Transfer of the Catabolic Plasmid Pjp4 in Bacterial Biofilms in Lab-Scale Continuous Flow-Through Cells

Limited information is available on how external environmental factors (e.g., the type of carbon source) affect biofilm architecture, conjugative transfer of the plasmid pJP4 and xenobiotic degradation in biofilms. The aim of this project was to investigate the influence of glucose and 2,4-dichlorophenoxyacetic acid, two different carbon sources which represent the absence and presence of selective pressure, respectively, on the combined effect of biofilm architecture, transfer of the plasmid pJP4 in soil derived mixed culture biofilms and consequent biodegradation of 2,4-D. The pJP4 plasmid was transferred to soil-derived mixed culture recipients in plate mating experiments and isolated transconjugant colonies were characterized as Comamonas testosteroni. Donor and transconjugant cells were not detected microscopically in biofilms and no transconjugant colonies were isolated; however, gfp, dsRed, and tfdB genes were detected in biofilm effluents with and without selective pressure. Heterogeneous biofilm architecture was observed for both with and without selective pressure.

Transfer of the Catabolic Plasmid Pjp4 in Bacterial Biofilms in Lab-Scale Continuous Flow-Through Cells

Limited information is available on how external environmental factors (e.g., the type of carbon source) affect biofilm architecture, conjugative transfer of the plasmid pJP4 and xenobiotic degradation in biofilms. The aim of this project was to investigate the influence of glucose and 2,4-dichlorophenoxyacetic acid, two different carbon sources which represent the absence and presence of selective pressure, respectively, on the combined effect of biofilm architecture, transfer of the plasmid pJP4 in soil derived mixed culture biofilms and consequent biodegradation of 2,4-D. The pJP4 plasmid was transferred to soil-derived mixed culture recipients in plate mating experiments and isolated transconjugant colonies were characterized as Comamonas testosteroni. Donor and transconjugant cells were not detected microscopically in biofilms and no transconjugant colonies were isolated; however, gfp, dsRed, and tfdB genes were detected in biofilm effluents with and without selective pressure. Heterogeneous biofilm architecture was observed for both with and without selective pressure.

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.

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2

ABSTRACT Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism. IMPORTANCE The transfer of information between DNA, RNA, and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware, i.e., the nucleoid, the RNA polymerase, and the ribosomes, are habitually taken for granted. In this work, we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida strain KT2440 through the nonhomogeneous milieu of the bacterial cytoplasm. The results expose that—similarly to computers—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code and accompanies the environmental lifestyle of this bacterium.

The subcellular architecture of the xyl gene expression flow of the TOL catabolic plasmid of Pseudomonas putida mt-2

ABSTRACTDespite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they co-localize with ribosome-rich domains of the intracellular milieu. This arrangement was kept even when the xyl genes were artificially relocated at different chromosomal locations. The same happened when genes were expressed through a heterologous T7 polymerase-based system, which originated mRNA foci outside the DNA. In contrast, rifampicin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs intrinsically run away from their initiation sites to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest that the distinct subcellular motion of xyl mRNAs results both from innate properties of the sequence at stake and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed on the background of current knowledge on the 3D organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.IMPORTANCEThe transfer of information between DNA, RNA and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware i.e. the nucleoid, the RNA polymerase and the ribosomes, are habitually taken for granted. In this work we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida KT2440 through the non-homogenous milieu of the bacterial cytoplasm. The results expose that— similarly to computers also—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code that accompanies the environmental lifestyle of this bacterium.

Microbial Degradation of Pyridine: a Complete Pathway in Arthrobacter sp. Strain 68b Deciphered

ABSTRACT Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b. IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N-heterocyclic compounds in nature.

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.

A New Catabolic Plasmid in Xanthobacter andStarkeyaspp. from a 1,2-Dichloroethane-Contaminated Site

ABSTRACT1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genesdhlAanddhlBhave not been well defined. In this study, the genomic context ofdhlAanddhlBwas determined in three aerobic DCA-degrading bacteria (Starkeya novellastrain EL1,Xanthobacter autotrophicusstrain EL4, andXanthobacter flavusstrain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonicaldhlAgene fromXanthobactersp. strain GJ10 was present in all three isolates, and, in each case, thedhlAgene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of therepAreplication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from theAlphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1and ISPme1) which were likely to have mobilizeddhlAanddhlBand played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1anddhlB2) were detected in the GTP isolates;dhlB1was most likely chromosomal and was similar to the canonicaldhlBgene from strain GJ10, whiledhlB2was carried on pDCA and was not closely related todhlB1. Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination.IMPORTANCEEarlier studies on the DCA-degradingXanthobactersp. strain GJ10 indicated that the key dehalogenasesdhlAanddhlBwere carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degradingXanthobacterstrains from Australia, in which a relatively small circular plasmid (pDCA) carries bothdhlAanddhlBhomologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenasedhlB2in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes.