A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function

Heterogeneity in single-cell outgrowth of Listeria monocytogenes in half Fraser enrichment broth is affected by strain variability and physiological state

The behaviour of pathogens at the single-cell level can be highly variable and can thus affect the detection efficacy of enrichment-based detection methods. The outgrowth of single cells of three Listeria monocytogenes strains was monitored after fluorescence-activated single-cell sorting in non-selective brain heart infusion (BHI) broth and selective half Fraser enrichment broth (HFB) to quantify outgrowth heterogeneity and its effect on the detection probability. Single-cell heterogeneity was higher in HFB compared to non-selective BHI and heterogeneity increased further when cells were heat-stressed. The increase in heterogeneity was also strain-dependent because the fast-recovering strain Scott A showed less outgrowth heterogeneity than the slower-recovering strains EGDe and H7962. Modelling of the outgrowth kinetics during the primary enrichment demonstrated that starting at low cell concentrations could fail detection of L. monocytogenes at least partly due to cell heterogeneity. This highlights that it is important to take single-cell heterogeneity into account when optimizing enrichment formulations and procedures when L. monocytogenes contamination levels are low

Analysis of the Role of RsbV, RsbW, and RsbY in Regulating σ(B) Activity in Bacillus cereus

The alternative sigma factor σ(B) is an important regulator of the stress response of Bacillus cereus. Here, the role of the regulatory proteins RsbV, RsbW, and RsbY in regulating σ(B) activity in B. cereus is analyzed. Functional characterization of RsbV and RsbW showed that they act as an anti-sigma factor antagonist and an anti-sigma factor, respectively. RsbW can also act as a kinase on RsbV. These data are in line with earlier functional characterizations of RsbV and RsbW homologs in B. subtilis. The rsbY gene is unique to B. cereus and its closest relatives and is predicted to encode a protein with an N-terminal CheY domain and a C-terminal PP2C domain. In an rsbY deletion mutant, the σ(B) response upon stress exposure was almost completely abolished, but the response could be restored by complementation with full-length rsbY. Expression analysis showed that rsbY is transcribed from both a σ(A)-dependent promoter and a σ(B)-dependent promoter. The central role of RsbY in regulating the activity of σ(B) indicates that in B. cereus, the σ(B) activation pathway is markedly different from that in other gram-positive bacteria

Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage-encoded holin–lysin system

Gram-positive bacterial extracellular membrane vesicles (EVs) have been drawing more attention in recent years. However, mechanistic insights are still lacking on how EVs are released through the cell walls in Gram-positive bacteria. In this study, we characterized underlying mechanisms of EV production and provide evidence for a role of prophage activation in EV release using the Gram-positive bacterium Lactococcus lactis as a model. By applying a standard EV isolation procedure, we observed the presence of EVs in the culture supernatant of a lysogenic L. lactis strain FM-YL11, for which the prophage-inducing condition led to an over 10-fold increase in EV production in comparison with the non-inducing condition. In contrast, the prophage-encoded holin–lysin knockout mutant YL11ΔHLH and the prophage-cured mutant FM-YL12 produced constantly low levels of EVs. Under the prophage-inducing condition, FM-YL11 did not show massive cell lysis. Defective phage particles were found to be released in and associated with holin–lysin-induced EVs from FM-YL11, as demonstrated by transmission electron microscopic images, flow cytometry and proteomics analysis. Findings from this study further generalized the EV-producing phenotype to Gram-positive L. lactis, and provide additional insights into the EV production mechanism involving prophage-encoded holin–lysin system. The knowledge on bacterial EV production can be applied to all Gram-positive bacteria and other lactic acid bacteria with important roles in fermentations and probiotic formulations, to enable desired release and delivery of cellular components with nutritional values or probiotic effects

Identification of CdnL, a putative transcriptional regulator involved in repair and outgrowth of heat-damaged bacillus cereus spores

Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-Treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-Treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-Treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.</p

Gene profiling-based phenotyping for identification of cellular parameters that contribute to fitness, stress-tolerance and virulence of Listeria monocytogenes variants.

Microbial population heterogeneity allows for a differential microbial response to environmental stresses and can lead to the selection of stress resistant variants. In this study, we have used two different stress resistant variants of Listeria monocytogenes LO28 with mutations in the rpsU gene encoding ribosomal protein S21, to elucidate features that can contribute to fitness, stress-tolerance and host interaction using a comparative gene profiling and phenotyping approach. Transcriptome analysis showed that 116 genes were upregulated and 114 genes were downregulated in both rpsU variants. Upregulated genes included a major contribution of SigB-controlled genes such as intracellular acid resistance-associated glutamate decarboxylase (GAD) (gad3), genes involved in compatible solute uptake (opuC), glycerol metabolism (glpF, glpK, glpD), and virulence (inlA, inlB). Downregulated genes in the two variants involved mainly genes involved in flagella synthesis and motility. Phenotyping results of the two rpsU variants matched the gene profiling data including enhanced freezing resistance conceivably linked to compatible solute accumulation, higher glycerol utilisation rates, and better adhesion to Caco 2 cells presumably linked to higher expression of internalins. Also, bright field and electron microscopy analysis confirmed reduced flagellation of the variants. The activation of SigB-mediated stress defence offers an explanation for the multiple-stress resistant phenotype in rpsU variants

Array expression ratios (log2 values) of candidate genes displaying specific upregulation during germination and outgrowth of heat-treated <i>B</i>. <i>cereus</i> ATCC14579 spores and verified by qPCR.

<p>False Discovery Rates below 0.05 are indicated in bold.</p

Expression profiles (log2 values) of reported spore specific transcripts during germination and outgrowth of untreated (white diamonds) and heat-treated (black diamonds) <i>B</i>. <i>cereus</i> ATCC14579 spores.

<p>Expression ratio’s for untreated spores are relative to untreated germinating control spores at t10, and for heat-treated spores relative to heat-treated germinating spores at t50.</p

Surviving and mildly and severely heat damaged spore fractions in <i>B</i>. <i>cereus</i> ATCC1457 and its mutant derivative strain Δ<i>cdnL</i> (BC4714) upon exposure to wet heat, hydrogen peroxide and sodium hypochlorite treatments.

<p>Averages of three independent experiments are represented.</p