Role of IncP-1β Plasmids pWDL7::rfpand pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

Abstract

ABSTRACTBroad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfpand its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1β subgroup. The plasmids are almost identical, but whereas pWDL7::rfpcarries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster,dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. ThedcaA1A2Bgene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol inEscherichia coli. Slight differences in thedcapromoter region between the plasmids and lack of induction of transcription of the pNB8cdcagenes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfpaccelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities.