Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 14, 2000
Publication Date: N/A
Technical Abstract: Objectives of these studies were: 1. To isolate coumermycin- resistant (Cou**r) strains with defined mutations in their gyrB genes (encoding DNA gyrase subunit B); 2. To evaluate Cou**r for monitoring genetic exchanges between B. hyo. cells. B. hyo. B204 cells were mutagenized with a dose of UV light (3500 um Joules) lethal for > 99% of the cell population. Survivors were plated onto Trypticase soy blood agar plates containing 0.5 or 2.0 ug coumermycin A1/ ml (TSBC). Nine Cou**r strains were isolated and their gyrB gene sequences were compared to that of wild-type strain B204. Four Cou**r strains had single nucleotide mutations in gyrB and five strains had no mutations. The corresponding amino acid changes in the GyrB proteins of the four strains were Gly78 > Ser (2 strains), Gly78 > Cys, and Thr166 > Ala. To investigate whether or not coumermycin-resistance could be transferred between B. hyo. strains, strain 435A (Cou**r GyrB: Gly78 > Ser) and previously constructed strain SH (Km**rCm**r) were co-cultured. After overnight incubation, co-culture samples were plated onto TSB plates containing coumermycin A1 (10 ug/ml), kanamycin (200 ug/ml) and chloramphenicol (10 ug/ml). Co-cultures contained cells at population densities of 357 CFU/ml (2.1 x 10**-6 total bacteria) that were resistant to all three antibiotics. Seven triply-resistant colonies were randomly selected and shown to contain cells with genotypes of both strain 435A and SH. These findings indicate the usefulness of Cou**r as a selectable marker in studies of B. hyodysenteriae gene exchange, demonstrate gene transfer easily occurs in B. hyodysenteriae cultures.