Progress 09/01/02 to 08/31/05
Outputs
DNA microarrays were fabricated from shotgun libraries of size-selected genome fragments of a lineage I and a lineage II reference strain. Over 7,000 clones from each library were successfully amplified and used to fabricate a lineage I array and a lineage II array. These arrays were then used to probe genomic DNAs from 100 different strains representing the genetic diversity of lineage I, lineage II, and lineage III populations. A total of 47 lineage II-specific genes were identified and a total of 18 lineage I-specific genes were found. Functional classification of the lineage-specific genes showed that the largest categories of lineage-specific genes are associated with the cell surface or with transcription regulation. These findings imply that lineage I and II populations differ significantly in their cell surface decoration as well as their patterns of gene expression. To test the hypothesis that lineage-specific genome content can translate into significant
phenotypic traits, genetic analysis was conducted on two different lineage II-specific transcription factors, encoded by the lmo0422 and lmo0423. Expression studies of these two genes showed that they comprise an operon in conjunction with the downstream gene lmo0421. The operon is highly induced during different types of physical and chemical stress conditions, but is mot highly induced during temperature upshift. In-frame deletions in these genes were then constructed and introduced into the lineage II strain 10403S. The mutant derivatives showed significant thermal sensitivity and both mutants lost a significant proportion of their adaptive response to temperature upshift. Thus, these lineage-specific genes contribute significantly to thermal resistance characteristics of the lineage. These results strongly support our hypothesis that lineage-specific gene content translates into significant phenotypic characteristics.
Impacts
The catalogue of lineage-specific and serotype-specific genes that were discovered as part of this project now provide a roadmap for functional studies of candidate genes that confer lineage and serotype-specific characteristics. Understanding the function of these lineage-specific genes will provide insight into the biases observed in the distribution of lineage I and lineage II strains among food and environmental samples and human clinical samples.
Publications
- 1. Zhang, C., M. Zhang, J. Ju, J. Nietfeldt, J. Wise, P.M. Terry, M. Olson, S.D. Kachman, M. Wiedmann, M. Samadpour, and A.K. Benson. 2003. Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: Identification of segments unique to lineage II populations. J. Bacteriol. 185: 5573-5584
- 2. Zhang, C., J. Nietfeldt, M. Zhang, and A.K. Benson. 2005. Functional consequences of genome evolution in Listeria monocytogenes: the lmo0423 and lmo0422 genes encode sigma C and LstR, a lineage II-specific heat shock system. J. Bacteriol. 187: 7243-7253
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Progress 10/01/03 to 09/30/04
Outputs
The specific aims of this work are: 1. To fabricate representative lineage I and lineage II reference microarrays and systematically probe genome diversity among lineage I, II, and III strains 2. To determine the extend of the alterations that are unique to each lineages or which have interesting patterns of distribution 3. To determine the phenotypic impact of relevant alterations using knock out mutations To date, we have completed aims 1 and 2. Lineage I and II chips were fabricated and used to probe a large strain set (100 strains) comprising genetic diversity in lineage I, II, and III. From these studies, we have identified 13 genes that are unique to (and conserved in) lineage I strains, 57 genes that are unique to and conserved in lineage II strains, and an additional 16 genes that are missing from the lineage III genome. Among the lineage I and II specific genes, the most common functional category was genes encoding cell surface characteristics, genes
encoding transcription factors, and genes encoding transport systems. In addition to cataloguing the lineage-specific gene content, we also conducted computational analyses on the lineage-specific genes to determine if they were a consequence of acquisition in one lineage or loss in the other lineages. Our data were consistent with the hypothesis that most of the lineage-specific genes are ancestral to the species and were therefore lost in lineages where they are absent. We have now begun the last stage of this proposal, making knock out mutations in lineage-specific genes in order to gain insight into the types of phenotypes that they may confer on the different lineages. Thus far, we have constructed knock-out mutations in six of these genes and are in the process of constructing knock-outs in eight additional genes. These knock-out mutants will be tested for defects in virulence characteristics and in physiological characteristics.
Impacts
Completion of the first step of the project has now identified lineage-specific genes. Several of these genes are currently the focus of functional analysis. The functional studies will shed light on unique physiological and virulence characteristics of the three lineages.
Publications
- Zhang, C., M. Zhang, J. Ju, J. Nietfeldt, J. Wise, P.M. Terry, M. Olson, S.D. Kachman, M. Wiedmann, M. Samadpour, and A.K. Benson. 2003. Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: Identification of segments unique to lineage II populations. J. Bacteriol. 185: 5573-5584.
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Progress 10/01/02 to 09/30/03
Outputs
Thirteen different serotypes of Listeria monocytogenes can be distinguished on the basis variation in somatic and flagellar antigens. Although the known virulence genes are present in all serotypes, greater than 90% of human cases of listeriosis are caused by serotypes 1/2a, 1/2b, and 4b and nearly all outbreaks of foodborne listeriosis have been caused by serotype 4b strains. Phylogenetic analysis of these three common clinical serotypes places them into two different lineages, with serotypes 1/2b and 4b belonging to lineage I and 1/2a belonging to lineage II. To begin examining ontogeny of the genome in these serotypes, DNA microarray analysis was used to identify lineage-specific and serotype-specific differences in genome content. A set of 43 strains representing serotypes 1/2a, 1/2b, and 4b was probed with a shotgun DNA microarray constructed from the serotype 1/2a strain 10403s. Clones spanning forty-seven different genes in 16 different contiguous segments
relative to the lineage II 1/2a genome were found to be absent in all lineage I strains tested (serotype 4b and 1/2b) and an additional nine were altered exclusively in 4b strains. Southern hybridization confirmed that conserved alterations were, in all but two loci, due to absence of the segments from the genome. Genes within these contigs comprise five functional categories, including genes involved in synthesis of cell surface molecules and regulation of virulence gene expression. Phylogenetic reconstruction and examination of compositional bias in the regions of difference are consistent with a model in which the ancestor of the two lineages had the 1/2 somatic serotype and the regions absent in the lineage I genome arose by loss of ancestral sequences.
Impacts
Completion of the first step of the project has now identified lineage-specific genes. Several of these genes are currently the focus of functional analysis. The functional studies will shed light on unique physiological and virulence characteristics of the three lineages.
Publications
- Zhang, C., M. Zhang, J. Ju, J. Nietfeldt, J. Wise, P.M. Terry, M. Olson, S.D. Kachman, M. Wiedmann, M. Samadpour, and A.K. Benson. 2003. Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: Identification of segments unique to lineage II populations. J. Bacteriol. 185: 5573-5584
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Progress 09/01/02 to 09/30/02
Outputs
This project started September 1, 2002, therefore there is no progress for one month.
Impacts
(N/A)
Publications
- No publications reported this period
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