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

Abstract

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-1␤ 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.

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

“…2). Catechols are known to be the oxidation products from aniline, methylaniline, and chloroaniline (14,18,32,33), but this is the first report to have detected a major amount of catechol from aniline. Next, to prove the presence of ␥-glutamyl intermediates in aniline oxidation, we purified the GS-like protein AtdA1 and showed that it can produce ␥-GA from aniline and L-glutamate in the presence of ATP and MgCl 2 (Fig.…”

“…This is strong evidence demonstrating that ␥-GA formation is the starting point of the proposed aniline oxidation mechanism (pathway). Recently, Król et al (33) reported that recombinant E. coli cells harboring only the dioxygenase genes (dcaA1A2B) from the AD gene cluster (dcaQTA1A2B) of Comamonas testosteroni produced a small amount of 4-chlorocatechol from 3-chloroaniline. Therefore, this dioxygenase may be able to attack anilines directly without GS-like and GAT-like proteins.…”

“…To date, several AD genes have been cloned and characterized (14,18,23,(30)(31)(32)(33), but the aniline oxidation mechanisms have not been elucidated, that is, the concrete functions of the products of the AD genes in aniline oxidation are unknown. All the AD gene clusters found so far encode GS-like and GAT-like proteins, which have not been found in other dioxygenases, with one exception (34).…”

“…strain ANA-18 (31), Delftia sp. strain AN3 (32), and Comamonas testosteroni I2 (33). However, the mechanism of aniline oxidation has not been defined, as there is little information on the functions of the gene products involved in the process.…”

Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

“…The two hosts, E. coli and P. putida (both belonging to the Gammaproteobacteria) were selected because the behavior of RK2 in these hosts has been relatively well studied. In addition, since IncP-1 plasmids have been found in strains within the Betaproteobacteria (for example, in the genera Achromobacter, Bordetella, Comamonas, Cupriavidus, Delftia, and Neisseria) and Alphaproteobacteria (genus Sphingomonas) (10,26,29,37,47,67,68), we also included representative hosts from those two classes: the betaproteobacterium Cupria- and M124 indicate the start methionines of TrfA-33 in RK2 and TrfA2 in pBP136, respectively. QLSLF indicates the conserved amino acid sequence motif responsible for interaction with DNA polymerase III (33).…”