The mechanism by which methionine relieves the growth inhibition of Escherichia coli K-12 that is caused by organic weak acid food preservatives was investigated. In the presence of 8 mM acetate the specific growth rate of E. coli Frag1 (in MacIlvaine’s minimal medium pH 6·0) is reduced by 50%. Addition of methionine restores growth to 80% of that observed in untreated controls. Similar relief was seen with cultures treated with either benzoate or propionate. Mutants with an elevated intracellular methionine pool were almost completely resistant to the inhibitory effects of acetate, suggesting that the methionine pool becomes limiting for growth in acetate-treated cells. Measurement of the intracellular concentrations of pathway intermediates revealed that the homocysteine pool is increased dramatically in acetate-treated cells, suggesting that acetate inhibits a biosynthetic step downstream from this intermediate. Supplementation of the medium with homocysteine inhibits the growth of E. coli cells. Acetate inhibition of growth arises from the depletion of the intracellular methionine pool with the concomitant accumulation of the toxic intermediate homocysteine and this augments the effect of lowering cytoplasmic pH.

To determine the contribution of sigma B (ς^B) to survival of stationary-phaseListeria monocytogenescells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolarsigBnull mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigBand wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigBstrain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigBstrain. These results suggest the existence inL. monocytogenesof both a ς^B-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ς^Bcontributed to resistance to both oxidative stress and carbon starvation inL. monocytogenes. The ΔsigBstrain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigBstrain lost viability more rapidly than the parent strain. ς^Bcontributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ς^Bplays a role in protectingL. monocytogenesagainst environmental adversities.

Weak organic acids have been used for centuries to preserve foods, but only recently has the possible mechanism for bacterial growth inhibition been investigated. Although the lowering of internal pH was favored as the cause of growth inhibition, the emphasis has shifted to the anion and its specificity. There are a number of applications of weak organic acids to foods and in the food industry be they pre-or postharvest, However, there is concern that the ability of foodborne pathogens to adapt to these acids may allow longer survival in these commodities and also to better survive transit through the gastric acid barrier of the stomach. Genomic and proteomic approaches have been applied to the identification of genes and proteins that may allow prokaryotes to cope with organic acid stress. These technologies in combination with genetic approaches may provide better identification of genes essential for survival to organic acids. These acids may have other roles: they can induce phenotypic antibiotic resistance, and the high concentrations of these acids in the colon may signal a relationship to diet, colonic microflora, and human health.

The foodborne pathogen L. monocytogenes is a highly adaptable organism that can persist in a wide range of environmental and food-related niches. The consumption of contaminated ready-to-eat (RTE) foods can cause infections, termed listeriosis, in vulnerable humans, particularly those with weakened immune systems. Although these infections are comparatively rare they are associated with high mortality rates and therefore this pathogen has a significant impact on food safety. L. monocytogenes can adapt to and survive a wide range of stress conditions including low pH, low water activity, and low temperature, which makes it problematic for food producers who rely on these stresses for preservation. Stress tolerance in L. monocytogenes can be explained partially by the presence of the General Stress Response (GSR), a transcriptional response under the control of the alternative sigma factor sigma B (B) that reconfigures gene transcription to provide homeostatic and protective functions to cope with the stress. Within the host B also plays a key role in surviving the harsh conditions found in the gastrointestinal tract. As the infection progresses beyond the GI tract L. monocytogenes uses an intracellular infectious cycle to propagate, spread and remain protected from the host’s humoral immunity. Many of the virulence genes that facilitate this infectious cycle are under the control of a master transcriptional regulator called PrfA. In this review we consider the environmental reservoirs that enable L. monocytogenes to gain access to the food chain and discuss the stresses that the pathogen must overcome to survive and grow in these environments. The overlap that exists between stress tolerance and virulence is described. We review the principal measures that are used to control the pathogen and point to exciting new approaches that might provide improved means of control in the future.

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ ^B , which is encoded by sigB , to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes . At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and Δ sigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the Δ sigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both Δ sigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σ ^B independent. σ ^B -mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ ^B reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σ ^B does not appear to contribute to pH _i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH _i buffering by the GAD system under the conditions examined in this study. In summary, a functional σ ^B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.

Sigma B (σ ^B ) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the Δ sigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The Δ sigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σ ^B contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the Δ sigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σ ^B in L. monocytogenes , we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the Δ sigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σ ^B . It also demonstrated clear roles for σ ^B in both osmotic and low-pH stress tolerance and identified specific components of the σ ^B regulon that contribute to the responses observed.