Identification of sigmaB-dependent genes in Bacillus cereus by proteome and in vitro transcription analysis.
The alternative sigma factor sigmaB 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 sigmaB-dependent genes in B. cereus. Two-dimensional gel electrophoresis was performed with protein extracts from a sigmaB-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 sigmaB-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 sigmaB-dependent operons. The proteins that are predicted to be encoded by the sigmaB-dependent genes include an intracellular protease, a Mg2+ transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all sigmaB-dependent genes, with the exception of thiG. By searching the B. cereus genome for this conserved promoter sequence, five more candidate sigmaB-dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus sigmaB-RNA polymerase holoenzyme confirmed the sigmaB 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 sigmaB dependent. In conclusion, sigmaB of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.
van Schaik
Willem
W
Wageningen Centre for Food Sciences, Wageningen, The Netherlands.
Zwietering
Marcel H
MH
de Vos
Willem M
WM
Abee
Tjakko
T
D001409:Bacillus cereus / Q000235:genetics*; D001426:Bacterial Proteins / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic; D020543:Proteome; D012808:Sigma Factor / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D020543:Proteome; C079011:SigB protein, Bacteria; D012808:Sigma Factor