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Characterization of insertion mutations in Bacillus anthracis capsule plasmidpXO2
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Characterization of insertion mutations in Bacillus anthracis capsule plasmidpXO2
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Characterization of insertion mutations in Bacillus anthracis capsule plasmidpXO2
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Dissertation
Characterization of insertion mutations in Bacillus anthracis capsule plasmidpXO2
1993
Overview
Transposon-mediated insertional mutagenesis was used for the isolation of auxotrophic mutants in plasmid-free derivatives of B. anthracis. A number of auxotrophic mutants were isolated and confirmed by cotransduction to result from insertion of Tn917. These experiments showed Tn917 to be an effective tool for insertional mutagenesis in B. anthracis and prompted the investigation of the B. anthracis capsule plasmid pXO2. The capsule of B. anthracis, which is an important virulence factor, is composed mainly, if not entirely, of the D-isomer of glutamic acid. Previous work in our laboratory demonstrated that pXO2 is involved in capsule synthesis by B. anthracis. Wild-type B. anthracis strains harboring pXO2 produce capsule (Cap $\\sp+)$when grown in a CO $\\sb2$ -rich atmosphere in the presence of bicarbonate. Utilization of the temperature-sensitive transposition selection vector pTV1 has allowed the isolation of a collection of pXO2::Tn917 derivatives. One class of insertion mutants produces greater amounts of capsular material than does wild type in the presence of CO $\\sb2$and bicarbonate. A second class is Cap $\\sp+$when grown with or without CO $\\sb2$and bicarbonate. A third class is Cap $\\sp+$when grown in air or CO $\\sb2,$and growth is inhibited by bicarbonate. A fourth class is Cap $\\sp+$when grown in air, and growth of this class is inhibited by CO $\\sb2.$A fifth class requires CO $\\sb2$for growth and is Cap $\\sp+$in the presence of CO $\\sb2$and bicarbonate. A sixth class retains pXO2 and is Cap $\\sp-$under all conditions of growth. The phenotypes of some of these mutants suggest that Tn917 is inserted in sequences involved in regulation of capsule synthesis. Restriction and DNA-DNA hybridization analysis of a collection of pXO2 plasmids carrying Tn917 insertions have shown that the transposon is located at a number of different sites. Restriction analysis with different enzymes has revealed two regions of pXO2 that may be involved in regulation of capsule synthesis. Quantitative determinations of glutamyl polypeptide showed that members of the first class of mutants produce more capsular material than does the parent strain. Determination of the configuration of glutamic acid in glutamyl polypeptide produced by the wild-type strain and these insertion mutants showed that the polypeptide from mutant strains did not differ from that produced by the parent strain.
