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

Abstract

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.

Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51

“…The DNA probes were a 1.6-kb XhoI fragment of plasmid pCBA3, containing the chlorocatechol 1,2-dioxygenase gene tfdC present on pJP4 (6,13,14,26), and a 2-kb PstI fragment of plasmid p261. 39 either A. eutrophus JMP222 or P. putida KT2442, using a triparental mating with E. coli HB101(pRK2013) as the helper strain (5).…”

“…In recent years, several bacteria have been isolated that were able to degrade chlorinated aromatic compounds, such as chlorinated benzoic acids, chlorinated phenols (27,39), and even chlorinated benzenes and chlorinated biphenyls (12,24). Bacteria able to use chlorinated benzenes as sole carbon and energy substrates (15,25,30,35) were found to oxidize the chlorinated benzene to a chlorocatechol by the action of a dioxygenase enzyme and a dehydrogenase.…”

“…In the case of chlorinated aromatic compounds, gene clusters have been described that code for the conversion of chlorinated catechols, such as tfdCDEF, present on plasmid pJP4 (6, 13,14,26), and clcABD, present on plasmid pAC27 (4,10). Furthermore, genes have been identified and characterized that encode the conversion of chlorinated benzoic acids (38,39) and chlorinated biphenyls (12,24).…”

Comparative genetic organization of incompatibility group P degradative plasmids

“…The direct repeats border an 8-kb sequence which has no significant homology with pSS50. Instability of degradation plasmids is not uncommon, and large spontaneous deletion mutants have been described for pJP4 (6), pBR60 (32), and TOL (18 (32), and pSS50 is from Hooper et al (12). Putative repeat sequences in pSS50 and pSS60 are denoted and oriented by arrows.…”

“…The IncP backbone of pJP4 is described in greater detail, as well as two related degradative plasmids from environmental isolates and a previously described 3-CBA-degrading plasmid, pBR60 (32,33), that appears to have a restricted host range. The similarities to and differences from the resistance plasmids are examined, including the presence of the mercury resistance sequence found in the IncPP resistance plasmids.…”