Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway.
In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.
Institut national de la recherche scientifique-santé, Université du Québec, Pointe-Claire, Canada.
J
J
Bergeron
D
D
Ahmad
D
D
Barriault
A
A
Larose
M
M
Sylvestre
J
J
Powlowski
D001483:Base Sequence; D001673:Biodegradation, Environmental; D001713:Biphenyl Compounds / Q000378:metabolism*; D002874:Chromosome Mapping; D005296:Ferrous Compounds / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009876:Operon / Q000235:genetics; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D011548:Pseudomonadaceae; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
D001713:Biphenyl Compounds; D005296:Ferrous Compounds; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D009243:NAD; C020748:ferrous sulfate; D009249:NADP; D010105:Oxygenases; C074161:biphenyl-2,3-dioxygenase; C006983:4-chlorobiphenyl