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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

Transcription level of Pctc on genetic background of WT and MP2 mutant.Pctc activation measurements of WT and MP2 were performed under ethanol stress in LB medium (A), osmotic stress in MM medium with 10 mM K+ concentration (B), osmotic stress in MM medium with 0.5 mM K+ concentration (C), shift from MM medium with 10 mM K+ concentration to MM medium with 0.5 mM K+ concentration (D). Time 0 indicates application of stress. Details of transcription activity measurements, growth, and stress conditions are described in Material and Methods.
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pone-0112590-g002: Transcription level of Pctc on genetic background of WT and MP2 mutant.Pctc activation measurements of WT and MP2 were performed under ethanol stress in LB medium (A), osmotic stress in MM medium with 10 mM K+ concentration (B), osmotic stress in MM medium with 0.5 mM K+ concentration (C), shift from MM medium with 10 mM K+ concentration to MM medium with 0.5 mM K+ concentration (D). Time 0 indicates application of stress. Details of transcription activity measurements, growth, and stress conditions are described in Material and Methods.

Mentions: When direct estimation of the promoter activity of the PyxkO promoter had failed, we tested the impact of the YxkO on the induction of SigB-dependent genes. For this purpose, we generally used the well-characterized SigB-dependent Pctc promoter, which is transcribed under the most studied stress conditions, and the Pctc promoter fused to lacZ (Pctc-lacZ) beneficially monitors SigB activity in the WT promoter context, as was shown previously [11]. The promoter-probe strains in both genetic backgrounds were prepared as described in the Materials and Methods section, and the promoter activity of Pctc was monitored. The tested conditions were ethanol stress in complex media and salt stress when cultivated in high (10 mM) and low (0.5 mM) concentrations of K+, respectively. Results are shown in Figure 2.


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)

Transcription level of Pctc on genetic background of WT and MP2 mutant.Pctc activation measurements of WT and MP2 were performed under ethanol stress in LB medium (A), osmotic stress in MM medium with 10 mM K+ concentration (B), osmotic stress in MM medium with 0.5 mM K+ concentration (C), shift from MM medium with 10 mM K+ concentration to MM medium with 0.5 mM K+ concentration (D). Time 0 indicates application of stress. Details of transcription activity measurements, growth, and stress conditions are described in Material and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112590-g002: Transcription level of Pctc on genetic background of WT and MP2 mutant.Pctc activation measurements of WT and MP2 were performed under ethanol stress in LB medium (A), osmotic stress in MM medium with 10 mM K+ concentration (B), osmotic stress in MM medium with 0.5 mM K+ concentration (C), shift from MM medium with 10 mM K+ concentration to MM medium with 0.5 mM K+ concentration (D). Time 0 indicates application of stress. Details of transcription activity measurements, growth, and stress conditions are described in Material and Methods.
Mentions: When direct estimation of the promoter activity of the PyxkO promoter had failed, we tested the impact of the YxkO on the induction of SigB-dependent genes. For this purpose, we generally used the well-characterized SigB-dependent Pctc promoter, which is transcribed under the most studied stress conditions, and the Pctc promoter fused to lacZ (Pctc-lacZ) beneficially monitors SigB activity in the WT promoter context, as was shown previously [11]. The promoter-probe strains in both genetic backgrounds were prepared as described in the Materials and Methods section, and the promoter activity of Pctc was monitored. The tested conditions were ethanol stress in complex media and salt stress when cultivated in high (10 mM) and low (0.5 mM) concentrations of K+, respectively. Results are shown in Figure 2.

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