Browsing by Author "Allen, Caitilyn"

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    Evolution of a gene for pathogenicity: endo-pectate lyase
    Allen, Caitilyn (Virginia Polytechnic Institute and State University, 1987)
    Erwinia carotovora subsp. Carotovora (Ecc) and Erwinia carotovora subsp. Atroseptica (Eca) are plant pathogenic bacteria that cause soft rot disease of many plant species and blackleg disease of potatoes, respectively. Ecc and Eca attack plants by means of a group of extracellular plant tissue-degrading enzymes. which rapidly breaks down the pectic polymers that form a structurally important part of the plant cell wall, is considered central to soft rot pathogenesis. In this work, I isolated and studied the genes encoding this enzyme from Ecc and Eca. A clone library of Ecc strain EC14 was constructed using cosmid PLAFR3. This library contains 2,200 clones with an average insert size of 27 kilobases of DNA and included a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. The proteolytic clone was used to complement a Tn5-induced protease mutant of Ecc; the complemented mutant was restored to near-wild type phenotype. Six of the pectolytic clones hybridized to a probe from a. previously isolated extracellular endo-pectate-pectate lyase gene from Ecc; one pectolytic clone had homology to a previously isolated clone encoding endo-polygalacturonase: three clones showed no relationship to either of the previously characterized Ecc pectolytic enzyme genes. A clone encoding the major endo-pectate lyase gene from Ecc was chosen for subcloning and further study. I used the plasmid vector pBR322 to construct a clone bank of Eca strain SRB; of the 1700 clones screened, five were pectolytic. Two of the Eca pectolytic. clones had homology to the Ecc endo-pectate lyase gene; upon examination, they proved to contain the same insert in opposite orientations. The Ecc endo-pectate lyase had a pI of 9. 5 and a molecular weight of 33,000; the analogous Eca endo-pectate lyase had a pI of 9.2 and a molecular weight of 31,000. Both enzymes required a divalent cation for activity (preferring Ca2+ over Mg2+ over Mn²⁺. The restriction endonuclease maps of the two clones did not have any tested sites in common. These differences suggest that although these two genes may have originated from a common ancestral gene, considerable divergence has taken place. I analyzed the fine structure of the Ecc endo-pectate lyase gene by DNA sequencing. The coding region of the gene is preceded by E. coli-type -10 and -35 sequences and encodes an unmodified protein of 281 amine acids. A typical secretion signal peptide is not present.
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    Meta-analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology
    Sharma, Parul; Johnson, Marcela A.; Mazloom, Reza; Allen, Caitilyn; Heath, Lenwood S.; Lowe-Power, Tiffany M.; Vinatzer, Boris A. (Microbiology Society, 2022-03)
    Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem tissue and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures and physiological traits. To increase our understanding of the evolution, diversity and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4940 genes on average, and a pangenome analysis found that there are 3262 genes in the core genome (similar to 60 % of the mean RSSC genome) with 13 128 genes in the extensive flexible genome. A core genome phylogenetic tree and a whole-genome similarity matrix aligned with the previously named species (R. solanacearum, R. pseudosolanacearum, R. syzygii) and phylotypes (I-IV). These analyses also highlighted a third unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. We also used our genome- based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved, cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome- based identification of newly sequenced RSSC strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service.