Bacillus cereus ATCC 14579 : physiological and genetic responses to bile, bile salts and mucin

Abstract

ABSTRACT

The aim of this thesis was to investigate the in vitro physiological and genetic responses of Bacillus cereus ATCC 14579 to bile, bile salts and mucin, substances which would be encountered in the gastrointestinal tract.

B. cereus was grown in media containing bile/bile salts and growth only occurred at low concentrations (no growth observed on LB-agar plates with 0.02 % bile salts). Preincubation with bile salts at non-lethal concentrations did not increase the levels of bile salts tolerance.

Cultures of B. cereus were grown to the mid-exponential growth phase and shifted to media containing bile (0.03 %), bile salts (0.005 %) and mucin (0.25 %). Global expression patterns (shifts to bile and bile salts containing media) were determined by hybridization of total RNA to microarrays. The expression of selected genes, fibronectin binding protein and two internalin genes, in shift experiments to bile, bile salts and mucin containing media were investigated by RT-PCR. The microarray data indicated a general stress response to bile and bile salts. The RT-PCR indicated a down-regulation of a putative virulence factor, internalin, in bile salts cultures.

Proteins were extracted from the shift experiments (not mucin) 30 minutes after the shift in a LiCl-buffer and analyzed by 2-D protein electrophoresis. Protein spots showing differential expression were excised, trypsin digested and analysed by MALDI-TOF. Protein identification by protein mass fingerprinting showed that dihydrolipoamide dehydrogenase was up-regulated in both shift experimets. Enolase was up-regulated in bile cultures, but down-regulated in bile salts cultures

All off the findings in this study indicate that B. cereus ATCC 14579 vegetative cells would not survive in the intestinal environment. Down-regulation of virulence factors and induction of what seem to be a secondary stress response indicates that B. cereus ATCC 14579 does not use bile as a factor for sensing the intestinal environment.