Impact of pollutant as selection pressure on conjugative transfer of dioxin-catabolic plasmids harbored by Rhodococcus sp. strain p52

Plasmid-mediated bioaugmentation has potential application in the cleanup of recalcitrant environmental pollutants. In this study, we examined the influence of various contaminants (in different categories or different amounts) as a selection pressure on the transfer of catabolic plasmids within an activated sludge bacteria community bioaugmented with Rhodococcus sp. strain p52 harboring pDF01 and pDF02. The distinguishable genera of transconjugants were isolated under the stresses of phenanthrene, dibenzothiophene, and dibenzo-p-dioxin. The difference in genomic G + C content (5.0 ~ 27.5%) between transconjugants and strain p52 indicated the transfer of the catabolic plasmids crossing phylogenetic boundaries. The specific removal rates in activated sludge reactors for phenanthrene, dibenzothiophene, and dibenzo-p-dioxin were elevated in turn. The three contaminants exerted different degrees of influence on the activated sludge bacteria bearing the catabolic plasmids. The highest proportion of transconjugants was detected in the reactor fed with dibenzo-p-dioxin. Additionally, as dibenzo-p-dioxin from 10 to 80 mg/L was fed into the reactors, the proportion of transconjugants increased. Film mating tests demonstrated that the plasmid transfer frequency varied among recipients under the contaminant stresses of phenanthrene, dibenzothiophene, and dibenzo-p-dioxin. Our study provides a characterization of the recalcitrant contaminants as a selection pressure that can modulate catabolic plasmid transfer during genetic bioaugmentation for the removal of contaminants.

Conjugative Transfer of IncP-9 Catabolic Plasmids Requires a Previously Uncharacterized Gene, mpfK, Whose Homologs Are Conserved in Various MPFT-Type Plasmids

ABSTRACT Conjugative transfer of bacterial plasmids to recipient cells is often mediated by type IV secretion machinery. Experimental investigations into the minimal gene sets required for efficient conjugative transfer suggest that such gene sets are variable, depending on plasmids. We have been analyzing the conjugative transfer of Pseudomonas-derived and IncP-9 plasmids, NAH7 and pWW0, whose conjugation systems belong to the MPFT type. Our deletion analysis and synthetic biology analysis in this study showed that these plasmids require previously uncharacterized genes, mpfK (formerly orf34) and its functional homolog, kikA, respectively, for their efficient conjugative transfer. MpfK was localized in periplasm and had four cysteine residues whose intramolecular or intermolecular disulfide bond formation was suggested to be important for efficient conjugative transfer. The mpfK homologs were specifically carried by many MPFT-type plasmids, including non-IncP-9 plasmids, such as R388 and R751. Intriguingly, the mpfK homologs from the two non-IncP-9 plasmids were not required for conjugation of their plasmids, but were able to complement efficiently the transfer defect of the NAH7 mpfK mutant. Our results suggested the importance of the mpfK homologs for conjugative transfer of MPFT-type plasmids. IMPORTANCE IncP-9 plasmids are important mobile genetic elements for the degradation of various aromatic hydrocarbons. Elucidation of conjugative transfer of such plasmids is expected to greatly contribute to our understanding of its role in the bioremediation of polluted environments. The present study mainly focused on the conjugation system of NAH7, a well-studied and naphthalene-catabolic IncP-9 plasmid. Our analysis showed that the NAH7 conjugation system uniquely requires, in addition to the conserved components of the type IV secretion system (T4SS), a previously uncharacterized periplasmic protein, MpfK, for successful conjugation. Our findings collectively revealed a unique type of T4SS-associated conjugation system in the IncP-9 plasmids.

Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

ABSTRACT NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer (oriT)-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT (oriT N) region was located within a 430-bp fragment, and the strand-specific nicking (nic) site and its upstream sequences that were important for efficient conjugation in the oriT N region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriT N region, between two copies of the pWW0 oriT (oriT W) region (which is clearly different from the oriT N region), and between the oriT N and oriT W regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriT N-containing plasmid. Such a plasmid was, in the presence of the NAH7Δnic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system. IMPORTANCE Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the oriT region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT-relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought.

Comparison of Four Comamonas Catabolic Plasmids Reveals the Evolution of pBHB To Catabolize Haloaromatics

ABSTRACTComamonasplasmids play important roles in shaping the phenotypes of their hosts and the adaptation of these hosts to changing environments, and understanding the evolutionary strategy of these plasmids is thus of great concern. In this study, the sequence of the 119-kb 3,5-dibromo-4-hydroxybenzonitrile-catabolizing plasmid pBHB fromComamonassp. strain 7D-2 was studied and compared with those of three otherComamonashaloaromatic catabolic plasmids. Incompatibility group determination based on a phylogenetic analysis of 24 backbone gene proteins, as well as TrfA, revealed that these four plasmids all belong to the IncP-1β subgroup. Comparison of the four plasmids revealed a conserved backbone region and diverse genetic-load regions. The four plasmids share a core genome consisting of 40 genes (>50% similarities) and contain 12 to 50 unique genes each, most of which are xenobiotic-catabolic genes. Two functional reductive dehalogenase gene clusters are specifically located on pBHB, showing distinctive evolution of pBHB for haloaromatics. The higher catabolic ability of thebhbA2B2cluster than thebhbABcluster may be due to the transcription levels and the character of the dehalogenase gene itself rather than that of its extracytoplasmic binding receptor gene. The plasmid pBHB is riddled with transposons and insertion sequence (IS) elements, and ISs play important roles in the evolution of pBHB. The analysis of the origin of thebhbgenes on pBHB suggested that these accessory genes evolved independently. Our work provides insights into the evolutionary strategies ofComamonasplasmids, especially into the adaptation mechanism employed by pBHB for haloaromatics.