Catabolic instability, plasmid gene deletion and recombination in Alcaligenes sp. BR60

An Alcaligenes sp. BR60, isolated from surface runoff waters of the Hyde Park industrial landfill, contained a novel 85 kb catabolic plasmid (pBR60) functional in 3-chlorobenzoate (3Cba) degradation. The plasmid exhibited a spontaneous 3.2% frequency of deletion of a 14 kb fragment specifying 3Cba degradation. The deletion mutant BR 40 and mitomycin C cured strains were not able to grow on 3Cba and had reversion frequencies of less than 10(-10) cell-1 generation-1. Transformation or conjugation of pBR60 into cured strains restored catabolic activity. An EcoRI, BglII, HindIII and SalI restriction map of the deletion region was constructed, and EcoRI and HindIII fragments spanning the deletion region of the plasmid were cloned in pUC18. Conjugation of resistance plasmid R68.45 into Alcaligenes sp. BR 60, with selection on antibiotics, resulted in the elimination of pBR60 and maintenance of unaltered R68.45. In 30% of the exconjugants, 3Cba degradative capacity was retained, although variation in the regulation of 3Cba degradation was observed in these strains. Hybridization of deletion region fragments to BglII digested total DNA of BR60 and the R68.45 cured exconjugants revealed the presence of pBR60 deletion region sequences in the chromosome of exconjugants. Hybridization also revealed a repeated sequence flanking the deletion region of pBR60. Selection on 4-chlorobenzoate as a sole source of carbon and energy resulted in the isolation of 4Cba+ mutants of Alcaligenes sp. BR60.