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Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.

ABSTRACT: The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A(1), and clorobiocin were investigated. All three clusters contained a gyrB(R) resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A(1) clusters were found to contain an additional, similar gene, named parY(R). Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB(R) and likewise of parY(R) in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A(1), suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrB(R) or parY(R) and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A(1) cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A(1), suggesting that these genes may be involved in antibiotic transport.

SUBMITTER: Schmutz E

PROVIDER: S-EPMC149333 | biostudies-literature | 2003 Mar

REPOSITORIES: biostudies-literature

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Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.

Schmutz Elisabeth E Mühlenweg Agnes A Li Shu-Ming SM Heide Lutz L

Antimicrobial agents and chemotherapy 20030301 3

The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A(1), and clorobiocin were investigated. All three clusters contained a gyrB(R) resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A(1) clusters were found to contain an additional, similar gene, named parY(R). Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB(R) and likewise of parY(R) ...[more]

PMID: 12604514

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Project description: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.

Dataset Information

Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.

Publications

Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.

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