Date of Award
Doctor of Philosophy
Zoology and Genetics
John E. Mayfield
A periplasmic catalase has been identified and purified from Brucella abortus using a procedure which fractionates periplasmic B. abortus proteins from other cellular components while retaining biological activity. The biochemical characteristics of the purified enzyme were studied and the gene encoding the enzyme cloned and sequenced. SDS polyacrylamide gel electrophoresis of the purified enzyme revealed only a single subunit with a molecular weight of 59,000. Gel filtration indicates the native protein is a tetramer. The native protein has a visible absorption spectrum with maxima at 407, 501, and 628 nm which is typical of mammalian, but not some bacterial, catalases. The purified enzyme has a specific activity of 5 x 106 M-1sec-1, and exhibits activity over a broad pH range with greater than 30% of maximal activity from pH 4 to pH 12. Fractionation experiments suggest that the active enzyme is exclusively periplasmic with no identifiable cytoplasmic catalase activity. The sequence reveals strong homology with mammalian and other prokaryotic catalases, but little or no homology with enzymes exhibiting significant peroxidase activity. Southern blot data indicates that there is only one copy of the catalase gene in the B. abortus genome. A deletion plasmid, pCatDEL, was constructed by removing the catalase coding region from a B. abortus DNA fragment plasmid pCAT5 and inserting a 1.4 kb neo fragment into the excision site. The deletion plasmid, pCatDEL was introduced into B. abortus by electroporation and Kan r colonies were selected. Both PCR and Southern blot confirmed that the catalase gene was deleted in these colonies. To test the hypothesis that catalase enzyme activity might protect the bacterium from external sources of H2O2, both short and long term exposure to H2O2 experiments were conducted with different results. The H2O2 challenge in liquid culture experiment indicated that the catalase-null mutants were more susceptible to hydrogen peroxide killing than wild type cells for strain 19 but not for strain 2308. In filter disc halo experiments, catalase mutants of both B. abortus S19 and S2308 were more susceptible to hydrogen peroxide than wild type strains. The time course of murine infection of S2308 catalase mutant was compared with that of wild-type S2308. Although the bacterial counts from BALB/c mice infected with the mutant strain lagged behind those from wild-type infected mice early infection, especially at day 21, the level of infection of the 2308 catalase mutant eventually reached a similar level to that of wild-type S2308.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Zheng yu Sha
Sha, Zheng yu, "Purification, characterization and cloning of a periplasmic catalase from B abortus and the role it plays in the pathogenesis of Brucella " (1994). Retrospective Theses and Dissertations. 11320.