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. , 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)B to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, 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.

Listeria monocytogenes can respond rapidly to changing environmental conditions, as illustrated by its ability to transition from a saprophyte to an orally transmitted facultative intracellular pathogen. Differential associations between various alternative σ factors and a core RNA polymerase provide a transcriptional mechanism for regulating bacterial gene expression that is crucial for survival in rapidly changing conditions. Alternative σ factors are key components of complex L. monocytogenes regulatory networks that include multiple transcriptional regulators of stress-response and virulence genes, regulation of genes encoding other regulators, and regulation of small RNAs. In this article, the contributions of various σ factors to L. monocytogenes stress response and virulence are described.

Sigma B (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 the sigB operons of both Bacillus subtilis and Listeria monocytogenes. In B. subtilis, these proteins contribute to regulation of B activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of B activity in response to environmental stresses, while RsbV contributes to B activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in B -mediated responses to various stresses, in-frame deletions were created in rsbT and rsbV. Phenotypic characterization of the L. monocytogenes rsbT and rsbV null mutants revealed that both mutants were similar to the ⌬sigB strain 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 ⌬rsbT and ⌬rsbV showed survival reductions similar to that of the ⌬sigB strain. These observations suggest that the pathways for Rsb-dependent regulation of B activity differ between L. monocytogenes and B. subtilis. As B also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ⌬prfAP1rsbT and ⌬prfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ⌬prfAP1P2 and ⌬prfAP1sigB double mutants, i.e., reduced hemolysis activity and reduced plaque size in mouse fibroblast cells. These findings indicate that RsbT and RsbV both contribute to B activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.Listeria monocytogenes, a gram-positive, non-spore-forming rod-shaped bacterium, is recognized as a foodborne pathogen. This organism is capable of surviving in a broad range of ecological niches (e.g., in farm environments and food processing plants) and in a wide range of hosts, including humans and many species of animals. In L. monocytogenes, the alternative sigma factor B ( B ) contributes to survival under stressful environmental conditions, such as exposure to low pH, oxidizing conditions, and starvation (17, 18). Loss of B also reduces L. monocytogenes virulence in a murine model (32, 44).The sigB gene, which encodes B , lies seventh in the sigB operon. This operon also includes seven additional genes, which encode the following regulator of sigma B proteins: RsbR, RsbS, RsbT, RsbU, RsbV, RsbW, and RsbX (4,19,25,44,45). In B. subtilis, activation of B by the Rsb proteins is achieved through a complex phosphorylation/dephosphorylation cascade in response to various cellular stimuli, which have been catego...

Complex interactions between bacteriophages and their bacterial hosts play significant roles in shaping the structure of environmental microbial communities, not only by genetic transduction but also by modification of bacterial gene expression patterns. Survival of phages solely depends on their ability to infect their bacterial hosts, most importantly during phage entry. Successful dynamic adaptation of bacteriophages when facing selective pressures, such as host adaptation and resistance, dictates their abundance and diversification. Co-evolution of the phage tail fibers and bacterial receptors determine bacterial host ranges, mechanisms of phage entry, and other infection parameters. This review summarizes the current knowledge about the physical interactions between tailed bacteriophages and bacterial pathogens (e.g., Salmonella enterica and Pseudomonas aeruginosa) and the influences of the phage on host gene expression. Understanding these interactions can offer insights into phage–host dynamics and suggest novel strategies for the design of bacterial pathogen biological controls.

To measure B activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the B -dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The ⌬sigB, ⌬rsbT, and ⌬rsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and B were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and B dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or B dependent in the presence of either CCCP or salt. opuCA transcript 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 as sigB. Overall, our data show that while B activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the B regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.Listeria monocytogenes is a non-spore-forming, gram-positive, facultative intracellular pathogen. The emergence of this organism as a difficult-to-control food-borne pathogen is at least in part due to its ability to survive in a broad range of ecological niches (13) and in many different hosts, including both animals and humans (11,50). Contamination of foods with L. monocytogenes raises both public health and economic concerns (33, 57). Although rare, listeriosis is a severe disease that results in death in 20 to 30% of reported cases (33). Infection in humans occurs predominantly among pregnant women, newborns, the elderly, and immunocompromised adults.B is a stress-responsive alternative sigma factor that has been identified in various low-GϩC-content gram-positive bacteria, including the genera Bacillus, Staphylococcus, and Listeria. In L. monocytogenes, B contributes to cell survival under stress conditions, including exposure to low pH, oxidative stress, carbon starvation, and growth at low temperatures (14,15,35,54,55 plays a major role in mouse septic arthritis (24), although it is not essential for infection in the mouse abscess model, the mouse hematogenous pyelonephritis model, or the rat osteomyelitis ...

The ability of Listeria monocytogenes to grow at refrigeration temperatures is critical for transmission of this foodborne pathogen. We evaluated the contributions of different transcriptional regulators and two-component regulatory systems to L. monocytogenes cold adaptation and cold growth. L. monocytogenes parent strain 10403S and selected isogenic null mutants in genes encoding four alternative sigma factors (sigB, sigH, sigC, and sigL), two regulators of sigmaB (rsbT and rsbV), two negative regulators (ctsR and hrcA), and 15 two-component response regulators were grown in brain heart infusion broth at 4 degrees C with (i) a high-concentration starting inoculum (10(8) CFU/ml), (ii) a low-concentration starting inoculum (102 CFU/ml), and (iii) a high-concentration starting inoculum of cold-adapted cells. With a starting inoculum of 10(8) CFU/ml, null mutants in genes encoding selected alternative sigma factors (DeltasigH, DeltasigC, and DeltasigL), a negative regulator (DeltactsR), regulators of sigmaB (DeltarsbT and DeltarsbV), and selected two-component response regulators (DeltalisR, Deltalmo1172, and Deltalmo1060) had significantly reduced growth (P < 0.05) compared with the parent strain after 12 days at 4 degrees C. The growth defect for DeltasigL was limited and was not confirmed by optical density (OD600) measurement data. With a starting inoculum of 102 CFU/ml and after monitoring growth at 4 degrees C over 84 days, only the DeltactsR strain had a consistent but limited growth defect; the other mutant strains had either no growth defects or limited growth defects apparent at only one or two of the nine sampling points evaluated during the 84-day growth period (DeltasigB, DeltasigC, and Deltalmo1172). With a 10(8) CFU/ml starting inoculum of cold-adapted cells, none of the mutant strains that had a growth defect when inoculation was performed with cells pregrown at 37 degrees C had reduced growth as compared with the parent strain after 12 days at 4 degrees C, suggesting a specific defect in the ability of these mutant strains to adapt to 4 degrees C after growth at 37 degrees C. Our data indicate (i) selected sigma factors and two-component regulators may contribute to cold adaptation even though two-component regulatory systems, alternative sigma factors, and the negative regulators CtsR and HrcA appear to have limited contributions to L. monocytogenes growth at 4 degrees C in rich media, and (ii) inoculum concentration and pregrowth conditions affect the L. monocytogenes cold-growth phenotype.

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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.