Targeted Gene Disruption by Homologous Recombination in the Oral Spirochete Treponema dentico/a ATCC 35405
Hollandsworth, Anne M.
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Treponema denticola has been found to play a significant role in the progression of periodontal disease based on numerical prevalence, high association level and spatial location within infected sites. There are a number of virulence factors controlled by various genes that contribute to the role of T. denticola as a periodontal pathogen. This investigation attempted to mutate two potentially virulent genes in T. denticola: TDE1343, a homolog of the htrA/degP gene in E. coli which codes for a heat-shock protein, and TDE1658, a homolog of the surA gene in E. coli which codes for a periplasmic peptidyl-prolyl isomerase, using a gene transfer system. The mutation of TDE1343 involved the disruption of pCF440, which contained an internal fragment of TDE1343, using a selectable ermF/AM cassette. TDE1658 was mutated by inserting the ermF/AM cassette into pCF466, which contained a fragment of TOE1658. The results of this study revealed that the plasmid containing the mutated TDE 1343 was lethal to the E. coli used in the gene transfer system. However, a plasmid containing the mutated TDE 1658 was not lethal to E. coli. This study concluded that the method used for gene transfer in T. denticola is ineffective for constructing a plasmid containing a mutated TDE1343 gene, but effective for the construction of a plasmid containing a disrupted TDE1658 gene. However, the incorporation of the mutated TDE1658 into the T. denticola genome through electroporation and homologous recombination has not yet been successful.