Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance

Abstract

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml. The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204. Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala. When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.

FIG. 1

Comparison of amino acid sequences of the GyrB proteins of B. hyodysenteriae (BhyoGyrB), B. burgdorferi (BbGyrB), and E. coli (EcoGyrB) and the ParE protein of E. coli (EcoParE). B. hyodysenteriae GyrB exhibits overall sequence identities of 56, 42, and 37% with B. burgdorferi GyrB, E. coli GyrB, and E. coli ParE, respectively. In regions A to D, the B. hyodysenteriae GyrB protein exhibits the following sequence identities with B. burgdorferi GyrB, E. coli GyrB, and E. coli ParE, respectively: region A, 44, 48, and 22%; region B, 50, 58, and 34%; region C, 60, 54, and 33%; and region D, (D) 58, 60, and 21%. B. hyodysenteriae Cn^r strains isolated in this study have amino acid mutations at positions Gly₇₈ and Thr₁₆₆, (indicated by asterisks).

FIG. 1

Comparison of amino acid sequences of the GyrB proteins of B. hyodysenteriae (BhyoGyrB), B. burgdorferi (BbGyrB), and E. coli (EcoGyrB) and the ParE protein of E. coli (EcoParE). B. hyodysenteriae GyrB exhibits overall sequence identities of 56, 42, and 37% with B. burgdorferi GyrB, E. coli GyrB, and E. coli ParE, respectively. In regions A to D, the B. hyodysenteriae GyrB protein exhibits the following sequence identities with B. burgdorferi GyrB, E. coli GyrB, and E. coli ParE, respectively: region A, 44, 48, and 22%; region B, 50, 58, and 34%; region C, 60, 54, and 33%; and region D, (D) 58, 60, and 21%. B. hyodysenteriae Cn^r strains isolated in this study have amino acid mutations at positions Gly₇₈ and Thr₁₆₆, (indicated by asterisks).

FIG. 2

Genotype analyses of antibiotic resistance determinants of strains SH (Km^r Cm^r), 435A (Cn^r), and QM-1 (Cn^r Km^r Cm^r). Six other triply resistant strains (QM-2 to QM-7) were examined and gave results identical to those obtained with strain QM-1. (A) PCR amplification to detect the kan gene (lanes 1, 3, and 5) and the cat gene (lanes 2, 4, and 6). (B) DNA sequence analysis to detect the gyrB nucleotide change (corresponding amino acid change) associated with coumermycin resistance.