Functional Consequences of Genome Evolution in Listeria Monocytogenes

NON-TECHNICAL SUMMARY: Listeria monocytogenes is a physiologically robust bacterium that causes the foodborne illness listeriosis. Because listeriosis has a high associated morbidity and mortality and because the organism can occur widely in food production settings, it poses a significant threat to the safety of the food supply. The purpose of this study is to understand how Listeria monocytogenes populations respond to and survive food processing, preservation, and sanitation measures.

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APPROACH: Genetic and physiological characterization of the sigC, lmo0422, and lmo0421 genes will be performed in the lineage II background using strains with in-frame deletions in each of the three genes and strains in which the genes are placed under control of the P-SPAC prmoter. Physiological conseuqneces will be determined by examining growth and survial characteristics of the mutants while genetic characteristics of the mutants will be examined by identification of target genes that subject to regulation by sigC and lmo0422. Once target genes are identified in the lineage II genetic background, expression patterns and promoter mapping studies will be conducted in both lineage I and lineage II backgrounds to determine how they are controlled in these two different phylogenetic lineages.

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PROGRESS: 2005/09 TO 2007/09<BR>
OUTPUTS: The comparative genome data demonstrated that transcription factors were among the most common functional category of lineage-specific genes, implying that patterns of gene expression may differ between the phylogenetic lineages of L. monocytogenes and could therefore contribute to the asymmetric ecological and virulence characteristics. The preliminary work on lmo0421-lmo0423 further showed that these lineage-specific genes can have a profound impact on not only the transcription patterns, but also significant phenotypic traits. This grant therefore extends this work to further define function of lmo0421-lmo0423 and to determine how lineage I strains have evolved pathways to mitigate loss of lmo0421-lmo0423. Specifically, the aims are to: 1. Characterize function of the sigC, lmo0422, lmo0421, genes 2. Determine how lineage I strains compensate for the absence of these genes. Genetic studies of the sigC(lmo0423)-lstR(lmo0422)-lmo0421 operon revealed that the operon encodes a novel regulatory system that responds to a number of physical and environmental stress conditions. The sigC gene encodes a secondary sigma subunit of RNA polymerase that is autocatalytic and mediates temperature-inducible expression of the operon. The lstR gene encodes a padR-like transcription factor that presumably functions controls transcription of a heat shock regulon. DNA microarray analyses comparing heat shcok-induction in the 10403S parental strain and the sigC and lstR mutants identified a large set of genes (n=271) whose expression is effected by sigC and lstR. These genes include the entire sigB operon and several members of the sigB regulon, as well as a significant number of ribosome proteins and translation factors. Genetic studies subsequently showed that sigC is necessary for sigB activation in lineage II strains and that sigB function accounts for only part of the sigC thermal resistance characteristics. sigB function in lineage I strains, which naturally lack sigC, is thermally-inducible as are many of the genes that are sigC-dependent in lineage II strains, implying that lineage I has evolved new pathways for temperature-dependent activation of sigB and likely other regulatory pathways. Subsequent genetic studies in which a PSPAC-controlled sigC-lstR-lmo0421 operon was introduced into lineage I strains also showed that these strains actually became temperature-sensitive, implying that sigC-lstR function are incompatible with the regulatory pathways present in lineage I. <BR> PARTICIPANTS: Andrew K. Benson, Principal Investigator Stephen D. Kachman, Dept. of Statistics, University of Nebraska, Collaborator Chaomei Zhang, Senior Research Associate, Dept. of Food Science and Technology, University of Nebraska <BR> TARGET AUDIENCES: 1. Academic Professionals and the Listeria researcher community (pu 2. Food production industry (through presentations at conferences) 3. Graduate and undergraduate students (workshops on DNA microarray analysis using Listeria as the model system)
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IMPACT: 2005/09 TO 2007/09<BR>
This work has now provided a huge leap forward in our understanding of lineage-specific physiological and virulence characteristics, demonstrating that a single genomic event (loss of sigC-lstR) was likely to have been a key driving force in reshaping the expression patterns of sigB-dependent genes. Because sigB plays an important role in stress adaptation and in expression of virulence genes, our findings further imply that selective pressure for evolution of a new pathway(s) to control sigB during divergence of lineage I strains may have brought about changes in physiological and virulence characteristics.