Sigma B (σ^B) is a stress-responsive alternative sigma factor that has been identified in various gram-positive bacteria. Seven different regulators of sigma B (Rsbs) are located in thesigBoperons of bothBacillus subtilisandListeria monocytogenes. InB. subtilis, these proteins contribute to regulation of σ^Bactivity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of σ^Bactivity in response to environmental stresses, while RsbV contributes to σ^Bactivation under both environmental and energy stresses inB. subtilis. To probeL. monocytogenesRsb roles in σ^B-mediated responses to various stresses, in-frame deletions were created inrsbTandrsbV. Phenotypic characterization of theL. monocytogenes rsbTandrsbVnull mutants revealed that both mutants were similar to the ΔsigBstrain in their abilities to survive under environmental stress conditions (exposure to synthetic gastric fluid, pH 2.5, acidified brain heart infusion broth [BHI], or oxidative stress [13 mM cumene hydroperoxide]). Under energy stress conditions (carbon starvation in defined media, entry into stationary phase, or reduced intracellular ATP), both ΔrsbTand ΔrsbVshowed survival reductions similar to that of the ΔsigBstrain. These observations suggest that the pathways for Rsb-dependent regulation of σ^Bactivity differ betweenL. monocytogenesandB. subtilis. As σ^Balso activates transcription of theL. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ΔprfAP1rsbTand ΔprfAP1rsbVdouble mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ΔprfAP1P2 and ΔprfAP1sigBdouble mutants, i.e., reduced hemolysis activity and reduced plaque size in mouse fibroblast cells. These findings indicate that RsbT and RsbV both contribute to σ^Bactivation inL. monocytogenesduring exposure to environmental and energy stresses as well as during tissue culture infection.

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. monocytogenes ; the seven regulatory proteins addressed include all four L. monocytogenes alternative sigma factors (σ ^B , σ ^C , σ ^H , and σ ^L ), the virulence gene regulator PrfA, and the heat shock-related negative regulators CtsR and HrcA. Whole-genome microarray analyses, used to identify regulons for each of these 7 transcriptional regulators, showed considerable overlap among regulons. Among 188 genes controlled by more than one regulator, 176 were coregulated by σ ^B , including 92 genes regulated by both σ ^B and σ ^H (with 18 of these genes coregulated by σ ^B , σ ^H , and at least one additional regulator) and 31 genes regulated by both σ ^B and σ ^L (with 10 of these genes coregulated by σ ^B , σ ^L , and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i) σ ^B to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, σ ^B , and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH , sigL , or sigC ) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.

To measure σ^Bactivation inListeria monocytogenesunder environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the σ^B-dependentopuCAandclpCgenes in strains having null mutations in genes encodingregulator ofsigmaBproteins (rsbTandrsbV) and sigma B (sigB) and in theL. monocytogeneswild-type 10403S strain under different stress conditions. The ΔsigB, ΔrsbT, and ΔrsbVstrains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels forhlywere also determined. RsbT, RsbV, and σ^Bwere all required foropuCAexpression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP). Expression ofclpCwas RsbT, RsbV, and σ^Bdependent in the presence of CCCP but not under the other conditions.hlyexpression was not RsbT, RsbV, or σ^Bdependent in the presence of either CCCP or salt.opuCAtranscript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such assigB. Overall, our data show that while σ^Bactivation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the σ^Bregulon (e.g.,clpC) appears to require networks involving multiple transcriptional regulators.

Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are major foodborne pathogens of concern worldwide. Bacteriophage applications have gained more interest for biocontrol in foods. This study isolated 36 Salmonella phages from several animal farms in Thailand and tested them on 47 Salmonella strains from several sources, including farms, seafood processing plant and humans in Thailand and USA. Phages were classified into three major groups. The estimated phage genome size showed the range from 50 ± 2 to 200 ± 2 kb. An effective phage cocktail consisting of three phages was developed. Approximately 4 log CFU/mL of S. Enteritidis and S. Typhimurium could be reduced. These phages revealed a burst size of up to 97.7 on S. Enteritidis and 173.7 PFU/cell on S. Typhimurium. Our phage cocktail could decrease S. Enteritidis on chicken meat and sunflower sprouts by 0.66 log CFU/cm² and 1.27 log CFU/g, respectively. S. Typhimurium on chicken meat and sunflower sprouts were decreased by 1.73 log CFU/cm² and 1.17 log CFU/g, respectively. Overall, animal farms in Thailand provided high abundance and diversity of Salmonella phages with the lysis ability on Salmonella hosts from various environments and continents. A developed phage cocktail suggests a potential biocontrol against Salmonella in fresh foods.

The stress-responsive alternative sigma factor σ^Bis conserved across diverse Gram-positive bacterial genera. InListeria monocytogenes, σ^Bregulates transcription of >150 genes, including genes contributing to virulence and to bacterial survival under host-associated stress conditions, such as those encountered in the human gastrointestinal lumen. An inhibitor ofL. monocytogenesσ^Bactivity was identified by screening ~57,000 natural and synthesized small molecules using a high-throughput cell-based assay. The compound fluoro-phenyl-styrene-sulfonamide (FPSS) (IC₅₀= 3.5 µM) downregulated the majority of genes previously identified as members of the σ^Bregulon inL. monocytogenes10403S, thus generating a transcriptional profile comparable to that of a 10403S ΔsigBstrain. Specifically, of the 208 genes downregulated by FPSS, 75% had been identified previously as positively regulated by σ^B. Downregulated genes included key virulence and stress response genes, such asinlA,inlB,bsh,hfq,opuC, andbilE. From a functional perspective, FPSS also inhibitedL. monocytogenesinvasion of human intestinal epithelial cells and bile salt hydrolase activity. The ability of FPSS to inhibit σ^Bactivity in bothL. monocytogenesandBacillus subtilisindicates its utility as a specific inhibitor of σ^Bacross multiple Gram-positive genera.IMPORTANCEThe σ^Btranscription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission ofL. monocytogenesand other Gram-positive bacteria. Regulation of σ^Bactivity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σ^Bacross Gram-positive bacterial genera, i.e., in bothListeria monocytogenesandBacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks inL. monocytogenesand other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission.