Assembly and Characterization of a Pathogen Strain Collection for Produce Safety Applications: Pre-growth Conditions Have a Larger Effect on Peroxyacetic Acid Tolerance Than Strain Diversity

Effective control of foodborne pathogens on produce requires science-based validation of interventions and control strategies, which typically involves challenge studies with a set of bacterial strains representing the target pathogens or appropriate surrogates. In order to facilitate these types of studies, a produce-relevant strain collection was assembled to represent strains from produce outbreaks or pre-harvest environments, including Listeria monocytogenes (n = 11), Salmonella enterica (n = 23), shiga-toxin producing Escherichia coli (STEC) (n = 13), and possible surrogate organisms (n = 8); all strains were characterized by whole genome sequencing (WGS). Strain diversity was assured by including the 10 most common S. enterica serotypes, L. monocytogenes lineages I–IV, and E. coli O157 as well as selected “non-O157” STEC serotypes. As it has previously been shown that strains and genetic lineages of a pathogen may differ in their ability to survive different stress conditions, a subset of representative strains for each “pathogen group” (e.g., Salmonella, STEC) was selected and assessed for survival of exposure to peroxyacetic acid (PAA) using strains pre-grown under different conditions including (i) low pH, (ii) high salt, (iii) reduced water activity, (iv) different growth phases, (v) minimal medium, and (vi) different temperatures (21°C, 37°C). The results showed that across the three pathogen groups pre-growth conditions had a larger effect on bacterial reduction after PAA exposure as compared to strain diversity. Interestingly, bacteria exposed to salt stress (4.5% NaCl) consistently showed the least reduction after exposure to PAA; however, for STEC, strains pre-grown at 21°C were as tolerant to PAA exposure as strains pre-grown under salt stress. Overall, our data suggests that challenge studies conducted with multi-strain cocktails (pre-grown under a single specific condition) may not necessarily reflect the relevant phenotypic range needed to appropriately assess different intervention strategies

Cell Envelope Stress Response And Antimicrobial Resistance In Bacillus Subtilis

The cell envelope of bacteria is of pivotal importance for growth and survival, and hence it is often the target of antimicrobial compounds. One of the main components involved in CESRs are extracytoplasmic function (ECF) [sigma] factors. The genome of B. subtilis encodes for seven ECF [sigma] factors, [sigma]M, [sigma]W, [sigma]X, [sigma]Y, [sigma]V, [sigma]Z and [sigma]YlaC. Several studies have been conducted to understand the role that these ECF [sigma] factors play in CESR in B. subtilis, one of the challenges found is that they display significant redundancy within their regulons. In this study, we have performed an in depth analysis of one of the ECF [sigma] factors of B. subtilis, [sigma]V, which had been previously uncharacterized. We have described the regulon of [sigma]V, the role that it plays in lysozyme resistance, and provided evidence for a novel promoter element important for [sigma]V recognition. Additionally, we have studied the role that [sigma]M plays in moenomycin resistance, and discovered a previously uncharacterized gene, ypmB, that seems to play an important role in cell envelope synthesis. Altogether, this dissertation takes further steps into understanding of the role that ECF [sigma] factors play in regulating the stress response triggered by cell envelope acting antimicrobials in B. subtilis

Resilience in the face of uncertainty: Sigma Factor B fine-tunes gene expression to support homeostasis in gram-positive bacteria

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 σ(B) σ(B) has been characterized in a subset of Gram-positive bacteria, including the genera Bacillus, Listeria, and Staphylococcus Recent insight from next-generation-sequencing results indicates that σ(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 σ(B) to resilience in Bacillus, Listeria, and Staphylococcus and illustrates recently described regulatory functions of σ(B)

The Listeria monocytogenes Bile Stimulon under Acidic Conditions Is Characterized by Strain-Specific Patterns and the Upregulation of Motility, Cell Wall Modification Functions, and the PrfA Regulon

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

Home Alone: Elimination of All but One Alternative Sigma Factor in Listeria monocytogenes Allows Prediction of New Roles for σB

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

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

The σ-like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute σ-region 2 and σ-region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the β-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in σ-region 2 and binds to the N-terminal coiled-coil element in the RNAP β′-subunit previously implicated in interaction with region 2 of σ-factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial σ-factor

Additional file 1: Table S1. of An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes

Genes identified as differentially expressed between L. monocytogenes overexpressing sigH (10403S::ΔsigBCHL P rha -sigH) and a ΔsigBCHL control strain, based on the RNA-seq coverage data calculated for complete ORFs. (DOCX 15 kb

Additional file 2: Table S2. of An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes

Listeria genomes used for comparative analysis of SigmaH-dependent promoters. (XLSX 10 kb