Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp 1 and prfAp 2 ) or from the upstream plcA promoter. We demonstrate that prfAp 2 is a functional B -dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor B contributes to virulence in L. monocytogenes.Bacterial survival within a host and establishment of infection depend in part on the ability of pathogenic bacteria to modulate gene expression in response to environmental conditions encountered during infection. Associations between alternative sigma factors and core RNA polymerase provide one mechanism for timely alterations in gene expression by directing transcription of different regulons in response to cellular signals (17). Alternative sigma factors have been demonstrated to contribute to cellular survival under adverse conditions. For example, the stress-responsive alternative sigma factor B contributes to the ability of stationary-phase Listeria monocytogenes cells to adapt to and resume growth at reduced temperatures (3).B also has been shown to contribute to L. monocytogenes survival under in vitro conditions of oxidative stress, starvation, and reduced pH (9). In addition to its presence in L. monocytogenes, B has been identified in diverse gram-positive organisms, including Listeria innocua, Staphylococcus aureus, Bacillus subtilis, Bacillus anthracis, Bacillus licheniformis, and Staphylococcus epidermidis (2,5,10,13,14,15,18,23,24). Preliminary evidence suggests that B may contribute to virulence in gram-positive pathogens (8,10,15,16,23 Loss of B impairs L. monocytogenes virulence in a murine model. In previous work, we found modest effects of a ⌬sigB mutation on L. monocytogenes virulence in the murine model. Specifically, when the wild-type and ⌬sigB strains were inoculated separately into individual animals, the ⌬sigB strain was found to be impaired in its ability to spread to murine liver and spleen (23). In this report, we describe the application of a competitive-index method (1), which is designed to control for multiple experimental variables, including interanimal variability (23), to sensitively assess virulence differences between bacterial strains. For competitive-index studies, equal numbers of two different strains, one of which must bear a selectable marker, are inoculated into the same animal, and then both strains are enumerated at multiple postinfection time points (1). To ensure that the presence of the selectable marker does not confer a competitive advantage or disadvantage, a control mixture, which is comprised of the strain bearing the marker and an otherwise isogenic strain lacking the marker, is tested in parallel.The relative virulence of a ⌬sigB null mutant was determined using a competitive-index strategy in a murine model (Table 1) were grown in brain heart infusion broth (BHI; Becton Dickinson, Sparks, Md.) at 37°C with...

Listeria monocytogenes

ABSTRACTThe food-borne pathogenListeria monocytogenesexperiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. During transmission,L. monocytogenesis likely to experience osmotic stress at different temperatures and may adapt to osmotic stress in a temperature-dependent manner. To understand the impact of temperature on the responses this pathogen uses to adapt to osmotic stress, we assessed genome-wide changes in theL. monocytogenesH7858 transcriptome during short-term and long-term adaptation to salt stress at 7°C and 37°C. At both temperatures, the short-term response to salt stress included increased transcript levels ofsigBand SigB-regulated genes, as well asmrpABCDEFG, encoding a sodium/proton antiporter. This antiporter was found to play a role in adaptation to salt stress at both temperatures; ΔmrpABCDEFGhad a significantly longer lag phase than the parent strain in BHI plus 6% NaCl at 7°C and 37°C. The short-term adaptation to salt stress at 7°C included increased transcript levels of two genes encoding carboxypeptidases that modify peptidoglycan. These carboxypeptidases play a role in the short-term adaptation to salt stress only at 7°C, where the deletion mutants had significantly different lag phases than the parent strain. Changes in the transcriptome at both temperatures suggested that exposure to salt stress could provide cross-protection to other stresses, including peroxide stress. Short-term exposure to salt stress significantly increased H2O2resistance at both temperatures. These results provide information for the development of knowledge-based intervention methods against this pathogen, as well as provide insight into potential mechanisms of cross-protection.

BackgroundWhile, traditionally, regulation of gene expression can be grouped into transcriptional, translational, and post-translational mechanisms, some mechanisms of rapid genetic variation can also contribute to regulation of gene expression, e.g., phase variation.ResultsWe show here that prokaryotes evolved to include homopolymeric tracts (HTs) within coding genes as a system that allows for efficient gene inactivation. Analyses of 81 bacterial and 18 archaeal genomes showed that poly(A) and poly(T) HTs are overrepresented in these genomes and preferentially located at the 5' end of coding genes. Location of HTs at the 5' end is not driven by a preferential placement of aminoacids encoded by the AAA and TTT codons at the N-terminal of proteins. The inlA gene of the pathogen L. monocytogenes was used as a model to further study the role of HTs in reversible gene inactivation. In a number of L. monocytogenes strains, inlA harbors a 5' poly(A) HT, which regularly shows frameshift mutation leading to expression of a truncated 8 aa InlA protein. Translational fusions of the inlA 5' end allowed us to estimate that the frequency of variation in this HT is about 1,000 fold higher than the estimated average point mutation frequency.ConclusionsAs frameshift mutations in HTs can occur at high frequencies and enable efficient gene inactivation, hypermutable HTs appear to represent a universal system for regulation of gene expression in prokaryotes. Combined with other studies indicating that HTs also enable rapid diversification of both coding and regulatory genetic sequences in eukaryotes, our data suggest that hypermutable HTs represent a general and rapid evolutionary mechanism facilitating adaptation and gene regulation across diverse organisms.

Listeria monocytogenes prfA, encoding positive regulatory factor A, is transcribed from three promoters (prfAP1, prfAP2, and PplcA). The prfAP2 promoter was previously proposed to be sigma B (sigma(B))-dependent. This hypothesis was tested by creating prfA promoter-gus transcriptional fusions in both L. monocytogenes wild-type (wt) and DeltasigB backgrounds and then measuring (i) beta-glucuronidase (GUS) activities; (ii) gus mRNA transcript levels; and (iii) the presence or absence of GUS in cells by immunofluorescence staining. prfAP2-directed expression increased as the wt L. monocytogenes strain entered stationary phase, whereas prfAP2-directed expression was greatly reduced in the DeltasigB strain, confirming both growth phase- and sigma(B)-dependent transcription of prfAP2. We conclude that prfAP2 is directly regulated by sigma(B).