|Matson, Eric - IOWA STATE UNIV.,AMES,IA.|
Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: October 16, 1999
Publication Date: N/A
Technical Abstract: Brachyspira (Serpulina) hyodysenteriae (B. hyo.) is an anaerobic spirochete and the etiologic agent of swine dysentery. The objectives of these studies were to sequence the gyrB gene of B. hyo. B204 and to isolate B. hyo. strains resistant to coumermycin, an antibiotic that binds to the GyrB protein. A 1688 bp portion of the gyrB gene was amplified from B204 chromosomal DNA using primers complementary to regions of the gene conserved in other bacteria. Subsequently, a clone containing gyrB was isolated from a genomic library of B204 DNA in vector Lambda ZAPII. The B204 gyrB sequence was determined by sequencing these two DNA sources. The gene is 1911 bp and encodes a putative protein sharing 59.9% and 43.1% amino acid sequence identity, respectively, with the GyrB proteins of Treponema pallidum and E. coli. To isolate Cour strains, B204 cells were exposed to UV light (3500 æJoules; 99 99.9% lethality) and survivors were plated on Trypticase soy blood agar containing 0.5 or 2.0 æg coumermycin / ml (TSBC plates). (The coumermycin MIC for B. hyo. B204 is 0.1 æg/ml). Cells not exposed to UV light were cultured on TSBC plates to obtain spontaneous mutants. Of nine Cour strains that were isolated, four had single nucleotide base changes in gyrB. The deduced amino acid substitutions in gyrB of the mutant strains are Gly78 > Ser (2 strains), Gly78 > Cys, and Thr166 > Ala. The availability of these Cour strains will facilitate investigations of gene transfer between B. hyodysenteriae cells.