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Browsing by Author "Shah, Nehal Rajendra"

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    Towards Novel Methods of Mutagenesis for Histophilus somni
    Shah, Nehal Rajendra (Virginia Tech, 2012-06-07)
    Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases in bovines, although nonpathogenic commensal strains also exist. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. To identify genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and may be enhanced by the presence of uptake signal sequences (USS) within the genome. We hypothesized that natural transformation occurs in H. somni because its genome is over-represented with USS and contains all the necessary genes for competence, except that ComD and ComE are mutated. For natural transformation, H. somni was grown to exponential phase, and then transferred to a non-growth defined medium to induce competence. H. somni strain 2336 was successfully transformed with homologous linear DNA (lob2A) containing an antibiotic marker gene, but at low efficiency. Shuttle vector pNS3K was also naturally transformed into H. somni at low efficiency. To attempt to improve transformation efficiency, comD and comE from H. influenzae were cloned into shuttle vector pNS3K to generate the plasmid pSScomDE. Although introduction of pSScomDE into H. somni was expected to increase the number and breadth of mutants generated by natural transformation, multiple attempts to electroporate pSScomDE into H. somni were unsuccessful. A native plasmid (pHS649) from H. somni strain 649 may prove to be a more efficient shuttle vector. Due to inefficiency in generating mutants by allelic exchange, transposon (Tn) mutagenesis with EZ