Non-Classical Protein Secretion and Transcriptome Studies during Stationary Phase of Bacillus Subtilis

A cloned esterase and several cytoplasmic proteins which lack a classical cleavable signal-peptide were secreted during late stationary phase in B. subtilis. Several lines of evidence indicate that secretion of enolase, SodA, and Est55 is not due to cell lysis. The extent of possible release of these proteins mediated by membrane vesicles into the medium was also found to be minimal. We have identified a hydrophobic α–helical domain within enolase that contributes to the secretion specificity. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid-encoded mutant enolases is totally blocked, while that of the wild-type chromosomal enolase is not affected in the same cultures. However, mutations on the conserved basic residues flanking the hydrophobic core region show no effect. GFP fusion experiments demonstrate that minimal length of N-terminus 140 amino acids and its tertiary structure are required to serve as a functional signal for the export of enolase. Transcriptome analysis revealed several interesting patterns in gene expression when the cell growth switches from exponential phase into stationary phase. As expected, once cell growth enters the stationary phase, expressions of most SigA-dependent house-keeping genes (for syntheses of ATP, amino acids, nucleotides, ribosomes), and surprisingly secY and yidC homolog in the Sec-dependent general protein secretion system were significantly decreased; however, secA and sipT were found progressively induced in the stationary phase. The sigB gene and the SigB regulon exhibited a distinct pattern of transient induction with a peak in transition phase. A total of 62 genes were induced by three fold after cessation of SigB-dependent surge, which includes sigW and many of SigW-depedent genes specifically for antitoxin resistant genes, and some unknown function genes. In addition, oxidative stress response and damage repair genes also dominantly induced in stationary phase implied a high level of oxidant or thio-depleting agents in stationary phase. Besides, induction of fruRAB at T40 and gap operon at T100 suggested a sequential switch of carbon utilization from glucose to fructose. These results indicate a complex adaptation physiology as Bacillus cells change from the fast growing exponential phase toward the stationary phase. INDEX WORDS: Est55, signal-less protein, enolase, secretion, EM domain, hydrophobicity, sigma factor, microarray, Bacillus subtilis NON-CLASSICAL PROTEIN SECRETION AND TRANSCRIPTOME STUDIES DURING STATIONARY PHASE OF BACILLUS SUBTILIS