Comprehensive Characterization of the Contribution of Individual SigB-Dependent General Stress Genes to Stress Resistance ofBacillus subtilis

Abstract

TheB -dependent general stress regulon of Bacillus subtilis comprises more than 150 members. Induction of this regulon by imposition of environmental or metabolic stress confers multiple, nonspecific, and preemptive stress resistance to nongrowing, nonsporulated cells of B. subtilis. In this study we performed a regulonwide phenotypic screening analysis to determine the stress sensitivity profiles of 94 mutants defective in candidate members of the general stress regulon that were previously identified in our transcriptional profiling study of the general stress response of B. subtilis. The phenotypic screening analysis included analysis of adaptation to a growth-inhibiting concentration of ethanol (10%, vol/vol) or NaCl (10%, wt/vol), severe heat shock (54°C), and low temperature (survival at 4°C and growth at 12.5°C). Surprisingly, 85% of the mutants tested displayed increased sensitivity at an ␣ confidence level of <0.01 to at least one of the four stresses tested, and 62% still exhibited increased sensitivity at an ␣ of <0.001. In essence, we were able to assign 63 genes (28 genes with an ␣ of <0.001) to survival after ethanol shock, 37 genes (28 genes with an ␣ of <0.001) to protection from NaCl shock, 34 genes (24 genes with an ␣ of <0.001) to survival at 4°C, and 10 genes (3 genes with an ␣ of <0.001) to management of severe heat shock. Interestingly, there was a substantial overlap between the genes necessary for survival during ethanol shock and the genes necessary for survival at 4°C, and there was also an overlap between genes required for survival during ethanol shock and genes required for survival during NaCl shock. Our data provide evidence for the importance of the B regulon at low temperatures, not only for growth but also for survival. Moreover, the data imply that a secondary oxidative stress seems to be a common component of the severe stresses tested.

A Comprehensive Proteomics and Transcriptomics Analysis ofBacillus subtilisSalt Stress Adaptation

“…The initial response of B. subtilis to a sudden and strong osmotic upshift is characterized by a strong and immediate activation of the B regulon, leading to synthesis and accumulation of proteins conferring a broad stress resistance to B. subtilis cells (21). The B regulon comprises at least 37 proteins that are critical for providing osmostress resistance, as judged by the salt-sensitive phenotype of their structural genes (28). Almost all of these B -dependent salt stress protective proteins are significantly upregulated either early at the mRNA level or in later adaptation stages of the osmotically challenged cells on the protein level (Fig.…”

“…Two routes for proline biosynthesis from its precursor glutamate operate in B. subtilis (4): ProB, ProG, and ProI catalyze anabolic proline biosynthesis, and at least two paralogue proteins (ProH and ProJ) are presumed to be responsible for high-level proline synthesis under hyperosmotic conditions (4,7,28). The ProA enzyme is used by both proline biosynthetic routes (4,7).…”

“…subtilis (65), is synthesized via a dedicated osmostress-responsive pathway that comprises at least two paralogue enzymes of the vegetative proline biosynthetic pathway (4,28). These paralogue proline biosynthetic enzymes are ProH, a pyrroline-5-carboxylate reductase, and ProJ, a glutamate 5-kinase.…”

“…These paralogue proline biosynthetic enzymes are ProH, a pyrroline-5-carboxylate reductase, and ProJ, a glutamate 5-kinase. Additionally, YerD, a putative ferredoxin-dependent glutamate synthase, might be involved in providing the precursor for proline biosynthesis subsequent to a severe osmotic upshift (28). B. subtilis possesses five osmotically regulated transport systems (OpuA to OpuE) for the acquisition of a broad spectrum of compatible solutes from environmental sources, and the uptake of these compounds provides a considerable degree of osmostress resistance (7).…”

“…Such a phenotype is also exhibited by a sigB mutant and by the disruption of yerD. Among the B -controlled genes are those coding for the import systems for the compatible solutes glycine betaine and proline, OpuD and OpuE (28). Transcription of both the opuE and opuD genes is under simultaneous control of B and the vegetative sigma factor A , thereby linking the nonspecific general stress response with the salt-specific adaptive response of B. subtilis (7,21).…”