The seven extracytoplasmic function (ECF) sigma () factors of Bacillus subtilis are broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of V as including at least 20 operons, many of which are also regulated by M , X , or W . The V regulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance of B. subtilis to lysozyme. Strains with null mutations in either sigV or all seven ECF factor genes (⌬7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of V in the ⌬7ECF background restores lysozyme resistance, whereas induction of M , X , or W does not. Lysozyme resistance results from the ability of V to activate the transcription of two operons: the autoregulated sigV-rsiV-oatA-yrhK operon and dltABCDE. Genetic analyses reveal that oatA and dlt are largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas an oatA dltA double mutant is as sensitive as a sigV null strain. Moreover, the sigV oatA dltA triple mutant is no more sensitive than the oatA dltA double mutant, indicating that there are no other V -dependent genes necessary for lysozyme resistance. Thus, we suggest that V confers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA and D-alanylation of teichoic acids by DltABCDE.

Gram-positive bacteria are ubiquitous and diverse microorganisms that can survive and sometimes even thrive in continuously changing environments. The key to such resilience is the ability of members of a population to respond and adjust to dynamic conditions in the environment. In bacteria, such responses and adjustments are mediated, at least in part, through appropriate changes in the bacterial transcriptome in response to the conditions encountered. Resilience is important for bacterial survival in diverse, complex, and rapidly changing environments and requires coordinated networks that integrate individual, mechanistic responses to environmental cues to enable overall metabolic homeostasis. In many Gram-positive bacteria, a key transcriptional regulator of the response to changing environmental conditions is the alternative sigma factor B . B has been characterized in a subset of Gram-positive bacteria, including the genera Bacillus, Listeria, and Staphylococcus. Recent insight from nextgeneration-sequencing results indicates that B -dependent regulation of gene expression contributes to resilience, i.e., the coordination of complex networks responsive to environmental changes. This review explores contributions of B to resilience in Bacillus, Listeria, and Staphylococcus and illustrates recently described regulatory functions of B .

SUMMARY The foodborne pathogen Listeria monocytogenes can modulate its transcriptome and proteome to ensure its survival during transmission through vastly differing environmental conditions. While L. monocytogenes utilizes a large array of regulators to achieve survival and growth in different intra- and extrahost environments, the alternative sigma factor σB and the transcriptional activator of virulence genes protein PrfA are two key transcriptional regulators essential for responding to environmental stress conditions and for host infection. Importantly, emerging evidence suggests that the shift from extrahost environments to the host gastrointestinal tract and, subsequently, to intracellular environments requires regulatory interplay between σB and PrfA at transcriptional, posttranscriptional, and protein activity levels. Here, we review the current evidence for cross talk and interplay between σB and PrfA and their respective regulons and highlight the plasticity of σB and PrfA cross talk and the role of this cross talk in facilitating successful transition of L. monocytogenes from diverse extrahost to diverse extra- and intracellular host environments.

Among the alternative sigma factors of Listeria monocytogenes, σ B controls the largest regulon. The aim of this study was to perform a comprehensive review of σ B -regulated genes, and the functions they confer. Materials & methods: A systematic search of PubMed and Web of Knowledge was carried out to identify members of the σ B regulon based on experimental evidence of σ B -dependent transcription and presence of a consensus σ B -dependent promoter. Results: The literature review identified σ B -dependent transcription units encompassing 304 genes encoding different functions including stress response and virulence. Conclusion: Our review supports the well-known roles of σ B in virulence and stress response and provides new insight into novel roles for σ B in metabolism and overall resilience of L. monocytogenes.

Listeria monocytogenes uses a variety of transcriptional regulation strategies to adapt to the extra-host environment, the gastrointestinal tract, and the intracellular host environment. While the alternative sigma factor SigB has been proposed to be a key transcriptional regulator that facilitates L. monocytogenes adaptation to the gastrointestinal environment, the L. monocytogenes' transcriptional response to bile exposure is not well-understood. RNA-seq characterization of the bile stimulon was performed in two L. monocytogenes strains representing lineages I and II. Exposure to bile at pH 5.5 elicited a large transcriptomic response with ~16 and 23% of genes showing differential transcription in 10403S and H7858, respectively. The bile stimulon includes genes involved in motility and cell wall modification mechanisms, as well as genes in the PrfA regulon, which likely facilitate survival during the gastrointestinal stages of infection that follow bile exposure. The fact that bile exposure induced the PrfA regulon, but did not induce further upregulation of the SigB regulon (beyond that expected by exposure to pH 5.5), suggests a model where at the earlier stages of gastrointestinal infection (e.g., acid exposure in the stomach), SigB-dependent gene expression plays an important role. Subsequent exposure to bile induces the PrfA regulon, potentially priming L. monocytogenes for subsequent intracellular infection stages. Some members of the bile stimulon showed lineage- or strain-specific distribution when 27 Listeria genomes were analyzed. Even though sigB null mutants showed increased sensitivity to bile, the SigB regulon was not found to be upregulated in response to bile beyond levels expected by exposure to pH 5.5. Comparison of wildtype and corresponding ΔsigB strains newly identified 26 SigB-dependent genes, all with upstream putative SigB-dependent promoters.

Among Listeria monocytogenes' four alternative σ factors, σB controls the largest regulon. As σB-dependent transcription of some genes may be masked by overlaps among regulons, and as some σB-dependent genes are expressed only under very specific conditions, we hypothesized that the σB regulon is not yet fully defined. To further extend our understanding of the σB regulon, we used RNA-seq to identify σB-dependent genes in an L. monocytogenes strain that expresses σB following rhamnose induction, and in which genes encoding the other alternative sigma factors have been deleted. Analysis of RNA-seq data with multiple bioinformatics approaches, including a sliding window method that detects differentially transcribed 5′ untranslated regions (UTRs), identified 105 σB-dependent transcription units (TUs) comprising 201 genes preceded by σB-dependent promoters. Of these 105 TUs, 7 TUs comprising 15 genes had not been identified previously as σB-dependent. An additional 23 genes not reported previously as σB-dependent were identified in 9 previously recognized σB-dependent TUs. Overall, 38 of these 201 genes had not been identified previously as members of the L. monocytogenes σB regulon. These newly identified σB-dependent genes encode proteins annotated as being involved in transcriptional regulation, oxidative and osmotic stress response, and in metabolism of energy, carbon and nucleotides. In total, 18 putative σB-dependent promoters were newly identified. Interestingly, a number of genes previously identified as σB-dependent did not show significant evidence for σB-dependent transcription in our experiments. Based on promoter analyses, a number of these genes showed evidence for co-regulation by σB and other transcriptional factors, suggesting that some σB-dependent genes require additional transcriptional regulators along with σB for transcription. Over-expression of a single alternative sigma factor in the absence of all other alternative sigma factors allowed us to: (i) identify new σB-dependent functions in L. monocytogenes, such as regulation of genes involved in 1,2-propanediol utilization (LMRG_00594-LMRG_00611) and biosynthesis of pyrimidine nucleotides (LMRG_00978-LMRG_00985); and (ii) identify new σB-dependent genes involved in stress response and pathogenesis functions. These data further support that σB not only regulates stress response functions, but also plays a broad role in L. monocytogenes homeostasis and resilience.

The growth of Listeria monocytogenes on refrigerated, ready-to-eat food products is a major health and economic concern. The natural antimicrobial nisin targets the bacterial cell wall and can be used to inhibit L. monocytogenes growth on cheese. Cell wall composition and structure, and therefore the efficacy of cell wall acting control strategies, can be severely affected by environmental and stress conditions. The goal of this study was to determine the effect of a range of pH and temperatures on the efficacy of nisin against several strains of L. monocytogenes in a lab-scale, cheese model. Cheese was made with or without the addition of nisin at different pH and then inoculated with L. monocytogenes; L. monocytogenes numbers were quantified after 1, 7, and 14 days of incubation at 6, 14, or 22 • C. While our data show that nisin treatment is able to reduce L. monocytogenes numbers, at least initially, growth of this pathogen can occur even in the presence of nisin, especially when cheese is stored at higher temperatures. Several environmental factors were found to affect nisin efficacy against L. monocytogenes. For example, nisin is more effective when cheese is stored at lower temperatures. Nisin is also more effective when cheese is made at higher pH (6 and 6.5), compared to cheese made at pH 5.5, and this effect is at least partially due to the activity of cell envelope modification genes dltA and mprF. Serotype was also found to affect nisin efficacy against L. monocytogenes; serotype 4b strains showed lower susceptibility to nisin treatment compared to serotype 1/2 strains. Overall, our results highlight the importance of considering environmental conditions specific to a food matrix when developing and applying nisin-based intervention strategies against L. monocytogenes.

The ability of bacteria to adapt to stress depends on the conditional expression of specific sets of genes. Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma (σ) factors that regulate functions important for survival under conditions eliciting cell envelope stress. Of these, four have been studied in detail: σM, σW, σX and σV. These four σ factors recognize overlapping sets of promoters, although the sequences that determine this overlapping recognition are incompletely understood. A major role in promoter selectivity has been ascribed to the core −10 and −35 promoter elements. Here, we demonstrate that a homopolymeric T-tract motif, proximal to the −35 element, functions in combination with the core promoter sequences to determine selectivity for ECF sigma factors. This motif is most critical for promoter activation by σV, and contributes variably to activation by σM, σX and σW. We propose that this motif, which is a feature of the deduced promoter consensus for a subset of ECF σ factors from many species, imparts intrinsic DNA curvature to influence promoter activity. The differential effect of this region among ECF σ factors thereby provides a mechanism to modulate the nature and extent of regulon overlap.