Prediction and validation of novel SigB regulon members in Bacillus subtilis and regulon structure comparison to Bacillales members.

BACKGROUND: Sigma factor B (SigB) is the central regulator of the general stress response in Bacillus subtilis and regulates a group of genes in response to various stressors, known as the SigB regulon members. Genes that are directly regulated by SigB contain a promotor binding motif (PBM) with a previously identified consensus sequence. RESULTS: In this study, refined SigB PBMs were derived and different spacer compositions and lengths (N12-N17) were taken into account. These were used to identify putative SigB-regulated genes in the B. subtilis genome, revealing 255 genes: 99 had been described in the literature and 156 genes were newly identified, increasing the number of SigB putative regulon members (with and without a SigB PBM) to > 500 in B. subtilis. The 255 genes were assigned to five categories (I-V) based on their similarity to the original SigB consensus sequences. The functionalities of selected representatives per category were assessed using promoter-reporter fusions in wt and ΔsigB mutants upon exposure to heat, ethanol, and salt stress. The activity of the PrsbV (I) positive control was induced upon exposure to all three stressors. PytoQ (II) showed SigB-dependent activity only upon exposure to ethanol, whereas PpucI (II) with a N17 spacer and PylaL (III) with a N16 spacer showed mild induction regardless of heat/ethanol/salt stress. PywzA (III) and PyaaI (IV) displayed ethanol-specific SigB-dependent activities despite a lower-level conserved - 10 binding motif. PgtaB (V) was SigB-induced under ethanol and salt stress while lacking a conserved - 10 binding region. The activities of PygaO and PykaA (III) did not show evident changes under the conditions tested despite having a SigB PBM that highly resembled the consensus. The identified extended SigB regulon candidates in B. subtilis are mainly involved in coping with stress but are also engaged in other cellular processes. Orthologs of SigB regulon candidates with SigB PBMs were identified in other Bacillales genomes, but not all showed a SigB PBM. Additionally, genes involved in the integration of stress signals to activate SigB were predicted in these genomes, indicating that SigB signaling and regulon genes are species-specific. CONCLUSION: The entire SigB regulatory network is sophisticated and not yet fully understood even for the well-characterized organism B. subtilis 168. Knowledge and information gained in this study can be used in further SigB studies to uncover a complete picture of the role of SigB in B. subtilis and other species.

SigB modulates expression of novel SigB regulon members via Bc1009 in non-stressed and heat-stressed cells revealing its alternative roles in Bacillus cereus.

BACKGROUND: The Bacillus cereus Sigma B (SigB) dependent general stress response is activated via the two-component RsbKY system, which involves a phosphate transfer from RsbK to RsbY. It has been hypothesized that the Hpr-like phosphocarrier protein (Bc1009) encoded by bc1009 in the SigB gene cluster may play a role in this transfer, thereby acting as a regulator of SigB activation. Alternatively, Bc1009 may be involved in the activation of a subset of SigB regulon members. RESULTS: We first investigated the potential role of bc1009 to act as a SigB regulator but ruled out this possibility as the deletion of bc1009 did not affect the expression of sigB and other SigB gene cluster members. The SigB-dependent functions of Bc1009 were further examined in B. cereus ATCC14579 via comparative proteome profiling (backed up by transcriptomics) of wt, Δbc1009 and ΔsigB deletion mutants under heat stress at 42 °C. This revealed 284 proteins displaying SigB-dependent alterations in protein expression levels in heat-stressed cells, including a subgroup of 138 proteins for which alterations were also Bc1009-dependent. Next to proteins with roles in stress defense, newly identified SigB and Bc1009-dependent proteins have roles in cell motility, signal transduction, transcription, cell wall biogenesis, and amino acid transport and metabolism. Analysis of lethal stress survival at 50 °C after pre-adaptation at 42 °C showed intermediate survival efficacy of Δbc1009 cells, highest survival of wt, and lowest survival of ΔsigB cells, respectively. Additional comparative proteome analysis of non-stressed wt and mutant cells at 30 °C revealed 96 proteins with SigB and Bc1009-dependent differences in levels: 51 were also identified under heat stress, and 45 showed significant differential expression at 30 °C. This includes proteins with roles in carbohydrate/ion transport and metabolism. Overlapping functions at 30 °C and 42 °C included proteins involved in motility, and ΔsigB and Δbc1009 cells showed reduced motility compared to wt cells in swimming assays at both temperatures. CONCLUSION: Our results extend the B. cereus SigB regulon to > 300 members, with a novel role of SigB-dependent Bc1009 in the activation of a subregulon of  > 180 members, conceivably via interactions with other transcriptional regulatory networks.

A web-based microbiological hazard identification tool for infant foods.

An integrated approach to identify and assess Microbiological Hazards (MHs) and mitigate risks in infant food chains is crucial to ensure safe foods for infants and young children. A systematic procedure was developed to identify MHs in specific infant foods. This includes five major steps: 1) relevant hazard-food pairing, 2) process inactivation efficiency, 3) recontamination possibility after processing, 4) MHs growth opportunity, and 5) MHs-food association level. These steps were integrated into an online tool called the Microbiological Hazards IDentification (MiID) decision support system (DSS), targeting food companies, governmental agencies and academia users, and is accessible at https://foodmicrobiologywur.shinyapps.io/Microbial_hazards_ID/. The MiID DSS was validated in four case studies, focussing on infant formula, fruit puree, cereal-based meals, and fresh fruits, each representing distinct products and processing characteristics. The results obtained through the application of the MiID DSS, compared with identification by food safety experts, consistently identified the top MHs in these food products. This process affirms its effectiveness in systematic hazard identification. The introduction of the MiID DSS helps to structure the first steps in HACCP (hazard analysis) and in risk assessment (hazard identification) to follow a structured and well-documented procedure, balancing the risk of overlooking relevant MHs or including too many irrelevant MHs. It is a valuable addition to risk analysis/assessment in infant food chains and has the potential for future extension. This includes the incorporation of newly acquired data related to infant foods via a semi-publicly hosted platform, or it can be adapted for hazard identification in general food products using a similar framework.

Disentangling the contributions of initial heterogeneities and dynamic stress adaptation to nonlinearities in bacterial survival curves.

The deviations from log-linearity that are often observed in bacterial survivor curves can be explained using different arguments, both biological and experimental. In this study, we used Bacillus subtilis as a model organism to demonstrate that the generally accepted vitalistic arguments (initial heterogeneities in the stress resistance of the cells in the population) may fail to describe microbial inactivation in some situations. In this sense, we showed how dynamic stress acclimation during an isothermal treatment provides an alternative explanation for survivor curves with an upwards curvature. We also provided an innovative experimental approach based on preadaptation experiments to evaluate which hypothesis is more suitable for the bacterial response. Furthermore, we used our experimental results to define bounds for the possible stress acclimation that may take place during dynamic treatments, concluding that the magnitude of stress acclimation may be larger for dynamic treatments than for isothermal experiments. We also evaluated the contribution of the SigB general stress response system to heat resistance by comparing the heat survival of wt and the ΔsigB mutant. Both strains survived better in 51, 52.5 and 55 °C when cells were pre-adapted at 48 °C than non-pre-adapted cells. However, ΔsigB was less resistant to heat than wt due to the missing SigB general stress system. Although these conclusions were based on B. subtilis as a model organism, this study can be the first step towards the development of a novel methodology able to estimate dynamic effects using only isothermal experiments. This would improve the models developed within the predictive microbiology community, improving our ability to predict microbial inactivation during industrial treatments, which are most often dynamic.

Lichenysin Production by Bacillus licheniformis Food Isolates and Toxicity to Human Cells.

Bacillus licheniformis can cause foodborne intoxication due to the production of the surfactant lichenysin. The aim of this study was to measure the production of lichenysin by food isolates of B. licheniformis in LB medium and skimmed milk and its cytotoxicity for intestinal cells. Out of 11 B. licheniformis isolates tested, most showed robust growth in high salt (1M NaCl), 4% ethanol, at 37 or 55°C, and aerobic and anaerobic conditions. All strains produced lichenysin (in varying amounts), but not all strains were hemolytic. Production of this stable compound by selected strains (high producers B4094 and B4123, and type strain DSM13 T ) was subsequently determined using LB medium and milk, at 37 and 55°C. Lichenysin production in LB broth and milk was not detected at cell densities < 5 log10 CFU/ml. The highest concentrations were found in the stationary phase of growth. Total production of lichenysin was 4-20 times lower in milk than in LB broth (maximum 36 µg/ml), and ∼10 times lower in the biomass obtained from milk agar than LB agar. Under all conditions tested, strain B4094 consistently yielded the highest amounts. Besides strain variation and medium composition, temperature also had an effect on lichenysin production, with twofold lower amounts of lichenysin produced at 55°C than at 37°C. All three strains produced lichenysin A with varying acyl chain lengths (C11-C18). The relative abundance of the C14 variant was highest in milk and the C15 variant highest in LB. The concentration of lichenysin needed to reduce cell viability by 50% (IC50) was 16.6 µg/ml for Caco-2 human intestinal epithelial cells and 16.8 µg/ml for pig ileum organoids. Taken together, the presence of low levels (<5 log10 CFU/ml) of B. licheniformis in foods is unlikely to pose a foodborne hazard related to lichenysin production. However, depending on the strain present, the composition, and storage condition of the food, a risk of foodborne intoxication may arise if growth to high levels is supported and such product is ingested.