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NAD(P)H-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

Petrovova M, Tkadlec J, Dvoracek L, Streitova E, Licha I - PLoS ONE (2014)

Bottom Line: The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress.We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme.We show that such enzymes could play a significant role in the survival of stressed cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic.

ABSTRACT

Background: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon.

Methods and results: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress.

Conclusion: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

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Related in: MedlinePlus

Growth characterization of WT, LD1, and MP2 mutants of Bacillus subtilis in long-term cultivation and in response to stress.For long-term growth measurements, the cells were grown in LB medium (A). Effect of MM medium and K+ limitation (0.5 mM K+) to growth rate of mutant (LD1) (B). Osmotic stress performed only for MP2 mutant in MM medium and K+ limitation is demonstrated (C). Effect of ethanol stress was measured in MM medium and K+ limitation for both mutants (LD1, MP2) (D). (WT - Bacillus subtilis 168 or SG4, LD1 and MP2– yxkO knock-out mutants, for details see Table 1). Exposure of stress is marked by arrows. Measurements were done with cells synchronized in exponential growth, and stress conditions were set up as is described in Material and Methods. The typical growth rate curve is shown from measurements made in triplicate for each condition.
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pone-0112590-g001: Growth characterization of WT, LD1, and MP2 mutants of Bacillus subtilis in long-term cultivation and in response to stress.For long-term growth measurements, the cells were grown in LB medium (A). Effect of MM medium and K+ limitation (0.5 mM K+) to growth rate of mutant (LD1) (B). Osmotic stress performed only for MP2 mutant in MM medium and K+ limitation is demonstrated (C). Effect of ethanol stress was measured in MM medium and K+ limitation for both mutants (LD1, MP2) (D). (WT - Bacillus subtilis 168 or SG4, LD1 and MP2– yxkO knock-out mutants, for details see Table 1). Exposure of stress is marked by arrows. Measurements were done with cells synchronized in exponential growth, and stress conditions were set up as is described in Material and Methods. The typical growth rate curve is shown from measurements made in triplicate for each condition.

Mentions: Firstly, to verify the yxkO gene phenotype previously described in a sporulation-proficient strain (spo0F221) [15], we transduced the cassette of mini-Tn10 from the L-42 mutant using PBS1 bacteriophage to Bacillus subtilis 168, and in parallel, we constructed an insertional mutant using the pMUTIN4 plasmid, as well (see Materials and Methods). Both mutants revealed the previously described phenotype, an increase in generation time from 55 min to 115 min and osmosensitivity in low K+ conditions (15) but also lower viability, even under stress conditions, as we demonstrated in the long-term growth measurements (Figure 1A).


NAD(P)H-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

Petrovova M, Tkadlec J, Dvoracek L, Streitova E, Licha I - PLoS ONE (2014)

Growth characterization of WT, LD1, and MP2 mutants of Bacillus subtilis in long-term cultivation and in response to stress.For long-term growth measurements, the cells were grown in LB medium (A). Effect of MM medium and K+ limitation (0.5 mM K+) to growth rate of mutant (LD1) (B). Osmotic stress performed only for MP2 mutant in MM medium and K+ limitation is demonstrated (C). Effect of ethanol stress was measured in MM medium and K+ limitation for both mutants (LD1, MP2) (D). (WT - Bacillus subtilis 168 or SG4, LD1 and MP2– yxkO knock-out mutants, for details see Table 1). Exposure of stress is marked by arrows. Measurements were done with cells synchronized in exponential growth, and stress conditions were set up as is described in Material and Methods. The typical growth rate curve is shown from measurements made in triplicate for each condition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231035&req=5

pone-0112590-g001: Growth characterization of WT, LD1, and MP2 mutants of Bacillus subtilis in long-term cultivation and in response to stress.For long-term growth measurements, the cells were grown in LB medium (A). Effect of MM medium and K+ limitation (0.5 mM K+) to growth rate of mutant (LD1) (B). Osmotic stress performed only for MP2 mutant in MM medium and K+ limitation is demonstrated (C). Effect of ethanol stress was measured in MM medium and K+ limitation for both mutants (LD1, MP2) (D). (WT - Bacillus subtilis 168 or SG4, LD1 and MP2– yxkO knock-out mutants, for details see Table 1). Exposure of stress is marked by arrows. Measurements were done with cells synchronized in exponential growth, and stress conditions were set up as is described in Material and Methods. The typical growth rate curve is shown from measurements made in triplicate for each condition.
Mentions: Firstly, to verify the yxkO gene phenotype previously described in a sporulation-proficient strain (spo0F221) [15], we transduced the cassette of mini-Tn10 from the L-42 mutant using PBS1 bacteriophage to Bacillus subtilis 168, and in parallel, we constructed an insertional mutant using the pMUTIN4 plasmid, as well (see Materials and Methods). Both mutants revealed the previously described phenotype, an increase in generation time from 55 min to 115 min and osmosensitivity in low K+ conditions (15) but also lower viability, even under stress conditions, as we demonstrated in the long-term growth measurements (Figure 1A).

Bottom Line: The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress.We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme.We show that such enzymes could play a significant role in the survival of stressed cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic.

ABSTRACT

Background: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon.

Methods and results: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress.

Conclusion: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

Show MeSH
Related in: MedlinePlus