Catabolic Plasmid Research Articles

Removing multiple refractory organic pollutants in wastewater by cell bioaugmentation and genetic bioaugmentation with Rhodococcus sp. strain p52

Bioaugmentation is an appealing remediation strategy to treat refractory organic pollutants in contaminated environments. In this study, activated sludge was bioaugmented with Rhodococcus sp. strain p52 to treat synthetic wastewater containing multiple refractory organic pollutants (dibenzofuran, dibenzo-p-dioxin, and phenanthrene). Bioaugmentation of activated sludge reactors with strain p52 alleviated the adverse effects of refractory pollutants on the chemical oxygen demand and ammonia nitrogen removal capacity of the reactors and enhanced the removal of the refractory organic pollutants, especially highly refractory compound dibenzo-p-dioxin. Toxicity of the refractory pollutants to activated sludge bacteria was attenuated due to contaminant degradation by strain p52. Genetic bioaugmentation mediated by catabolic plasmid transfer from strain p52 to activated sludge bacteria and cell bioaugmentation occurred along with successful colonization and proliferation of strain p52. In addition, a potential metabolically mutualistic relationship existed between strain p52 and activated sludge bacteria for refractory pollutants degradation. Both refractory organic pollutants input and strain p52 bioaugmentation profoundly influenced the structure of the activated sludge bacterial community and drove bacterial community shifts in different directions. This study provides insights into the activities of bioaugmented bacteria and bioaugmentation technology for environmental contaminant remediation.

Different Roles of Dioxin-Catabolic Plasmids in Growth, Biofilm Formation, and Metabolism of Rhodococcus sp. Strain p52.

Microorganisms harbor catabolic plasmids to tackle refractory organic pollutants, which is crucial for bioremediation and ecosystem health. Understanding the impacts of plasmids on hosts provides insights into the behavior and adaptation of degrading bacteria in the environment. Here, we examined alterations in the physiological properties and gene expression profiles of Rhodococcus sp. strain p52 after losing two conjugative dioxin-catabolic megaplasmids (pDF01 and pDF02). The growth of strain p52 accelerated after pDF01 loss, while it decelerated after pDF02 loss. During dibenzofuran degradation, the expression levels of dibenzofuran catabolic genes on pDF01 were higher compared to those on pDF02; accordingly, pDF01 loss markedly slowed dibenzofuran degradation. It was suggested that pDF01 is more beneficial to strain p52 under dibenzofuran exposure. Moreover, plasmid loss decreased biofilm formation, especially after pDF02 loss. Transcriptome profiling revealed different pathways enriched in upregulated and downregulated genes after pDF01 and pDF02 loss, indicating different adaptation mechanisms. Based on the transcriptional activity variation, pDF01 played roles in transcription and anabolic processes, while pDF02 profoundly influenced energy production and cellular defense. This study enhances our knowledge of the impacts of degradative plasmids on native hosts and the adaptation mechanisms of hosts, contributing to the application of plasmid-mediated bioremediation in contaminated environments.

Exploration of the catabolic plasmid genes profile of crude oil degrading bacteria isolated from aged oil contaminated soils of Anambra State

This study was undertaken to explore the catabolic plasmid genes profile and molecular identification of crude oil degrading bacteria isolated from aged oil contaminated soils of Anambra State. Enrichment technique, turbidometric test, plasmid profiling and curing tests, biodegradation capability testing as well asmolecular identification method were carried out using standard procedures.A total of sixty (60) bacterial isolates were encountered in the four different soil samples. Of the total bacterial isolates, 22 strains demonstrated high crude oil degrading potentials (A600nm > 0.3). The plasmid DNA was present in 8 out of the 22 bacterial strains such that bacteria strains G2, H4, and K4 had one plasmid each with molecular weight of 9416 base pairs while bacteria strains C3, D1, J3 and L2 had two plasmids each with molecular weight of 2111 base pairs. The bacterial strains C1 and J3 recorded the highest growths on the solid media after the plasmid removal. The isolates belong to various species which are Bacillus cereus C12 (100 % similarity), Pseudomonas aeruginosa KAVK01 (100% similarity), Bacillus licheniformis 126 (96 % similarity), Ochrobacteriumintermedium E85b (97% similarity), Bacillus subtillis SDDlas (100 % similarity), Bacillus subtillis LK4.5 (97 % similarity),Enterobacter cloacae GEBRI III (97 % similarity) and Bacillus cereus So24 (100 % similarity). Therefore, plasmid-borne Pseudomonas aeruginosa and Ochrobacteriumintermediumare recommended for use in bioremediation because of their resilience to the crude oil contaminant and degradation capacities.

Roles of n-hexadecane in the degradation of dibenzofuran by a biosurfactant-producing bacterium Rhodococcus sp.

The hydrophobic nature of dioxins is a major obstacle to their bioremediation. The present study attempted to improve the bioaccessibility of dioxins in two-phase partitioning bioreactors and activated sludge reactors. Dibenzofuran and n-hexadecane were used as model compounds for dioxins and non-aqueous phase liquids (NAPLs), respectively. First, biosurfactants produced by dioxin-degrading Rhodococcus sp. strain p52 were analyzed. Then, the role of n-hexadecane during the biodegradation of dibenzofuran was evaluated. The results showed that high NAPL ratios (n-hexadecane ≥40 g/L) in the two liquid-phase systems favored dibenzofuran removal, while the cell surface of strain p52 became extremely hydrophobic (>97%), which promoted its adhesion to NAPLs and subsequent transport of dibenzofuran from NAPL to the cells. In addition, a bioaugmented activated sludge reactor with n-hexadecane as the supplemented carbon source could not only facilitate the aerobic granular sludge formation but also improve dibenzofuran removal and the activated sludge settling properties. Simultaneously, conjugative transfer of catabolic plasmids was promoted. This study demonstrated the potential application of strain p52 with NAPLs for the bioremediation of dioxins.

Genetic bioaugmentation with triclocarban-catabolic plasmid effectively removes triclocarban from wastewater

Triclocarban, one of the emerging pollutants, has been accumulating, and it is frequently detected in wastewater. Due to its toxicity and persistence, the efficient removal of triclocarban from wastewater systems is challenging. Genetic bioaugmentation with transferable catabolic plasmids has been considered to be a long-lasting method to clean up pollutants in continuous flow wastewater treatment systems. In this study, bioaugmentation with Pseudomonas putida KT2440, harboring the transferrable triclocarban-catabolic plasmid pDCA-1-gfp-tccA2, rapidly converted 50 μM triclocarban in wastewater into 3,4-dichloroaniline and 4-chloroaniline, which are further mineralized more easily. RT-qPCR results showed that the ratio of the copy number of pDCA-1-gfp-tccA2 to the cell number of strain KT2440 gradually increased during genetic bioaugmentation, suggesting horizontal transfer and proliferation of the plasmid. By using DNA stable isotope probing (SIP) and amplicon sequencing, OTU86 (Escherichia-Shigella), OTU155 (Citrobacter), OTU5 (Brucella), and OTU15 (Enterobacteriaceae) were found to be the potential recipients of the plasmid pDCA-1-gfp-tccA2 in the wastewater bacterial community. Furthermore, three transconjugants in the genera of Escherichia, Citrobacter, and Brucella showing triclocarban-degrading abilities were isolated from the wastewater. This study develops a new method for removing triclocarban from wastewater and provides insights into the environmental behavior of transferrable catabolic plasmids in bacterial community in wastewater systems.

Engineering of the chloroaniline-catabolic plasmid pDCA-1 and its potential for genetic bioaugmentation

Plasmids disseminate pollutant-catabolic genes through horizontal transfer and contribute to the catabolic potential of the host microbial community. Genetic bioaugmentation of conjugative catabolic plasmids has recently been considered an effective and novel approach for long-term bioremediation of contaminated sites. In this study, we engineered an IncP-1β-2 subgroup plasmid pDCA-1 from Achromobacter sp. ANB-1 for its potential application in genetic bioaugmentation. A dcaA1A2B cluster responsible for the deamination of chloroaniline to chlorocatechol and a 1,2-dioxygenase gene (ccdC) for the ring cleavage of chlorocatechol were found to locate on the broad host range plasmid pDCA-1. Insertion of an amidase gene (phh or tccA2) at the accessory region of plasmid pDCA-1 greatly expanded its catabolic substrate spectrum from chloroaniline (3-chloroaniline and 3,4-dichloroaniline) to herbicides (linuron, propanil, propham, and chlorpropham), bacteriostatic agent (triclocarban), plant regulator (forchlorfenuron), and insecticide (diflubenzuron). Taking advantages of high-throughput cell sorting and 16S rRNA gene-based amplicon sequencing, we depicted the diversity of bacterial recipients for the engineered plasmid pDCA-1-gfp-phh in a soil bacterial consortium, showing an excellent conjugative transfer capacity of the pDCA-1 derivative to various Gram negative and even positive strains in natural environment. All in all, the engineered plasmid pDCA-1 had a great potential in genetic bioaugmentation of the sites contaminated with chloroanilines and their derivatives.

Characterization of the Agrobacterium octopine-cucumopine catabolic plasmid pAtAg67

In addition to tumor-inducing agrobacteria, non-pathogenic strains are often isolated from crown gall tumors. Such non-pathogenic strains sometimes contain catabolic plasmids that allow them to take advantage of the opines produced in the tumors. Here we characterize for the first time an octopine catabolic plasmid, pAtAg67, which is derived from an Agrobacterium strain isolated from a grapevine tumor in Crete. By sequence analysis, we deduce that pAtAg67 enables its host to catabolize not only octopine, but also cucumopine and agrocinopine-like compounds. We found that a highly similar set of catabolic genes was present in the Ti plasmids of tumorigenic octopine-cucumopine grapevine strains such as pTiAg57. However, the catabolic genes in octopine-cucumopine Ti plasmids were interrupted by a T-DNA segment. As no T-DNA remnants, virulence genes or border repeats were found in pAtAg67, catabolic plasmid pAtAg67 does not appear to be a degenerate octopine-cucumopine Ti plasmid. In line, plasmid pAtAg67 was found to be compatible with incRh1 octopine Ti plasmids, but to be incompatible with the incRh2 agropine Ti plasmid pTiBo542, forming cointegrates by recombination in the homologous trb genes.

The 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 spread 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 three contaminants exerted different degrees of influence on the activated sludge bacteria bearing the catabolic plasmids. The relatively high ratios of transconjugant-bearing catabolic plasmids were detected in the reactor fed with dibenzo-p-dioxin. As dibenzo-p-dioxin from 10 to 80 mg/L was fed into the reactors, the ratios of transconjugant-bearing catabolic plasmids increased. Additionally, levels of ROS and extracellular LDH of activated sludge bacteria in the contaminants-fed reactors increased, comparing with that in the control reactor, indicating that the contaminants exerted toxicity which promoted the cell membrane permeability of the activated sludge bacteria. 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.

Conjugal transfer of catabolic plasmids by damaged bacterial cells: Rescuing genes for pesticide biodegradation

Bacterial conjugative plasmids could carry genes involved in pesticide biodegradation. However, its conjugal transfer is affected by stressors such as metals. Our aim was to determine if bacterial cells exposed to copper can transfer by conjugation the catabolic plasmid pRO101 and assessed the expression of traK gene. Cupriavidus pinatubonensis AEO106 and Pseudomonas putida KT2440 were used as donor and recipient strains, respectively. Mating assays were carried out in presence of Cu2+ (0.3, 0.8, 1.6, 3.9 or 7.9 mM) or H2O2 (0.1, 0.2, 0.5, 1, 2 or 5 mM). The expression of traK gene and the effect of Cu2+ on donor cells was determined by RTqPCR and flow cytometry, respectively. Cytoplasmic membrane of 90% of donor cells was damaged by Cu2+, but the transference of pRO101 plasmid occurred at a proportion similar to the control. Moreover, traK gene expression was reduced (c.a. 50%) by Cu2+. This demonstrated that damaged donor cells were still able to transfer the pRO101 plasmid, allowing a rescue of these genetic material and contribute to self-purifying processes in the environment. Although without ruling out the possibility that transformation occurred, the expression of the traK gene suggested that conjugation occurred between damaged donor cells and recipient cells.

Genome analysis of the metabolically versatile Pseudomonas umsongensis GO16: the genetic basis for PET monomer upcycling into polyhydroxyalkanoates.

The throwaway culture related to the single-use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET-derived terephthalic acid (TA) into PHA and performed an in-depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.

H-NS Family Proteins Drastically Change Their Targets in Response to the Horizontal Transfer of the Catabolic Plasmid pCAR1.

H-NS family proteins regulate the expression of many genes by preferably binding to AT-rich genomic regions and altering DNA topology. They are found in both bacterial chromosomes and plasmids, and plasmid-encoded H-NS family proteins have sometimes been suggested to act as a molecular backup of the chromosomally encoded ones. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1, which belongs to incompatibility P-7 group. We have investigated the function of Pmr in Pseudomonas putida KT2440, where two H-NS family proteins (TurA and TurB) encoded on the chromosome are expressed predominantly. Previous transcriptome analyses suggested that TurA, TurB, and Pmr cooperatively regulate numerous genes, but the differentially transcribed genes in KT2440ΔturA(pCAR1), KT2440ΔturB(pCAR1), and KT2440(pCAR1Δpmr) compared with those in KT2440(pCAR1) were somewhat different. Here, we performed RNA sequencing analyses to compare the differentially transcribed genes after the deletion of turA or turB in KT2440, and turA, turB or pmr in KT2440(pCAR1). Three pCAR1-free strains (KT2440, KT2440ΔturA, KT2440ΔturB) and four pCAR1-harboring strains [KT2440(pCAR1), KT2440ΔturA(pCAR1), KT2440ΔturB(pCAR1), KT2440(pCAR1Δpmr)], grown until the log and stationary phases, were used. In KT2440, TurA was the major H-NS family protein regulating a large number and wide range of genes, and both TurA and TurB were suggested to functionally compensate each other, particularly during the stationary phase. In KT2440(pCAR1), the numbers of differentially transcribed genes after the deletion of turA or turB drastically increased compared to those in KT2440. Notably, more than half of the differentially transcribed genes in KT2440ΔturA and KT2440ΔturB did not overlap with those in KT2440ΔturA(pCAR1) and KT2440ΔturB(pCAR1). This dynamic change could be explained by the acquisition of pCAR1 itself and the expression of Pmr. After pCAR1 was transferred into the host, TurA and TurB could be detached from the chromosome of KT2440 and they could newly bind to pCAR1. Moreover, Pmr could reconstitute the chromosome-binding heteromeric oligomers which were formed by TurA and TurB. Our study revealed that horizontal transfer of a plasmid changes the transcriptional network of the chromosomally encoded H-NS family proteins.

Conjugative transfer of Megaplasmids pND6-1 and pND6-2 enhancing naphthalene degradation in aqueous environment: characterization and bioaugmentation prospects.

Naphthalene catabolic strain Pseudomonas putida ND6 harbors two megaplasmids pND6-1 and pND6-2; naphthalene-degrading associated genes are located on pND6-1, while plasmid pND6-2 possesses potential coding genes for conjugative transfer. In this study, the recombinant ND6 in which pND6-1 and pND6-2 were labeled with gentamicin and kanamycin-encoding genes, respectively, was constructed to investigate the conjugative transfer behavior of the two plasmids in distinct aqueous matrices. The results indicated that both pND6-1 and pND6-2 plasmids could transfer from donor strain ND6 to a recipient strain P. putida KT2440, while the transfer frequency of pND6-2 (1.90 × 10- 2) was significantly higher than that of pND6-1 (3.12 × 10- 9). Furthermore, the concomitant transfer of pND6-1 and pND6-2 was confirmed at a lower frequency of 10- 9 colonies per recipient similar with that of pND6-1. The conjugative transfer efficiency was obviously affected by the initial inoculum and the stability of microbes. Moreover, more than 90% of the transconjugants lost the plasmids after 20 generations cultivation without resistance pressure, suggesting the importance of selective pressure to maintain the plasmid stability. Finally, the naphthalene degradation analysis by mixed ND6 and KT2440 revealed that conjugative transfer of catabolic plasmids contributed to the rapid dispersion of the degradation genes on plasmids and enhanced the naphthalene removal.

Inheritance of IncP-9 Catabolic Plasmids in Pseudomonas Bacteria

This work investigated the capability of the conjugative transfer of epsilon-caprolactam biodegradation (CAP) plasmids, belonging to incompatibility (Inc) groups P-2, P-7, P-9, and IncP-9 naphthalene biodegradation (NAH) plasmids to various species of Pseudomonas bacteria, including strains of plant growth-promoting rhizobacteria (PGPR), and the character of plasmid inheritance in these strains. The frequency of the conjugative transfer of all CAP and NAH plasmids (irrespective of their Inc groups) into Pseudomonas PGPR was shown to be lower than that into P. putida soil strains by more than an order of magnitude. The low frequency of conjugative transfer was found not to be caused by secretion of antibiotically active metabolites by Pseudomonas PGPR strains. Stability of inheritance of catabolic plasmids under nonselective conditions was established to depend on their Inc group and bacterial host strain. Thus, all investigated plasmids were stably maintained in P. putida KT2442 and BS394 strains for at least 100 generations. However, only CAP plasmids of IncP-2 and IncP-7 groups were stably maintained in Pseudomonas PGPR cells. CAP and NAH plasmids of different IncP-9 subgroups were eliminated from cells of rhizosphere pseudomonads within several generations. Thus, the work first revealed inheritance features of IncP-9 catabolic plasmids in Pseudomonas PGPR cells.

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

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.

Negative effect of copper nanoparticles on the conjugation frequency of conjugative catabolic plasmids

Due to their antimicrobial properties, copper nanoparticles (CuNPs) have been proposed to be used in agriculture for pest control. Pesticides removal is mainly done by microorganisms, whose genes usually are found in conjugative catabolic plasmids (CCP). The aim of this work was to evaluate if CuNPs at subinhibitory concentrations modify the conjugation frequency (CF) of two CCP (pJP4 and pADP1). CuNPs were characterized by scanning electron microscopy with an X-ray detector, dynamic light scattering and X-ray diffraction. Mating assays were done in LB broth supplemented with CuNPs (10, 20, 50 and 100 µg mL−1) or equivalent concentrations of CuSO4. Interestingly, we observed that in LB, Cu+2 release from CuNPs is fast as evaluated by atomic absorption spectrophotometry. Donor and recipient strains were able to grow in all copper concentrations assayed, but CF of mating pairs was reduced to 10% in the presence of copper at 20 or 50 µg Cu mL−1 compared to control. Thus, our results indicated that both copper forms, CuNPs or CuSO4, negatively affected the transfer of catabolic plasmids by conjugation. Since dissemination of degradative genes by conjugation contribute to degradation of pesticides by microorganisms, this work improves our understanding of the risks of using copper in agriculture soils, which could affect the biodegradative potential of microbial communities.

Biotechnological Potential of Rhodococcus Biodegradative Pathways.

The genus Rhodococcus is a phylogenetically and catabolically diverse group that has been isolated from diverse environments, including polar and alpine regions, for its versatile ability to degrade a wide variety of natural and synthetic organic compounds. Their metabolic capacity and diversity result from their diverse catabolic genes, which are believed to be obtained through frequent recombination events mediated by large catabolic plasmids. Many rhodococci have been used commercially for the biodegradation of environmental pollutants and for the biocatalytic production of high-value chemicals from low-value materials. Recent studies of their physiology, metabolism, and genome have broadened our knowledge regarding the diverse biotechnological applications that exploit their catabolic enzymes and pathways.

Catabolism of the groundwater micropollutant 2,6-dichlorobenzamide beyond 2,6-dichlorobenzoate is plasmid encoded in Aminobacter sp. MSH1.

Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as sole source of carbon and energy. In the first step, MSH1 converts BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) by means of the BbdA amidase encoded on the IncP-1β plasmid pBAM1. Information about the genes and degradation steps involved in 2,6-DCBA metabolism in MSH1 or any other organism is currently lacking. Here, we show that the genes for 2,6-DCBA degradation in strain MSH1 reside on a second catabolic plasmid in MSH1, designated as pBAM2. The complete sequence of pBAM2 was determined revealing that it is a 53.9kb repABC family plasmid. The 2,6-DCBA catabolic genes on pBAM2 are organized in two main clusters bordered by IS elements and integrase genes and encode putative functions like Rieske mono-/dioxygenase, meta-cleavage dioxygenase, and reductive dehalogenases. The putative mono-oxygenase encoded by the bbdD gene was shown to convert 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoate (3-OH-2,6-DCBA). 3-OH-DCBA was degraded by wild-type MSH1 and not by a pBAM2-free MSH1 variant indicating that it is a likely intermediate in the pBAM2-encoded DCBA catabolic pathway. Based on the activity of BbdD and the putative functions of the other catabolic genes on pBAM2, a metabolic pathway for BAM/2,6-DCBA in strain MSH1 was suggested.

Petroleum contamination and bioaugmentation in bacterial rhizosphere communities from Avicennia schaueriana.

Anthropogenic activity, such as accidental oil spills, are typical sources of urban mangrove pollution that may affect mangrove bacterial communities as well as their mobile genetic elements. To evaluate remediation strategies, we followed over the time the effects of a petroleum hydrocarbon degrading consortium inoculated on mangrove tree Avicennia schaueriana against artificial petroleum contamination in a phytoremediation greenhouse experiment. Interestingly, despite plant protection due to the inoculation, denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene fragments amplified from the total community DNA indicated that the different treatments did not significantly affect the bacterial community composition. However, while the bacterial community was rather stable, pronounced shifts were observed in the abundance of bacteria carrying plasmids. A PCR-Southern blot hybridization analysis indicated an increase in the abundance of IncP-9 catabolic plasmids. Denaturing gradient gel electrophoresis of naphthalene dioxygenase (ndo) genes amplified from cDNA (RNA) indicated the dominance of a specific ndo gene in the inoculated petroleum amendment treatment. The petroleum hydrocarbon degrading consortium characterization indicated the prevalence of bacteria assigned to Pseudomonas spp., Comamonas spp. and Ochrobactrum spp. IncP-9 plasmids were detected for the first time in Comamonas sp. and Ochrobactrum spp., which is a novelty of this study.

Genetic Bioaugmentation of Activated Sludge with Dioxin-Catabolic Plasmids Harbored by Rhodococcus sp. Strain p52.

Horizontal transfer of catabolic plasmids is used in genetic bioaugmentation for environmental pollutant remediation. In this study, we examined the effectiveness of genetic bioaugmentation with dioxin-catabolic plasmids harbored by Rhodococcus sp. strain p52 in laboratory-scale sequencing batch reactors (SBRs). During 100 days of operation, bioaugmentation decreased the dibenzofuran content (120 mg L-1) in the synthetic wastewater by 32.6%-100% of that in the nonbioaugmented SBR. Additionally, dibenzofuran was removed to undetectable levels in the bioaugmented SBR, in contrast, 46.8 ± 4.1% of that in the influent remained in the nonbioaugmented SBR after 96 days. Moreover, transconjugants harboring pDF01 and pDF02 were isolated from the bioaugmented SBR after 2 days, and their abilities to degrade dibenzofuran were confirmed. After 80 days, the copy numbers of strain p52 decreased by 3 orders of magnitude and accounted for 0.05 ± 0.01% of the total bacteria, while transconjugants were present at around 106 copies mL-1 sludge and accounted for 8.2 ± 0.3% of the total bacteria. Evaluation of the bacterial community profile of sludge by high-throughput 16S rRNA gene sequencing revealed that genetic bioaugmentation led to a bacterial community with an even distribution of genera in the SBR. This study demonstrates the promise of genetic bioaugmentation with catabolic plasmids for dioxins remediation.

Diversity of Oil-Degrading Microorganisms in the Gulf of Finland (Baltic Sea) in Spring and in Summer

Diversity of the oil-degrading microbial strains isolated from the water and sediments of the Gulf of Finland (Baltic Sea) in winter and in summer was studied. Substrate specificity of the isolates for aliphatic and aromatic hydrocarbons was studied. The isolates belonged to 32 genera of the types Proteobacteria (alpha-, beta-, and gammaproteobacteria), Actinobacteria,Firmicutes, and Bacteroidetes. Seasonal variations of the oil-degrading microbial communities was revealed. The presence of the known genes responsible for the degradation of oil aliphatic and aromatic hydrocarbons was determined. The alkB sequence of the alkane hydroxylase gene was found in ~16% of the studied strains. The sequence of the phnAc phenanthrene 3,4- dioxygenase was found in Sphingobacterium sp. and Arthrobacter sp. isolates retrieved in winter and summer. In five Pseudomonas sp. strains from winter samples, the classical operons of naphthalene degradation (nah) were localized in catabolic plasmids, of which three belonged to IncР-9, one, to IncР-7, and two to an unidentified incompatibility group. Burkholderia and Delftia strains contained the operons for naphthalene degradation via salicylate and gentisate (nag). The presence of nag genes has not been previously reported for Delftia spp. strains. The sequences of the nagG salicylate 5-hydroxylase gene were also found in Achromobacter, Sphingobacterium, and Stenotrophomonas strains.