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General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes.

ABSTRACT: The gene encoding the general stress transcription factor sigmaB in the gram-positive bacterium Listeria monocytogenes was isolated with degenerate PCR primers followed by inverse PCR amplification. Evidence for gene identification includes the following: (i) phylogenetic analyses of reported amino acid sequences for sigmaB and the closely related sigmaF proteins grouped L. monocytogenes sigmaB in the same cluster with the sigmaB proteins from Bacillus subtilis and Staphylococcus aureus, (ii) the gene order in the 2, 668-bp portion of the L. monocytogenes sigB operon is rsbU-rsbV-rsbW-sigB-rsbX and is therefore identical to the order of the last five genes of the B. subtilis sigB operon, and (iii) an L. monocytogenes sigmaB mutant had reduced resistance to acid stress in comparison with its isogenic parent strain. The sigB mutant was further characterized in mouse models of listeriosis by determining recovery rates of the wild-type and mutant strains from livers and spleens following intragastric or intraperitoneal infection. Our results suggest that sigmaB-directed genes do not appear to be essential for the spread of L. monocytogenes to mouse liver or spleen at 2 and 4 days following intragastric or intraperitoneal infection.

SUBMITTER: Wiedmann M

PROVIDER: S-EPMC107335 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes.

Wiedmann M M Arvik T J TJ Hurley R J RJ Boor K J KJ

Journal of bacteriology 19980701 14

The gene encoding the general stress transcription factor sigmaB in the gram-positive bacterium Listeria monocytogenes was isolated with degenerate PCR primers followed by inverse PCR amplification. Evidence for gene identification includes the following: (i) phylogenetic analyses of reported amino acid sequences for sigmaB and the closely related sigmaF proteins grouped L. monocytogenes sigmaB in the same cluster with the sigmaB proteins from Bacillus subtilis and Staphylococcus aureus, (ii) th ...[more]

PMID: 9658010

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Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

Project description:Listeria monocytogenes is a public health and food safety challenge due to its virulence and natural stress resistance phenotypes. The variable distribution of L. monocytogenes molecular subtypes with respect to food products and processing environments and among human and animal clinical listeriosis cases is observed. Sixty-two clinical and food-associated L. monocytogenes isolates were examined through phenome and genome analysis. Virulence assessed using a zebrafish infection model revealed serotype and genotype-specific differences in pathogenicity. Strains of genetic lineage I serotype 4b and multilocus sequence type clonal complexes CC1, CC2, CC4, and CC6 grew and survived better and were more virulent than serotype 1/2a and 1/2c lineage II, CC8, and CC9 strains. Hemolysis, phospholipase activity, and lysozyme tolerance profiles were associated with the differences observed in virulence. Osmotic stress resistance evaluation revealed serotype 4b lineage I CC2 and CC4 strains as more osmotolerant, whereas serotype 1/2c lineage II CC9 strains were more osmo-sensitive than others. Variable tolerance to the widely used quaternary ammonium compound benzalkonium chloride (BC) was observed. Some outbreak and sporadic clinical case associated strains demonstrated BC tolerance, which might have contributed to their survival and transition in the food-processing environment facilitating food product contamination and ultimately outbreaks or sporadic listeriosis cases. Genome comparison uncovered various moderate differences in virulence and stress associated genes between the strains indicating that these differences in addition to gene expression regulation variations might largely be responsible for the observed virulence and stress sensitivity phenotypic differences. Overall, our study uncovered strain and genotype-dependent variation in virulence and stress resilience among clinical and food-associated L. monocytogenes isolates with potential public health risk implications. The extensive genome and phenotypic data generated provide a basis for developing improved Listeria control strategies and policies.

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General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes.

Publications

General stress transcription factor sigmaB and its role in acid tolerance and virulence of Listeria monocytogenes.

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