The Bacillus anthracis plasmid pXO1 carries genes for the synthesis of anthrax toxin. Wild-type strains produce maximal amounts of the toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF), only when grown in the presence of bicarbonate and CO$\sb2$. Previous work in this laboratory identified other pXO1-associated phenotypes in B. anthracis Weybridge A. These include improved growth on minimal medium, reduced frequency of sporulation, and reduced sensitivity to bacteriophages. To localize regions of pXO1 that confer these phenotypes, transposon mutagenesis utilizing pTV1 was used to generate Tn917 insertions in pXO1. Insertion mutants were isolated that (i) exhibited an increase in sensitivity to bacteriophage; (ii) produced LF and EF, but were deficient in production of PA; (iii) were deficient in production of all three toxin components; and (iv) overproduced toxin in the presence and absence of added bicarbonate and CO$\sb2$. The locations of Tn917 insertions in plasmids from the latter two types of mutants were outside of the toxin structural genes indicating that regulatory genes may have been interrupted. A gene was identified between pag and cya that encoded a trans-acting positive regulatory factor designated atxA. A region upstream of lef may be involved in negative and/or bicarbonate regulation of toxin synthesis. Restriction analysis of pXO1 from B. anthracis Weybridge A as well as from several other strains revealed restriction profiles which differed from that previously published for pXO1 from Sterne. A series of DNA-DNA hybridizations with pXO1 (Weybridge A) and pXO1 (Sterne) showed that an inversion of approximately 40 kb had occurred in the toxin-encoding region. The junctions encompassing these regions of pXO1 from Weybridge A and Sterne were cloned. Results from DNA-DNA hybridization studies with the cloned junction fragments suggested that inverted repeats of perfect or near perfect match may be present. Thus far, no differences in the plasmid-associated phenotypes have been found that can be attributed to the inversion.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8941 |
| Date | 01 January 1994 |
| Creators | Hornung, Jan Marie |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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