CodY, a pleiotropic regulator, influences multicellular behaviour and efficient production of virulence factors in Bacillus cereus

In response to nutrient limitation in the environment, the global transcriptional regulator CodY modulates various pathways in low G+C Gram-positive bacteria. In Bacillus subtilis CodY triggers adaptation to starvation by secretion of proteases coupled to the expression of amino acid transporters. Furthermore, it is involved in modulating survival strategies like sporulation, motility, biofilm formation, and CodY is also known to affect virulence factor production in pathogenic bacteria. In this study, the role of CodY in Bacillus cereus ATCC 14579, the enterotoxin-producing type strain, is investigated. A marker-less deletion mutant of codY (?codY) was generated in B. cereus and the transcriptome changes were surveyed using DNA microarrays. Numerous genes involved in biofilm formation and amino acid transport and metabolism were upregulated and genes associated with motility and virulence were repressed upon deletion of codY. Moreover, we found that CodY is important for efficient production of toxins and for adapting from nutrient-rich to nutrient-limited growth conditions of B. cereus. In contrast, biofilm formation is highly induced in the ?codY mutant, suggesting that CodY represses biofilm formation. Together, these results indicate that CodY plays a crucial role in the growth and persistence of B. cereus in different environments such as soil, food, insect guts and the human body

Role of Ureolytic Activity in Bacillus cereus Nitrogen Metabolism and Acid Survival▿

The presence and activities of urease genes were investigated in 49 clinical, food, and environmental Bacillus cereus isolates. Ten strains were shown to have urease genes, with eight of these strains showing growth on urea as the sole nitrogen source. Two of the urease-positive strains, including the sequenced strain ATCC 10987, could not use urea for growth, despite their capacities to produce active urease. These observations can be explained by the inability of the two strains to use ammonium as a nitrogen source. The impact of urea hydrolysis on acid stress resistance was subsequently assessed among the ureolytic B. cereus strains. However, none of the strains displayed increased fitness under acidic conditions or showed enhanced acid shock survival in the presence of urea. Expression analysis of urease genes in B. cereus ATCC 10987 revealed a low level of expression of these genes and a lack of pH-, nitrogen-, urea-, oxygen-, and growth phase-dependent modulation of mRNA transcription. This is in agreement with the low urease activity observed in strain ATCC 10987 and the other nine strains tested. Although a role for B. cereus ureolytic activity in acid survival cannot be excluded, its main role appears to be in nitrogen metabolism, where ammonium may be provided to the cells in nitrogen-limited, urea-containing environments

Comparative Transcriptomic and Phenotypic Analysis of the Responses of Bacillus cereus to Various Disinfectant Treatments▿ †

Antimicrobial chemicals are widely applied to clean and disinfect food-contacting surfaces. However, the cellular response of bacteria to various disinfectants is unclear. In this study, the physiological and genome-wide transcriptional responses of Bacillus cereus ATCC 14579 exposed to four different disinfectants (benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and peracetic acid) were analyzed. For each disinfectant, concentrations leading to the attenuation of growth, growth arrest, and cell death were determined. The transcriptome analysis revealed that B. cereus, upon exposure to the selected concentrations of disinfectants, induced common and specific responses. Notably, the common response included genes involved in the general and oxidative stress responses. Exposure to benzalkonium chloride, a disinfectant known to induce membrane damage, specifically induced genes involved in fatty acid metabolism. Membrane damage induced by benzalkonium chloride was confirmed by fluorescence microscopy, and fatty acid analysis revealed modulation of the fatty acid composition of the cell membrane. Exposure to sodium hypochlorite induced genes involved in metabolism of sulfur and sulfur-containing amino acids, which correlated with the excessive oxidation of sulfhydryl groups observed in sodium hypochlorite-stressed cells. Exposures to hydrogen peroxide and peracetic acid induced highly similar responses, including the upregulation of genes involved in DNA damage repair and SOS response. Notably, hydrogen peroxide- and peracetic acid-treated cells exhibited high mutation rates correlating with the induced SOS response

Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases

BACKGROUND: Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). RESULTS: Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. CONCLUSIONS: In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases

Phenotypic and Transcriptomic Analyses of Mildly and Severely Salt-Stressed Bacillus cereus ATCC 14579 Cells▿ †

Bacteria are able to cope with the challenges of a sudden increase in salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the pathogen Bacillus cereus ATCC 14579 were exposed to both mild (2.5% [wt/vol] NaCl) and severe (5% [wt/vol] NaCl) salt stress conditions. B. cereus continued to grow at a slightly reduced growth rate when it was shifted to mild salt stress conditions. Exposure to severe salt stress resulted in a lag period, and after 60 min growth had resumed, with cells displaying a filamentous morphology. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed that the expression of these cells overlapped with the expression of cells exposed to 5% salt stress, suggesting that the corresponding genes were involved in a general salt stress response. Upregulation of osmoprotectant, Na+/H+, and di- and tripeptide transporters and activation of an oxidative stress response were noticeable aspects of the general salt stress transcriptome response. Activation of this response may confer cross-protection against other stresses, and indeed, increased resistance to heat and hydrogen peroxide could be demonstrated after preexposure to salt. A temporal shift between the transcriptome response and several phenotypic responses of severely salt-stressed cells was observed. After resumption of growth, these cells showed cellular filamentation, reduced chemotaxis, increased catalase activity, and optimal oxidative stress resistance, which corresponded to the transcriptome response displayed in the initial lag period. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross-protection mechanisms

Measurement of the ambipolar carrier capture time in a gallium arsenide/aluminum gallium arsenide separate confinement heterostructure quantum well

The carrier capture in a separate confinement heterostructure quantum well has been studied both experimentally and theoretically. Our calculations show that the electron and hole capture time vary strongly as a function of the excess energy. At an excess energy of 40 meV, both capture times are equal resulting in an ambipolar capture process which allows a direct comparison between theory and experiment. We carried out subpicosecond luminescence spectroscopy experiments and deduce an ambipolar overall capture time of 20 ps, a number which for the first time is in agreement with theoretical predictions. The quantum mechanical overall capture time of 20 ps gives rise to a classical local capture time of 3 ps which is determined from a diffusion model