Abstract

BackgroundOne 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 ResultsWe 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.ConclusionWe 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.

Highlights

  • In an effort to understand the global adaptation network that evolved in Bacillus sp., several recent studies were carried out, focused on the genome-wide transcriptional profiling of the stress response of Bacillus subtilis 168 [1,2,3,4]

  • Confirmation of yxkO knock-out mutant phenotype Firstly, to verify the yxkO gene phenotype previously described in a sporulation-proficient strain [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

  • 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) and lower viability, even under stress conditions, as we demonstrated in the long-term growth measurements (Figure 1A)

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Summary

Introduction

In an effort to understand the global adaptation network that evolved in Bacillus sp., several recent studies were carried out, focused on the genome-wide transcriptional profiling of the stress response of Bacillus subtilis 168 [1,2,3,4]. Several physiological analyses of the Bacillus subtilis 168 proteome during the adaptation to various environmental stresses have been published as well [5,6,7] These studies identified stress specific regulons that are involved in stress function and confirm that the synthesis of most vegetative proteins is repressed, with the exception of enzymes that take part in adaptive responses. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; 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. 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
Results
Conclusion

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