Title page for ETD etd-07122012-112148


Type of Document Master's Thesis
Author Shah, Nehal Rajendra
Author's Email Address Nesmall2@vt.edu
URN etd-07122012-112148
Title Towards Novel Methods of Mutagenesis for Histophilus somni
Degree Master of Science
Department Biomedical Sciences and Pathobiology
Advisory Committee
Advisor Name Title
Inzana, Thomas J. Committee Chair
Schubot, Florian D. Committee Member
Subbiah, Elankumaran Committee Member
Keywords
  • Natural transformation
  • DNA uptake
  • in-vitro transposition
  • transposon
  • competence genes
  • Himar1
  • plasmid vector
Date of Defense 2012-06-07
Availability restricted
Abstract
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::Tn5™Tnp Transposome™ (Epicentre) was used to generate a bank of mutants, but the mutation efficiency was low. Therefore the mariner Tn element is being tested as a more efficient method for random mutagenesis of H. somni. The transposase, which is required for excision of the Tn, was over-expressed in Escherichia coli, and then purified using amylose resin. H. somni was then naturally transformed after in-vitro transposition using pMarStrep, which contains the mariner Tn with StrepR antibiotic gene marker, and a series of transposition and ligation components. However, mariner Tn mutants were not generated. Nonetheless, natural transformation and/or mariner Tn mutagenesis may still prove to be efficient methods for mutagenesis of H. somni. Through the use of more effective mutagenesis systems, genes responsible for the expression of virulence factors can be identified, and improved vaccine candidates can be developed.
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