NAD(P)H-Hydrate Dehydratase- A Metabolic Repair Enzyme and Its Role in <i>Bacillus subtilis</i> Stress Adaptation

<div><p>Background</p><p>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 <i>Bacillus</i> sp. The GSR is one of the largest regulon in <i>Bacillus</i> 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 <i>Bacillus subtilis</i>168 osmosensitive mutant, defective in the <i>yxkO</i> gene (encoding a putative ribokinase), which was recently assigned <i>in vitro</i> as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon.</p><p>Methods and Results</p><p>We show the impact of YxkO on the activity of SigB-dependent P<i>ctc</i> 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.</p><p>Conclusion</p><p>We performed the first characterization of a <i>Bacillus subtilis</i>168 knock-out mutant in the <i>yxkO</i> gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.</p></div

Comparative 2DE analysis of WT versus MP2 mutant exposed to osmotic and ethanol stress – metabolic enzymes.

<p>Proteins with protein level profiles that are similar for both stresses. For experimental conditions and data evaluation, see <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a></i>. Separate columns of the bar charts show the protein level of respective proteins, as calculated from the quantification of the spot volume by PDQuest 8.0 software; y-axes are scaled in intensity for each particular protein. Bars represent each strains and conditions, and there are in the same order as the protein level profiles are presented.</p

Comparative 2DE analysis of WT versus MP2 mutant exposed to osmotic and ethanol stress – stress adaptation and motility.

<p>Proteins with protein level profiles that are similar for both stresses. For experimental conditions and data evaluation, see <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a></i>. The picture description is same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#pone-0112590-g003" target="_blank">Figure 3</a>.</p

Transcription level of P<i>ctc</i> on genetic background of WT and MP2 mutant.

<p>P<i>ctc</i> 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 <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a>.</i></p

Comparative 2DE analysis of WT versus MP2 mutant exposed to osmotic stress.

<p>Proteins with protein level profiles that are unique for osmotic stress. For experimental conditions and data evaluation, see <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a></i>. The picture description is same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#pone-0112590-g003" target="_blank">Figure 3</a>.</p

Growth characterization of WT, LD1, and MP2 mutants of <i>Bacillus subtilis</i> in long-term cultivation and in response to stress.

<p>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 - <i>Bacillus subtilis</i> 168 or SG4, LD1 and MP2– <i>yxkO</i> knock-out mutants, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#pone-0112590-t001" target="_blank">Table 1</a>). 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 <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112590#s2" target="_blank">Material and Methods</a></i>. The typical growth rate curve is shown from measurements made in triplicate for each condition.</p

<i>Bacillus subtilis</i> strains, phage, and plasmids used in this study.

<p><i>Bacillus subtilis</i> strains, phage, and plasmids used in this study.</p

MALDI-TOF peptide mapping identification.

<p>Detailed data from MALDI-TOF peptide mapping identification of proteins, that differed in levels when exposed to osmotic and ethanol stress.</p><p>MALDI-TOF peptide mapping identification.</p