Chlorobenzoate inhibits growth and induces stress proteins in the PCB-degrading bacterium <Emphasis Type="Italic">Burkholderia xenovorans</Emphasis> LB400

Aerobic bacteria, such as Burkholderia xenovorans LB400, are able to degrade a wide range of polychlorobiphenyls (PCBs). Generally, these bacteria are not able to transform chlorobenzoates (CBAs), which accumulate during PCB degradation. In this study, the effects of CBAs on the growth, the morphology and the proteome of Burkholderia xenovorans LB400 were analysed. 4-CBA and 2-CBA were observed to inhibit the growth of strain LB400 on glucose. Strain LB400 exposed to 4-CBA exhibited increased number and size of electron-dense granules in the cytoplasm, which could be polyphosphates. Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to characterise the molecular response of strain LB400 to 4-CBA. This compound induced the enzymes BenD and CatA of benzoate and catechol catabolic pathways. The induction of molecular chaperones DnaK and HtpG by 4-CBA indicated that the exposure to this compound constitutes a stressful condition for this bacterium. Additionally, the induction of some Krebs cycle enzymes was observed, probably as response to cellular energy requirements. This study contributes to the knowledge on the effects of CBA on the PCB-degrader Burkholderia xenovorans LB400.     

Influence of chlorobenzoic acids on the growth and degradation potentials of PCB-degrading microorganisms

The biodegradation of polychlorinated biphenyls (PCBs) by diverse bacteria including those utilized in this study is often incomplete, a concomitant accumulation of chlorobenzoic acids (CBAs) are released as dead-end products. The build-up of these metabolites in the growth medium may result in feed-back inhibition and impede PCB biotransformation. In this investigation using GC-ECD and HPLC analyses, we confirmed that CBAs inhibit growth and PCB biodegradation potentials of five tropical bacteria namely, Pseudomonas aeruginosa SA-1, Enterobacter sp. SA-2, Ralstonia sp. SA-3, Ralstonia sp. SA-5 and Pseudomonas sp. SA-6. Among the four CBAs (2-CBA, 3-CBA, 4-CBA acids and 2,3-diCBA), 3-CBA was the strongest inhibitor followed by 4-CBA. Furthermore, we found that 3-CBA heavily inhibited growth of SA-3 and SA-6 on monochlorobiphenyls by 82–90% while elimination rate was inhibited by 71–88%. In the case of 2,3-diCBA, inhibition was generally less than 60%. However, effects of both acids were stronger in SA-3 than SA-6. We also found that 3-CBA and 2,3-diCBA completely inhibited carbon-chloride cleavage of 2-CB and 3-CB since cultivation in the absence of the acids resulted in recovery of 23–50% chloride in the culture fluids of organisms. These findings may therefore, have practical and ecological significance and are useful for improving the efficiency and the stability of some biological treatment processes.     

Biodegradation of mono-hydroxylated PCBs by Burkholderia xenovorans

Three hydroxylated derivatives of PCBs, 2′-hydroxy-4-chlorobiphenyl (2′-OH-4-CB), 3′-hydroxy-4-chlorobiphenyl (3′-OH-4-CB), and 4′-hydroxy-4-chlorobiphenyl (4′-OH-4-CB), were transformed by the PCB degrader, Burkholderia xenovorans. When the bacterium was growing on biphenyl (biphenyl pathway-inducing conditions), all three hydroxylated isomers were transformed. However, only 2′-OH-4-CB was transformed by the bacterium growing on succinate (conditions non-inductive of the biphenyl pathway). Gene expression analyses showed a strong induction of key genes of the biphenyl pathway (bph) when cells were grown on biphenyl, which is consistent with the transformation of the three isomers by biphenyl-grown cells. When cells were grown on succinate, only exposure to 2′-OH-4-CB resulted in expression of biphenyl pathway genes, which suggests that this isomer was capable of inducing the biphenyl pathway. These results provide the first evidence that bacteria are able to metabolize PCB derivatives hydroxylated on the non-chlorinated ring.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Sphingobium fuliginis HC3: A Novel and Robust Isolated Biphenyl- and Polychlorinated Biphenyls-Degrading Bacterium without Dead-End Intermediates Accumulation

Biphenyl and polychlorinated biphenyls (PCBs) are typical environmental pollutants. However, these pollutants are hard to be totally mineralized by environmental microorganisms. One reason for this is the accumulation of dead-end intermediates during biphenyl and PCBs biodegradation, especially benzoate and chlorobenzoates (CBAs). Until now, only a few microorganisms have been reported to have the ability to completely mineralize biphenyl and PCBs. In this research, a novel bacterium HC3, which could degrade biphenyl and PCBs without dead-end intermediates accumulation, was isolated from PCBs-contaminated soil and identified as Sphingobium fuliginis. Benzoate and 3-chlorobenzoate (3-CBA) transformed from biphenyl and 3-chlorobiphenyl (3-CB) could be rapidly degraded by HC3. This strain has strong degradation ability of biphenyl, lower chlorinated (mono-, di- and tri-) PCBs as well as mono-CBAs, and the biphenyl/PCBs catabolic genes of HC3 are cloned on its plasmid. It could degrade 80.7% of 100 mg L ⁻¹ biphenyl within 24 h and its biphenyl degradation ability could be enhanced by adding readily available carbon sources such as tryptone and yeast extract. As far as we know, HC3 is the first reported that can degrade biphenyl and 3-CB without accumulation of benzoate and 3-CBA in the genus Sphingobium, which indicates the bacterium has the potential to totally mineralize biphenyl/PCBs and might be a good candidate for restoring biphenyl/PCBs-polluted environments.     

Construction and Characterization of Two Recombinant Bacteria That Grow on ortho- and para-Substituted Chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISP_OHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Biodegradation of 4-chlorobiphenyl by using induced cells and cell extract of Burkholderia xenovorans

This study aimed to evaluate the efficiency of Burkholderia xenovorans LB400 cells and their cell extract to remediate 4-chlorobiphenyl (4-CB). The bacterium previously induced with 4-CB was able to degrade up to 98% of initial 50 mg L^?1 of 4-CB from mineral medium within 96 h of incubation. The degradation of 4-CB occurred through the formation of meta-cleavage product 2-hydroxy-6-oxo-6phenylhexa-2,4-dienoic acid (HOPDA), as revealed through enzymatic assay of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD). A derivative of 1,2-benzenedicarboxylic acid was observed as one of the major intermediate metabolites of 4-CB degradation. Time course production of 2,3-DHBD during growth corresponds with the degradation pattern of 4-CB by the bacterium. In vitro degradation of 4-CB using cell extract of B. xenovorans showed complete degradation of initial 25 mg L^?1 of 4-CB within 6 h of incubation. To the best of the authors' knowledge, this is the first report in which in vitro degradation of 4-CB using cell extract of Burkholderia xenovorans is presented.     

Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils

Contaminated sites in Lagos, Nigeria were screened for the presence of chlorobiphenyl-degrading bacteria. The technique of continual enrichment on Askarel fluid yielded bacterial isolates able to utilize dichlorobiphenyls (diCBs) as growth substrates and six were selected for further studies. Phenotypic typing and 16S rDNA analysis classified these organisms as species of Enterobacter, Ralstonia and Pseudomonas. All the strains readily utilized a broad spectrum of xenobiotics as sole sources of carbon and energy. Growth was observed on all monochlorobiphenyls (CBs), 2,2′-, 2,3-, 2,4′-, 3,3′- and 3,5-diCB as well as di- and trichlorobenzenes Growth was also sustainable on Askarel electrical transformer fluid and Aroclor 1221. Time-course studies using 100 ppm of 2-, 3- or 4-CB resulted in rapid exponential increases in cell numbers and CB transformation to respective chlorobenzoates (CBAs) within 70 h. Significant amounts of chloride were recovered in culture media of cells incubated with 2-CB and 3-CB, suggesting susceptibilities of both 2- and 3-chlorophenyl rings to attack, while the 4-CB was stoichiometrically transformed to 4-CBA. Extensive degradation of most of the congeners in Aroclor 1221 was observed when isolates were cultivated with the mixture as a sole carbon source. Aroclor 1221 was depleted by a minimum of 51% and maximum of 71%. Substantial amounts of chloride eliminated from the mixture ranged between 15 and 43%. These results suggest that some contaminated soils in the tropics may contain exotic micro-organisms whose abilities and potentials are previously unknown. An understanding of these novel strains therefore, may help answer questions about the microbial degradation of polychlorinated biphenyls (PCBs) in natural systems and enhance the potential use of bioremediation as an effective tool for cleanup of PCB-contaminated soils.     

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 M) and 3,4-CBA (50 M). Although 2,5-CBA and 2,6-CBA were not utilised as carbon sources by strain JHR22, they did not inhibit 2-CBA utilisation at the concentrations studied, whereas 2,4-CBA was co-metabolised with 2-CBA. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.Abbreviations PCB Polychlorinated biphenyl - CBA Chlorobenzoate     

Degradation of PCB congeners by bacterial strains

Biological in situ methods are options for the remediation of contaminated sites. An approach to quantify biodegradation by soil bacteria was developed, combining experiment with mathematical modelling. We performed in vitro assays to investigate the potential and kinetics of the wild-type degrader, Burkholderia sp. strain LB400 (expressing bph) and the genetically modified Pseudomonas fluorescens strains F113pcb and F113L::1180 (expressing bph under different promoters) to metabolise individual congeners of polychlorinated biphenyls (PCBs). Kinetics of metabolism was analysed using the Monod model. Results revealed similar patterns of degradable PCB congeners for LB400 and F113L::1180. The degree of PCB degradation was comparable for LB400 and F113L::1180 but was much lower for F113rifpcb. In additional mesocosm experiments with PCB-contaminated soil, the F113 derivatives demonstrated a good survival ability in willow (Salix sp.) rhizosphere. Strain F113L::1180 in combination with willow plants is expected to degrade a large spectrum of PCB congeners in soil. The data from the experiments were used to calculate the time scale of the degradation process in a PCB-contaminated soil. The uncertainty of the model predictions due to the uncertainties of experimental removal velocities and bacterial cell density in soil was quantified.     

Antioxidant compounds improved PCB-degradation by Burkholderia xenovorans strain LB400

Polychlorobiphenyls (PCBs) are toxic and persistent organic pollutants that are widely distributed in the environment. Burkholderia xenovorans LB400 is capable of degrading aerobically an unusually wide range of PCBs. However, during PCB-degradation B. xenovorans LB400 generates reactive oxygen species (ROS) that affect its viability. The aim of this study was to increase the efficiency of PCB-degradation of B. xenovorans LB400 by adding antioxidant compounds that could increase tolerance to oxidative stress. The effect of antioxidant compounds on the growth, morphology and PCB-degradation by B. xenovorans LB400 was evaluated. α-Tocopherol or vitamin E (vitE) and berry extract (BE) increased slightly the growth of strain LB400 on biphenyl, whereas in presence of ascorbic acid or vitamin C (vitC) an inhibition of growth was observed. The growth of B. xenovorans LB400 in glucose was inhibited by the addition of 4-chlorobiphenyl (4-CB). Interestingly, in presence of α-tocopherol the growth of strain LB400 was less affected by 4-CB. By transmission electronic microscopy it was observed that α-tocopherol preserved the cell membranes and improved cell integrity of glucose-grown LB400 cells exposed to 4-CB, suggesting a protective effect of α-tocopherol. Notably, α-tocopherol increased biphenyl and 4-CB degradation by B. xenovorans LB400 in an aqueous solution. The effect of antioxidants compounds on PCB-bioremediation was evaluated in agricultural soil spiked with 2-chlorobiphenyl (2-CB), 4-CB and 2,4'-chlorobiphenyl (2,4'-CB). For bioaugmentation, LB400 cells grown on biphenyl and subsequently incubated with pyruvate were added to the soil. Native soil microbiota was able to remove PCBs. Bioaugmentation with strain LB400 increased strongly the PCB-degradation rate. Bioaugmentation with strain LB400 and biostimulation with α-tocopherol or berry extract increased further the PCB degradation. Half-life of 2,4'-CB decreased by bioaugmentation from 24 days to 4 days and by bioaugmentation in presence of α-tocopherol and berry extract to 2 days. By bioaugmentation with strain LB400, 85% of 2,4'-CB was degraded in 20 days, whereas bioaugmentation with strain LB400 and biostimulation with α-tocopherol or berry extract reduced the time to less than 13 days. This indicates that antioxidant compounds stimulated PCB-degradation in soil. Therefore, the addition of antioxidant compounds constitutes an attractive strategy for the scale-up of aerobic PCB-bioremediation processes.     

Molecular genetics and evolutionary relationship of PCB-degrading bacteria

Biphenyl-utilizing soil bacteria are ubiquitously distributed in the natural environment. They cometabolize a variety of polychlorinated biphenyl (PCB) congeners to chlorobenzoic acids through a 2,3-dioxygenase pathway, or alternatively through a 3,4-dioxygenase system. Thebph genes coding for the metabolism of biphenyl have been cloned from several pseudomonads. The biochemistry and molecular genetics of PCB degradation are reviewed and discussed from the viewpoint of an evolutionary relationship.Abbreviations BP biphenyl - bph BP/PCB-degradative gene - 23DHBP 2,3-dihydroxybiphenyl - HPDA 2-hydroxy-6-oxo-6-phenylhexa 2,4-dienoic acid - KF707 P. pseudoalcaligenes strain KF707 - LB400 Pseudomonas sp. strain LB400 - PCB polychlorinated biphenyls - Q1 P. paucimobilis strain Q1tod; toluene catabolic gene     

Hybrid pseudomonads engineered by two-step homologous recombination acquire novel degradation abilities toward aromatics and polychlorinated biphenyls

Pseudomonas pseudoalcaligenes KF707 possesses a chromosomally encoded bph gene cluster responsible for the catabolism of biphenyl and polychlorinated biphenyls. Previously, we constructed chimeric versions of the bphA1 gene, which encodes a large subunit of biphenyl dioxygenase, by using DNA shuffling between bphA1 genes from P. pseudoalcaligenes KF707 and Burkholderia xenovorans LB400. In this study, we demonstrate replacement of the bphA1 gene with chimeric bphA1 sequence within the chromosomal bph gene cluster by two-step homologous recombination. Notably, some of the hybrid strains acquired enhanced and/or expanded degradation capabilities for specific aromatic compounds, including single aromatic hydrocarbons and polychlorinated biphenyls.     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Remediation of PCBs in soil by surfactant washing and biodegradation in the wash by Pseudomonas sp. LB400

Solutions from the washing of polychlorinated biphenyl (PCB)-contaminated soil with a variety of commercial nonionic or anionic surfactants were incubated with Pseudomonas sp. LB400 in an attempt to remediate the soil and destroy the PCBs. Nonionic surfactants washed more PCBs from the soil (up to 89%) but inhibited their biodegradation. Anionic surfactants washed less PCBs from the soil but were more effective in biodegradation tests, removing up to 67% of total PCBs.     

Cometabolic degradation of mono-chloro benzoic acids by <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. R04 grown on organic carbon sources

The aerobic cometabolism of chlorobenzoic acids (CBAs) by Rhodococcus sp. R04 was accomplished by augmenting the medium with organic carbon sources. In mineral medium supplemented with glucose (MMG), 0.5 mM 2-CBA was incompletely metabolized after the 5-day incubation, while the near-complete disappearance of 0.5 mM 4-CBA was monitored. Over the 5-day incubation period, the concentration of chloride increased to 0.17 mM in bottles containing 4-CBA, glucose and strain R04; whereas in cultivation with 2-CBA the chloride content was about 0.1 mM. After 5-day incubation, 28.5% 4-CBA was remained in mineral medium supplemented with ethanol (MME), and the relatively low values of chloride were released. To our knowledge, it is first report that the feasibility of using ethanol as an added substrate for cometabolic degradation of CBA by aerobic polychlorinated biphenyl (PCB)-degrading bacteria. The specific activities of (chloro)benzoate 1,2-dioxygenase and (chloro)catechol 1,2-dioxygenase activities were detected in cell-free extracts (CFEs) of strain R04. These results suggest that the initial degradation of CBAs occurred most likely prior to chloride release.     

Dechlorination of Pesticides by a Specific Bacterial Glutathione S‐transferase,BphKLB400: Potential for Bioremediation

The bphK gene located in the bph operon of Burkholderia xenovorans LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione‐S‐transferases (GSTs). GSTs are a superfamily of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Recently, BphK^LB400 was shown to catalyze the dechlorination of a number of toxic chlorinated organic compounds. Comparison of the amino acid sequence of BphK^LB400 with GSTs from other bacteria that degrade polychlorinated biphenyls identified a number of highly conserved amino acids in the C‐terminal region of the protein thought to be associated with substrate specificity. Mutating the conserved amino acid at position 180 of BphK^LB400 from an alanine to a proline residue resulted in an increase in GST activity of bacterial cell extracts towards a number of chlorinated organic substrates tested including commonly used pesticides. Laboratory scale plant protection experiments suggested that E. coli expressing BphK^LB400 [wildtype and mutant (Ala180Pro)] could protect pea plants from the effects of chloromequat chloride. Therefore, BphK^LB400, identified as having dechlorination activity towards toxic chlorinated organic compounds used in the environment, could have potential in bioremediation.     

A β-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.     

The Complete Multipartite Genome Sequence of Cupriavidus necator JMP134, a Versatile Pollutant Degrader

Background

Cupriavidus necator JMP134 is a Gram-negative β-proteobacterium able to grow on a variety of aromatic and chloroaromatic compounds as its sole carbon and energy source.

Methodology/Principal Findings

Its genome consists of four replicons (two chromosomes and two plasmids) containing a total of 6631 protein coding genes. Comparative analysis identified 1910 core genes common to the four genomes compared (C. necator JMP134, C. necator H16, C. metallidurans CH34, R. solanacearum GMI1000). Although secondary chromosomes found in the Cupriavidus, Ralstonia, and Burkholderia lineages are all derived from plasmids, analyses of the plasmid partition proteins located on those chromosomes indicate that different plasmids gave rise to the secondary chromosomes in each lineage. The C. necator JMP134 genome contains 300 genes putatively involved in the catabolism of aromatic compounds and encodes most of the central ring-cleavage pathways. This strain also shows additional metabolic capabilities towards alicyclic compounds and the potential for catabolism of almost all proteinogenic amino acids. This remarkable catabolic potential seems to be sustained by a high degree of genetic redundancy, most probably enabling this catabolically versatile bacterium with different levels of metabolic responses and alternative regulation necessary to cope with a challenging environment. From the comparison of Cupriavidus genomes, it is possible to state that a broad metabolic capability is a general trait for Cupriavidus genus, however certain specialization towards a nutritional niche (xenobiotics degradation, chemolithoautotrophy or symbiotic nitrogen fixation) seems to be shaped mostly by the acquisition of “specialized” plasmids.

Conclusions/Significance

The availability of the complete genome sequence for C. necator JMP134 provides the groundwork for further elucidation of the mechanisms and regulation of chloroaromatic compound biodegradation.