywaC ΔyjbM) and expression of only remaining (p)ppGpp synthase activity was placed under control of the IPTG-inducible promoter (PSpac::relA). This strain formed isolated colonies on LB plates without and with IPTG 1mM at permissive temperature (23°C). However, in absence of SMC (Δsmc) in this background, IPTG was absolutely required to form isolated colonies. Note that only residual growth in the mass of the streak is observed without IPTG.
Mentions: The combination of the four deletions was attempted by transforming the triple mutant Δsmc ΔyjbM ΔywaC with chromosomal DNA from ΔrelA ΔyjbM ΔywaC strain and selecting for the antibiotic resistance marker associated with ΔrelA under permissive conditions (LB at 23°C). A total of 30 transformants were obtained from four independent experiments, and their chromosome integrity at the 4 loci was verified (see Materials and Methods). All transformants except one had recovered the wild type smc locus. The transformant which carried the ΔrelA Δsmc ΔyjbM ΔywaC deletions exhibited a plating efficiency reduced nearly 10-fold relative to Δsmc under permissive conditions (S4 Table), suggesting that (p)ppGpp synthesis is important for the fitness of the Δsmc mutant. However, since we cannot rule out that the quadruple deletion mutant has acquired compensatory mutation(s), we cannot conclude from these results that (p)ppGpp synthesis is essential for Δsmc viability. Thus, we placed the relA gene under control of the inducible pSpac promoter in the ΔyjbM ΔywaC background (see Materials and Methods). The resulting strain did not require IPTG for growth (Fig 5). The Δsmc allele was introduced in the presence of IPTG. The resulting strain Δsmc ΔyjbM ΔywaC pSpac::relA grew similarly to the Δsmc and Δsmc ΔyjbM ΔywaC strains under permissive conditions (LB + IPTG 1mM at 23°C) (Fig 5). In sharp contrast, when relA expression was not induced, the Δsmc ΔyjbM ΔywaC pSpac::relA strain could not form isolated colonies. Our results strongly support that an active (p)ppGpp synthase is required for the viability of the Δsmc.

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