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

Department of Biological Sciences, Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, Idaho, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 11/2011; 78(3):828-38. DOI: 10.1128/AEM.07480-11
Source: PubMed


Broad-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::rfp and 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::rfp carries 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. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli. Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfp accelerated 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.

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Available from: Stefan Wuertz, Oct 03, 2015
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    • "Similarly, C. testosteroni 12 metabolizes 3-chloroaniline via the meta-cleavage pathway (Boon et al., 2000). Król et al. (2012) showed that chloroaniline dioxygenase of C. testosteroni WDL7 is a multicomponent enzyme consisting of large and small subunits of dioxygenase (encoded by dcaA1, dcaA2), and a reductase (encoded by dcaA3). "
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    Frontiers in Microbiology 07/2015; 6. DOI:10.3389/fmicb.2015.00820 · 3.99 Impact Factor
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    • "However, in a later bioaugmentation experiment, pI2 endowed reactors with the capability to degrade 3-CA completely (Bathe et al. 2005). At the genomic scale, the upper pathway enzymes of pWDL7::rfp were found to be able to convert 3-CA to 4-chlorocatechol (4-CC) (Król et al. 2012). As the upper pathway enzymes from these two plasmids share a high similarity, pI2 may also be able to convert 3-CA to 4- CC. "
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    • "Quantification of 3-CA Samples from biodegradation experiments were taken at predetermined time points and filtered through 0.20 μm filters . The supernatant was analyzed for 3-CA concentration using reverse-phase HPLC, where an Acsentis C18 column (100 mm×2.1 mm, 5 μm particle size), a mobile phase of methanol/water (50/50), a flow rate of 0.6 mL/min and a UV detector set at 254 nm were used (Król et al. 2012). "
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