Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site

Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.     

Evolution of a Pathway for Chlorobenzene Metabolism Leads to Natural Attenuation in Contaminated Groundwater

Complete metabolism of chlorinated benzenes is not a feature that is generally found in aerobic bacteria but is thought to be due to a novel recombination of two separate gene clusters. Such a recombination could be responsible for adaptation of a natural microbial community in response to contamination with synthetic chemicals. This hypothesis was tested in a chlorobenzene (CB)-contaminated aquifer. CB-degrading bacteria from a contaminated site were characterized for a number of years by examining a combination of growth characteristics and DNA-DNA hybridization, PCR, and DNA sequence data. The genetic information obtained for the CB pathway of the predominant microorganism, Ralstonia sp. strain JS705, revealed a unique combination of (partially duplicated) genes for chlorocatechol degradation and genes for a benzene-toluene type of aromatic ring dioxygenase. The organism was detected in CB-polluted groundwater by hybridizing colonies cultivated on low-strength heterotrophic media with probes for the CB pathway. Southern hybridizations performed to determine the organization of the CB pathway genes and the 16S ribosomal DNA indicated that CB-degrading organisms isolated from different wells at the site were identical to JS705. Physiological characterization by the Biolog test system revealed some differences. The genes for the aromatic ring dioxygenase and dihydrodiol dehydrogenase of JS705 were detected in toluene and benzene degraders from the same site. Our results suggest that recent horizontal gene transfer and genetic recombination of existing genes between indigenous microorganisms were the mechanisms for evolution of the catabolic pathway. Evolution of the CB pathway seems to have created the capacity for natural attenuation of CB at the contaminated site.     

Characterization of the novel HCH-degrading strain, Microbacterium sp. ITRC1

A gram-positive Microbacterium sp. strain, ITRC1, that was able to degrade the persistent and toxic hexachlorocyclohexane (HCH) isomers was isolated and characterized. The ITRC1 strain has the capacity to degrade all four major isomers of HCH present in both liquid cultures and aged contaminated soil. DNA fragments corresponding to the two initial genes involved in γ-HCH degradative pathway, encoding enzymes for γ-pentachlorocyclohexene hydrolytic dehalogenase (linB) and a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (linC), were amplified by PCR and sequenced. Their presence in the ITRC1 genomic DNA was also confirmed by Southern hybridization. Sequencing of the amplified DNA fragment revealed that the two genes present in the ITRC1 strain were homologous to those present in Sphingomonas paucimobilis UT26. Both 16S rRNA sequencing and phylogenetic analysis resulted in the identification of the bacteria as a Microbacterium sp. We assume that these HCH-degrading bacteria evolved independently but possessed genes similar to S. paucimobilis UT26. The reported results indicate that catabolic genes for γ-HCH degradation are highly conserved in diverse genera of bacteria, including the gram-positive groups, occurring in various environmental conditions.     

Evaluation of strains isolated by growth on naphthalene and biphenyl for hybridization of genes to dioxygenase probes and polychlorinated biphenyl-degrading ability.

Approximately equal numbers of bacteria were isolated from primarily tropical soils by growth on biphenyl and naphthalene to compare their competence in polychlorinated biphenyl (PCB) degradation. The strains isolated by growth on biphenyl catalyzed more extensive PCB degradation than the strains isolated by growth on naphthalene, suggesting that naphthalene cocontamination may be only partially effective in stimulating the cometabolism of lower chlorinated PCBs. Probes were made from the bph, nah, and tod genes encoding the large iron iron sulfur protein of the dioxygenase complex and hybridized to 19 different strains. The hybridization patterns did not correlate well with the substrates of isolation, suggesting that there is considerable diversity in these genes in nature and that probe hybridization is not a reliable indication of catabolic capacity. The strains with the most extensive PCB degradation capacity did strongly hybridize to the bph probe, but a few strains that exhibited strong hybridization had poor PCB-degrading ability. Of the 19 strains studied, 5 hybridized to more than one probe and 2, including one strong PCB degrader, hybridized to all three probes. Southern blots showed that the bph and nah probes hybridized to separate bands, suggesting that multiple dioxygenases were present. Multiple dioxygenases may be an important feature of competitive decomposers in nature and hence may not be rare. Most of the isolates identified were members of the beta subgroup of the Proteobacteria, a few were gram positive, and none were true Pseudomonas species.     

Anaerobic ammonium-oxidizing (anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

Biofilm Formation by Salmonella Enterica Serovar 1,4,[5],12:i:- Portuguese Isolates: A Phenotypic,Genotypic, and Socio-geographic Analysis

Biofilm-forming ability is well established as an important virulence factor. However, there are no studies available regarding biofilm formation of Salmonella Typhimurium 1,4,[5],12:i:-, the new pandemic serovar in Europe. To address this problem, biofilm expression by Salmonella 1,4,[5],12:i:- was evaluated using 133 isolates from clinical, environmental and animal origins, collected in Portugal from 2006 to 2011. Biofilm detection was performed by phenotypic and genotypic methods, such growth characterization in agar and broth medium, optical density determination by microtiter assays and direct observation by fluorescent in situ hybridization. Biofilm-related genes adrA, csgD and gcpA were detected by PCR. A socio-geographic characterization of strains as biofilm producers was also performed. Results showed that biofilm formation in monophasic Salmonella is widely distributed in Portuguese isolates and could be one of the reasons for its dissemination in this country. Biofilm expression varies between locations, showing that isolates from some regions like Lisboa or Ponta Delgada have an increased ability to persist in the environment due to an enhanced biofilm production. Biofilm formation also varies between risk groups, with a higher prevalence in isolates from salmonellosis infections in women. Therefore, the analysis of the socio-geographic distribution of biofilm-forming bacteria should be considered for the establishment of more adequate regulatory measures or therapeutics regimens, especially important due to the continuous increase of infections caused by antimicrobial resistant microorganisms.     

Distribution of Dehalococcoidia in the Anaerobic Deep Water of a Remote Meromictic Crater Lake and Detection of Dehalococcoidia-Derived Reductive Dehalogenase Homologous Genes

Here we describe the natural occurrence of bacteria of the class Dehalococcoidia (DEH) and their diversity at different depths in anoxic waters of a remote meromictic lake (Lake Pavin) using 16S rRNA gene amplicon sequencing and quantitative PCR. Detected DEH are phylogenetically diverse and the majority of 16S rRNA sequences have less than 91% similarity to previously isolated DEH 16S rRNA sequences. To predict the metabolic potential of detected DEH subgroups and to assess if they encode genes to transform halogenated compounds, we enriched DEH-affiliated genomic DNA by using a specific-gene capture method and probes against DEH-derived 16S rRNA genes, reductive dehalogenase genes and known insertion sequences. Two reductive dehalogenase homologous sequences were identified from DEH-enriched genomic DNA, and marker genes in the direct vicinity confirm that gene fragments were derived from DEH. The low sequence similarity with known reductive dehalogenase genes suggests yet-unknown catabolic potential in the anoxic zone of Lake Pavin.     

Detection of bioterror agents in air samples using real-time PCR

Aims:  To use real-time PCR for the detection of bacterial bioterror agents in a liquid air sample containing potential airborne interferences, including bacteria, without the need for DNA extraction.
Methods and Results:  Bacteria in air were isolated after passive sedimentation onto R2A agar plates and characterized by 16S rRNA sequencing. Real-time PCR was used to identify different bacterial bioterror agents in an artificial air sample consisting of a liquid air sample and a mixture of miscellaneous airborne bacteria showing different colony morphology on R2A agar plates. No time-consuming DNA extraction was performed. Specifically designed fluorescent hybridization probes were used for identification.
Conclusions:  Fourteen different bacterial genera were classified by 16S rRNA gene sequencing of selected bacterial colonies showing growth on R2A agar plates. Real-time PCR amplification of all the bacterial bioterror agents was successfully obtained in the artificial air sample containing commonly found airborne bacteria and other potential airborne PCR interferences.
Significance and Impact of the Study:  Bacterial bioterror agents can be detected within 1 h in a liquid air sample containing a variety of commonly found airborne bacteria using real-time PCR. Airborne viable bacteria at Kjeller (Norway) were classified to the genera level using 16S rRNA gene sequencing.     

Microbial diversity of biofilms in dental unit water systems

We investigated the microbial diversity of biofilms found in dental unit water systems (DUWS) by three methods. The first was microscopic examination by scanning electron microscopy (SEM), acridine orange staining, and fluorescent in situ hybridization (FISH). Most bacteria present in the biofilm were viable. FISH detected the beta and gamma, but not the alpha, subclasses of Proteobacteria: In the second method, 55 cultivated biofilm isolates were identified with the Biolog system, fatty acid analysis, and 16S ribosomal DNA (rDNA) sequencing. Only 16S identified all 55 isolates, which represented 13 genera. The most common organisms, as shown by analyses of 16S rDNA, belonged to the genera Afipia (28%) and Sphingomonas (16%). The third method was a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA. This method revealed 40 genera: the most common ones included Leptospira (20%), Sphingomonas (14%), Bacillus (7%), Escherichia (6%), Geobacter (5%), and Pseudomonas (5%). Some of these organisms may be opportunistic pathogens. Our results have demonstrated that a biofilm in a health care setting may harbor a vast diversity of organisms. The results also reflect the limitations of culture-based techniques to detect and identify bacteria. Although this is the greatest diversity reported in DUWS biofilms, other genera may have been missed. Using a technique based on jackknife subsampling, we projected that a 25-fold increase in the number of subclones sequenced would approximately double the number of genera observed, reflecting the richness and high diversity of microbial communities in these biofilms.     

Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the alpha-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the beta-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the beta-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3, 4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the beta-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3, 4-dioxygenase and the beta-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.     

Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

Variations in the abundance and identity of class II aromatic ring-hydroxylating dioxygenase genes in groundwater at an aromatic hydrocarbon-contaminated site

The abundance of genes encoding aromatic ring-hydroxylating dioxygenases (RHDs) in the groundwater at an aromatic hydrocarbon-contaminated landfill near Sydney, Australia, was determined by quantitative DNA-DNA hybridization using class II RHD genes as probes. There were marked differences in hybridization signal intensity against DNA extracted from the groundwater at seven different locations across this heterogeneous site. This was interpreted as indicating variation in RHD gene abundance. Clone libraries of polymerase chain reaction (PCR)-amplified RHD gene fragments were constructed from DNA from each of the groundwater samples. The libraries from the samples with greater RHD gene abundance were dominated by a group of bacterial class II RHD genes, designated the S-cluster, that has yet to be found in cultured isolates. These groundwater samples contained no detectable petroleum hydrocarbons. A second group of class II RHD gene sequences, designated the T-cluster, dominated RHD gene clone libraries prepared from groundwater samples that contained detectable levels of total petroleum and aromatic hydrocarbons but lower RHD gene abundance. The hosts and in situ expression of these novel genes, and the substrates of the enzymes they encode, remain unknown. The scarcity of genes from known aromatic hydrocarbon-degrading bacteria and the numerical dominance of the novel genes suggest that the hosts of these novel genes may play an important role in aromatic hydrocarbon degradation at this site.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Coccidioidomycosis and blastomycosis: advances in molecular diagnosis

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.     

Real-time PCR detection of Fe-type nitrile hydratase genes from environmental isolates suggests horizontal gene transfer between multiple genera

Nitriles are widespread in the environment as a result of biological and industrial activity. Nitrile hydratases catalyse the hydration of nitriles to the corresponding amide and are often associated with amidases, which catalyze the conversion of amides to the corresponding acids. Nitrile hydratases have potential as biocatalysts in bioremediation and biotransformation applications, and several successful examples demonstrate the advantages. In this work a real-time PCR assay was designed for the detection of Fe-type nitrile hydratase genes from environmental isolates purified from nitrile-enriched soils and seaweeds. Specific PCR primers were also designed for amplification and sequencing of the genes. Identical or highly homologous nitrile hydratase genes were detected from isolates of numerous genera from geographically diverse sites, as were numerous novel genes. The genes were also detected from isolates of genera not previously reported to harbour nitrile hydratases. The results provide further evidence that many bacteria have acquired the genes via horizontal gene transfer. The real-time PCR assay should prove useful in searching for nitrile hydratases that could have novel substrate specificities and therefore potential in industrial applications.     

Molecular detection of catabolic genes for polycyclic aromatic hydrocarbons in the reed rhizosphere of Sunchon Bay

This study focused on detecting catabolic genes for polycyclic aromatic hydrocarbons (PAHs) distributed in the reed rhizosphere of Sunchon Bay, Korea. These marsh and mud environments were severely affected by human activities, including agriculture and fisheries. Our previous study on microbial roles in natural decontamination displayed the possibility that PAH-degrading bacteria, such as Achromobacter sp., Alcaligenes sp., Burkholderia sp. and Pseudomonas sp. play an important decontamination role in a reed rhizosphere. In order to gain further fundamental knowledge on the natural decontamination process, catabolic genes for PAH metabolism were investigated through PCR amplification of dioxygenase genes using soil genomic DNA and sequencing. Comparative analysis of predicted amino acid sequences from 50 randomly selected dioxygenase clones capable of hydroxylating inactivated aromatic nuclei indicated that these were divided into three groups, two of which might be originated from PAH-degrading bacteria. Amino acid sequences of each dioxygenase clone were a part of the genes encoding enzymes for initial catabolism of naphthalene, phenanthrene, or pyrene that might be originated from bacteria in the reed rhizosphere of Sunchon Bay.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis

Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.     

Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria

Pseudomonas putida BN210, carrying the self- transferable clc-element encoding degradation of 3-chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3-chlorobenzoate-contaminated model wastewater. Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3-chlorobenzoate mineralizing bacteria mainly belonging to the beta-proteobacteria. In contrast, in non-inoculated reactors, no 3-chlorobenzoate degradation was observed and no 3-chlorobenzoate degraders could be recovered. Southern blots hybridization of genomic DNA using clc-element-specific probes and FIGE analysis indicated the presence of the complete clc-element in one or more copies in the isolates. Moreover, the isolates could transfer the clc genes to Ralstonia metallidurans recipients. Two representative reactor isolates, Ralstonia sp. strains KP3 and KP9 demonstrated a higher growth rate on 3-chlorobenzoate than strain BN210 in batch cultures. When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3-chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3-chlorobenzoate degrading population in the reactor. Our data suggest that in situ horizontal transfer of the clc-element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3-chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.