Identification of σ B -Dependent Genes in Bacillus cereus by Proteome and In Vitro Transcription Analysis

Abstract

ABSTRACT The alternative sigma factor σ B of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells. This study describes the identification of σ B -dependent genes in B. cereus . Two-dimensional gel electrophoresis was performed with protein extracts from a σ B -overproducing B. cereus strain. Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing. The σ B -dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B. cereus ATCC 14579 and its sigB null mutant. Northern blot analysis also revealed that six other genes were part of σ B -dependent operons. The proteins that are predicted to be encoded by the σ B -dependent genes include an intracellular protease, a Mg 2+ transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all σ B -dependent genes, with the exception of thiG . By searching the B. cereus genome for this conserved promoter sequence, five more candidate σ B -dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus σ B -RNA polymerase holoenzyme confirmed the σ B dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also σ B dependent. In conclusion, σ B of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.