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.

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 (ΔcodY) 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 ΔcodY 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.

Genome-wide transcriptional profiling of the cell envelope stress response and the role of LisRK and CesRK in Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes is widely distributed in the environment and capable of causing food-borne infections in susceptible individuals. In this study, we investigated the cell envelope stress response in L. monocytogenes. Whole-genome transcriptional profiling was performed to investigate the response upon exposure to the cell wall antibiotic cefuroxime. Differential expression (at least twofold) of 558 genes was observed, corresponding to 20 % of the L. monocytogenes genome. The majority of genes that were strongly induced by cefuroxime exposure have cell-envelope-related functions, including the dlt operon and the gene encoding penicillin-binding protein PBPD2. A large overlap was observed between the cefuroxime stimulon and genes known to be induced in L. monocytogenes in blood and during intracellular infection, indicating that the cell envelope stress response is active at various stages of the infectious process. We analysed the roles of the two-component systems LisRK and CesRK in the cell envelope response, showing that activation of the most highly cefuroxime-induced genes was LisR- and CesR-dependent. In addition, multiple VirRS- and LiaSR-regulated genes were found to be induced in response to cefuroxime exposure. In total, 53 % of the genes upregulated at least fourfold by cefuroxime exposure are under positive control by one of the four two-component systems. Using genetic analyses, we showed that several genes of the cefuroxime stimulon contribute to the innate resistance of L. monocytogenes to cefuroxime and tolerance to other cell-envelope-perturbing conditions. Collectively, these findings demonstrate central roles for two-component systems in orchestrating the cell envelope stress response in L. monocytogenes.

Heat stress leads to superoxide formation in Bacillus cereus detected using the fluorescent probe MitoSOX.

Bacillus cereus is a food-borne human pathogen and food spoilage organism. Spores and vegetative cells of B. cereus can be found almost everywhere and therefore often end up in food processing equipment and food products. To remove spores and vegetative cells from food or equipment, harsh treatments such as high temperatures are applied. The heat stress response of B. cereus and other organisms has been studied and it has been shown that reactive oxygen species may be involved in inactivating the bacterial cells. Using a novel approach with the fluorescent probe MitoSOX, the formation of superoxide in B. cereus cells upon exposure to heat has been confirmed. MitoSOX can be used in combination with other probes, including, SYTOX green, CYTO 9, and CFDA, showing superoxide formation in combination with damaged cell membranes, intact cell membranes, and esterase activity in cells with intact membranes, respectively. MitoSOX in combination with flow cytometry-assisted sorting showed three distinct populations, a low fluorescent population that was still viable, a highly fluorescent population that could not be recovered on agar plates, and a low fluorescent non-viable population that appeared after prolonged exposure to heat. This third population may include dead cells where MitoSOX binds to DNA without reacting with superoxide. Superoxide formation during exposure to lethal temperatures by B. cereus shows that superoxide plays a role in bacterial cell death and its generation may thus contribute to the efficiency of food preservation conditions.

Bacillus cereus responses to acid stress.

Coping with acid environments is one of the prerequisites for the soil saprophytic and human pathogenic lifestyle of Bacillus cereus. This minireview highlights novel insights in the responses displayed by vegetative cells and germinating spores of B. cereus upon exposure to low pH as well as organic acids, including acetic acid, lactic acid and sorbic acid. Insights regarding the possible acid-inflicted damage, physiological responses and protective mechanisms have been compiled based on single cell fluorescence microscopy, flow cytometry and transcriptome analyses.

Physiological parameters of Bacillus cereus marking the end of acid-induced lag phases.

During lag phases microbial cells adapt to their environment and prepare to proliferate. Physiological parameters of B. cereus cells upon exposure to near-growth-boundary acid stress were investigated and markers for the transition between lag phase and growth were identified using fluorescent probes combined with flow cytometry. Determination of cell counts and optical density revealed lag phases of 1h, 2h and 5h, in cultures shifted to pH 7, pH 5.3 (set with lactic acid) and pH 4.9 (set with sulfuric acid), respectively. The obtained lag phases fitted the trends in ATP levels, which were constant during the lag phase and increased after the onset of growth. Both the percentage of PI-stained cells and cells with a significant membrane potential decreased during the lag phase. This points to repair of membrane damage and the loss of membrane potential. However, both trends extended in the growth phase, thus not suitable to mark the onset of growth. The activity of the electron transfer chain and esterases did allow for assessment of transition between lag and growth phase. These activities were generally low during the lag phase and increased after the onset of growth. Our results show that, independent of the duration of the lag phase, for different conditions the same physiological trends could be observed. The change in signal of selected probes can be used as a marker for transition from lag phase to the growth phase and may aid in identification of novel targets interfering with bacterial exit from lag phase.

Primary and secondary oxidative stress in Bacillus.

Coping with oxidative stress originating from oxidizing compounds or reactive oxygen species (ROS), associated with the exposure to agents that cause environmental stresses, is one of the prerequisites for an aerobic lifestyle of Bacillus spp. such as B. subtilis, B. cereus and B. anthracis. This minireview highlights novel insights in the primary oxidative stress response caused by oxidizing compounds including hydrogen peroxide and the secondary oxidative stress responses apparent upon exposure to a range of agents and conditions leading to environmental stresses such as antibiotics, heat and acid. Insights in the pathways and damaging radicals involved have been compiled based among others on transcriptome studies, network analyses and fluorescence techniques for detection of ROS at single cell level. Exploitation of the current knowledge for the control of spoilage and pathogenic bacteria is discussed.

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.

Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation.

Acid stress resistance of the food-borne human pathogen Bacillus cereus may contribute to its survival in acidic environments, such as encountered in soil, food and the human gastrointestinal tract. The acid stress responses of B. cereus strains ATCC 14579 and ATCC 10987 were analysed in aerobically grown cultures acidified to pH values ranging from pH 5.4 to pH 4.4 with HCl. Comparative phenotype and transcriptome analyses revealed three acid stress-induced responses in this pH range: growth rate reduction, growth arrest and loss of viability. These physiological responses showed to be associated with metabolic shifts and the induction of general stress response mechanisms with a major oxidative component, including upregulation of catalases and superoxide dismutases. Flow cytometry analysis in combination with the hydroxyl (OH.) and peroxynitrite (ONOO(-))-specific fluorescent probe 3'-(p-hydroxyphenyl) fluorescein (HPF) showed excessive radicals to be formed in both B. cereus strains in bactericidal conditions only. Our study shows that radicals can indicate acid-induced malfunctioning of cellular processes that lead to cell death.

Comparative analysis of transcriptional and physiological responses of Bacillus cereus to organic and inorganic acid shocks.

Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to pH 5.5 set with different acidulants including hydrochloric acid (HCl), lactic acid (HL) and acetic acid (HAc). Phenotypes observed included a decreased growth rate (with HCl), bacteriostatic and bactericidal conditions, with 2mM undissociated HAc or HL, and 15mM undissociated HAc, respectively. In the latter condition a concomitant decrease in intracellular ATP levels was observed. The transcriptome analyses revealed general and specific responses to the acidulants used. The general acid stress response includes modulation of pyruvate metabolism with activation of the butanediol fermentation pathway, and an oxidative stress response that was, however, more extensive in the bacteriostatic and bactericidal conditions. HL-specific and HAc-specific responses include modulation of metabolic pathways for amino acid metabolism. Activation of lactate, formate, and ethanol fermentation pathways, alternative electron-transport chain components and fatty acid biosynthesis genes was noted in the presence of 15mM undissociated HAc. In conclusion, our study has provided insights in phenotype-associated, and general and acidulant-specific responses in B. cereus.

The impact of oxygen availability on stress survival and radical formation of Bacillus cereus.

Both the growth and stress survival of two model Bacillus cereus strains, ATCC 14579 and ATCC 10987, were tested in three different conditions varying in oxygen availability, i.e., aerobic, microaerobic and anaerobic conditions. Both B. cereus strains displayed highest growth rates and yields under aerobic conditions, whereas the microaerobic and anaerobic cultures showed similar reduced growth performances. The cells grown and exposed microaerobically and anaerobically were more resistant to heat and acid than cells that were cultured and exposed aerobically. On the other hand, the anaerobically grown cells were more sensitive to hydrogen peroxide compared to the (micro)aerobically grown cells. The increased heat- and acid-induced inactivation in aerobic conditions appeared to be associated with intracellular accumulation of excess hydroxyl and/or peroxynitrite radicals, as determined by flow cytometry in combination with the fluorescent reporter dye 3'-(p-hydroxyphenyl) fluorescein. This suggests that radical formation may contribute to inactivation of bacteria in the presence of oxygen, such as in aerobic and microaerobic conditions. No evidence was found for radical formation upon exposure to salt and hydrogen peroxide. The increased resistance to heat and acid in microaerobic and anaerobic conditions shows that oxygen availability should be taken into account when behavior of bacteria, such as B. cereus, in food industry related conditions is investigated, because oxygen availability may affect the efficiency of food preservation conditions.

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.

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.

Metabolic capacity of Bacillus cereus strains ATCC 14579 and ATCC 10987 interlinked with comparative genomics.

Bacillus cereus is an important food-borne pathogen and spoilage organism. In this study, numerous phenotypes and the genomes of B. cereus strains ATCC 14579 and ATCC 10987 were analysed to compare their metabolic capacity and stress resistance potential. The growth performance of the two strains was assessed for nearly 2000 phenotypes, including use of nutrient sources, performance in acid and basic environments, osmo-tolerance and antibiotic resistance. Several food-relevant phenotypic differences were found between ATCC 14579 and ATCC 10987, such as differences in utilization of carbohydrates, peptides, amino acids and ammonia. Subsequently, the genomes of both strains were analysed with INPARANOID to search for strain-specific open reading frames (ORFs). B. cereus ATCC 14579 and ATCC 10987 were found to harbour 983 and 1360 strain-specific ORFs respectively. The strain-specific phenotypic features were interlinked with corresponding genetic features and for several phenotypic differences a related strain-specific genetic feature could be identified. In conclusion, the combination of phenotypic data with strain-specific genomic differences has led to detailed insight into the performance of the two B. cereus strains, and may supply indicators for the performance of these bacteria in different environments and ecological niches.

Role of a highly conserved bacterial protein in outer membrane protein assembly.

After transport across the cytoplasmic membrane, bacterial outer membrane proteins are assembled into the outer membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various outer membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of outer membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in outer membrane protein assembly.