Towards Novel Methods of Mutagenesis for Histophilus somni

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dc.contributor.authorShah, Nehal Rajendraen
dc.contributor.committeechairInzana, Thomas J.en
dc.contributor.committeememberSubbiah, Elankumaranen
dc.contributor.committeememberSchubot, Florian D.en
dc.contributor.departmentBiomedical Sciences and Pathobiologyen
dc.date.accessioned2014-03-14T21:40:23Zen
dc.date.adate2012-07-27en
dc.date.available2014-03-14T21:40:23Zen
dc.date.issued2012-06-07en
dc.date.rdate2012-07-27en
dc.date.sdate2012-07-12en
dc.description.abstractHistophilus 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 EZen
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-07122012-112148en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-07122012-112148/en
dc.identifier.urihttp://hdl.handle.net/10919/43708en
dc.publisherVirginia Techen
dc.relation.haspartShah_NR_T_2012.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectNatural transformationen
dc.subjectDNA uptakeen
dc.subjectin-vitro transpositionen
dc.subjecttransposonen
dc.subjectcompetence genesen
dc.subjectHimar1en
dc.subjectplasmid vectoren
dc.titleTowards Novel Methods of Mutagenesis for Histophilus somnien
dc.typeThesisen
thesis.degree.disciplineBiomedical Sciences and Pathobiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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