Cupriavidus Necator JMP134 Research Articles

Potential of preventive bioremediation to reduce environmental contamination by pesticides in an agricultural context: A case study with the herbicide 2,4-D

One of the major problems with pesticides is linked to the non-negligible proportion of the sprayed active ingredient that does not reach its intended target and contaminates environmental compartments. Here, we have implemented and provided new insights to the preventive bioremediation process based on the simultaneous application of the pesticide with pesticide-degrading microorganisms to reduce the risk of leaching into the environment. This study pioneers such a practice, in an actual farming context. The 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) and one of its bacterial mineralizing-strains (Cupriavidus necator JMP134) were used as models. The 2,4-D biodegradation was studied in soil microcosms planted with sensitive (mustard) and insensitive (wheat) plants. Simultaneous application of a 2,4-D commercial formulation (DAM®) at agricultural recommended doses with 105 cells.g−1 dw of soil of the JMP134 strain considerably accelerated mineralization of the herbicide since its persistence was reduced threefold for soil supplemented with the mineralizing bacterium without reducing the herbicide efficiency. Furthermore, the inoculation of the Cupriavidus necator strain did not significantly affect the α- and β-diversity of the bacterial community. By tackling the contamination immediately at source, the preventive bioremediation process proves to be an effective and promising way to reduce environmental contamination by agricultural pesticides.

Efficient Degradation of Phenoxyalkanoic Acid Herbicides by the Alkali-Tolerant Cupriavidus oxalaticus Strain X32.

Phenoxyalkanoic acid (PAA) herbicides are mainly metabolized by microorganisms in soils, but the degraders that perform well under alkaline environments are rarely considered. Herein, we report Cupriavidus oxalaticus strain X32, which showed encouraging PAA-degradation abilities, PAA tolerance, and alkali tolerance. In liquid media, without the addition of exogenous carbon sources, X32 could completely remove 500 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-chloro-2-methylphenoxyacetic acid within 3 days, faster than that with the model degrader Cupriavidus necator JMP134. Particularly, X32 still functioned at pH 10.5. Of note, with X32 inoculation, we observed 2,4-D degradation in soils and diminished phytotoxicity to maize (Zea mays). Furthermore, potential mechanisms underlying PAA biodegradation and alkali tolerance were then analyzed by whole-genome sequencing. Three modules of tfd gene clusters involved in 2,4-D catabolism and genes encoding monovalent cation/proton antiporters involved in alkali tolerance were putatively identified. Thus, X32 could be a promising candidate for the bioremediation of PAA-contaminated sites, especially in alkaline surroundings.

ХРОМОСОМНАЯ ЛОКАЛИЗАЦИЯ ГЕНОВ КАТАБОЛИЗМА ХЛОРФЕНОКСИУКСУСНЫХ КИСЛОТ ШТАММА PSEUDOMONAS AERUGINOSA 21SG

A small plasmid pPA21SG with a size of 6.2 kb was isolated from chlorophenoxyacetic acids-degrading strain Pseudomonas aeruginosa 21SG. The chromosomal location of the 4-CPA, 2,4-D and 2,4,5-T degradation genes was determined by the elimination of plasmid from 21SG strain with ethidium bromide (500 μg/ml). A PCRbased analysis revealed the absence in genome of 21SG strain the tfdA and tftA genes, that responsible for the initiation steps of 2,4-D and 2,4,5-T degradation by strains Cupriavidus necator JMP134 (pJP4) и Burkholderia phenoliruptrix АС1100.

Siderophore-Mediated Iron Acquisition Enhances Resistance to Oxidative and Aromatic Compound Stress in Cupriavidus necator JMP134.

Many bacteria secrete siderophores to enhance iron uptake under iron-restricted conditions. In this study, we found that Cupriavidus necator JMP134, a well-known aromatic pollutant-degrading bacterium, produces an unknown carboxylate-type siderophore named cupriabactin to overcome iron limitation. Using genome mining, targeted mutagenesis, and biochemical analysis, we discovered an operon containing six open reading frames (cubA-F) in the C. necator JMP134 genome that encodes proteins required for the biosynthesis and uptake of cupriabactin. As the dominant siderophore of C. necator JMP134, cupriabactin promotes the growth of C. necator JMP134 under iron-limited conditions via enhanced ferric iron uptake. Furthermore, we demonstrated that the iron concentration-dependent expression of the cub operon is mediated by the ferric uptake regulator (Fur). Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and resistance to oxidative and aromatic compound stress in C. necator JMP134. In conclusion, we identified a carboxylate-type siderophore named cupriabactin, which plays important roles in iron scavenging, bacterial motility, biofilm formation, and stress resistance.IMPORTANCE Since siderophores have been widely exploited for agricultural, environmental, and medical applications, the identification and characterization of new siderophores from different habitats and organisms will have great beneficial applications. Here, we identified a novel siderophore-producing gene cluster in C. necator JMP134. This gene cluster produces a previously unknown carboxylate siderophore, cupriabactin. Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and oxidative stress resistance. Most notably, this system also plays important roles in increasing the resistance of C. necator JMP134 to stress caused by aromatic compounds, which provide a promising strategy to engineer more efficient approaches to degrade aromatic pollutants.

Rapid Biodegradation of the Herbicide 2,4-Dichlorophenoxyacetic Acid by Cupriavidus gilardii T-1.

Phytotoxicity and environmental pollution of residual herbicides have caused much public concern during the past several decades. An indigenous bacterial strain capable of degrading 2,4-dichlorophenoxyacetic acid (2,4-D), designated T-1, was isolated from soybean field soil and identified as Cupriavidus gilardii. Strain T-1 degraded 2,4-D 3.39 times more rapidly than the model strain Cupriavidus necator JMP134. T-1 could also efficiently degrade 2-methyl-4-chlorophenoxyacetic acid (MCPA), MCPA isooctyl ester, and 2-(2,4-dichlorophenoxy)propionic acid (2,4-DP). Suitable conditions for 2,4-D degradation were pH 7.0-9.0, 37-42 °C, and 4.0 mL of inoculums. Degradation of 2,4-D was concentration-dependent. 2,4-D was degraded to 2,4-dichlorophenol (2,4-DCP) by cleavage of the ether bond and then to 3,5-dichlorocatechol (3,5-DCC) via hydroxylation, followed by ortho-cleavage to cis-2-dichlorodiene lactone (CDL). The metabolites 2,4-DCP or 3,5-DCC at 10 mg L-1 were completely degraded within 16 h. Fast degradation of 2,4-D and its analogues highlights the potential for use of C.gilardii T-1 in bioremediation of phenoxyalkanoic acid herbicides.

Simultaneous biodegradation of three mononitrophenol isomers by a tailor-made microbial consortium immobilized in sequential batch reactors.

Nitroaromatic compounds are readily spread in the environment and pose great potential toxicity concerns. Here, we report the simultaneous degradation of three isomers of mononitrophenol in a single system by employing a consortium of three bacteria, both in flasks and lab-scale sequential batch reactors. The results demonstrate that simultaneous biodegradation of three mononitrophenol isomers can be achieved by a tailor-made microbial consortium immobilized in sequential batch reactors, providing a pilot study for a novel approach for the bioremediation of mixed pollutants, especially isomers present in wastewater.

Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum.

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 μM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol.

Construction of an engineered strain capable of degrading two isomeric nitrophenols via a sacB- and gfp-based markerless integration system

In this study, a gfp-based novel markerless allelic exchange integration system was developed. By employing gfp gene and sacB gene as counter-selectable markers, an ortho-nitrophenol degradation operon (onpABC gene cluster) was successfully inserted into the chromosome of meta-nitrophenol utilizer Cupriavidus necator JMP134. Through two rounds of recombination, the engineered strain (strain JMP134-ONP) was directly selected from the plate by fluorescence screening and has the ability to degrade both ortho-nitrophenol and meta-nitrophenol, simultaneously. This relatively simple and efficient method can be used as an alternative strategy of allelic exchange insertion for the application of metabolic engineering in various bacterial strains, complementary to existing gene knock-in procedures.

Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4

Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100% removal against 200mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0% on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10%, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100% after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95%. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65% on day 76, almost 100% removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.

Bioaugmentation with a consortium of bacterial nitrophenol-degraders for remediation of soil contaminated with three nitrophenol isomers

A consortium consisting of para-nitrophenol utilizer Pseudomonas sp. strain WBC-3, meta-nitrophenol utilizer Cupriavidus necator JMP134 and ortho-nitrophenol utilizer Alcaligenes sp. strain NyZ215 was inoculated into soil contaminated with three nitrophenol isomers for bioaugmentation. Accelerated removal of all nitrophenols was achieved in inoculated soils compared to un-inoculated soils, with complete removal of nitrophenols in inoculated soils occurring between 2 and 16 days. Real-time polymerase chain reaction (PCR) targeting nitrophenol-degradation functional genes indicated that the three strains survived and were stable over the course of the incubation period. The abundance of total indigenous bacteria (measured by 16S rRNA gene real-time PCR) was slightly negatively impacted by the nitrophenol contamination. Denaturing gradient gel electrophoresis profiles of total and group-specific indigenous community suggested a dynamic change in species richness occurred during the bioaugmentation process. Furthermore, Pareto–Lorenz curves and Community organization parameters indicated that the bioaugmentation process had little impact on species evenness within the microbial community.

Effect of glucose on the fatty acid composition of Cupriavidus necator JMP134 during 2,4-dichlorophenoxyacetic acid degradation: implications for lipid-based stable isotope probing methods.

Combining lipid biomarker profiling with stable isotope probing (SIP) is a powerful technique for studying specific microbial populations responsible for the degradation of organic pollutants in various natural environments. However, the presence of other easily degradable substrates may induce significant physiological changes by altering both the rate of incorporation of the target compound into the biomass and the microbial lipid profiles. In order to test this hypothesis, Cupriavidus necator JMP134, a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium, was incubated with [(13)C]2,4-D, [(13)C]glucose, or mixtures of both substrates alternatively labeled with (13)C. C. necator JMP134 exhibited a preferential use of 2,4-D over glucose. The isotopic analysis showed that glucose had only a small effect on the incorporation of the acetic chain of 2,4-D into the biomass (at days 2 and 3) and no effect on that of the benzenic ring. The addition of glucose did change the fatty acid methyl ester (FAME) composition. However, the overall FAME isotopic signature reflected that of the entire biomass. Compound-specific individual isotopic analyses of FAME composition showed that the (13)C-enriched FAME profiles were slightly or not affected when tracing the 2,4-D acetic chain or 2,4-D benzenic ring, respectively. This batch study is a necessary step for validating the use of lipid-based SIP methods in complex environments.

Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase

Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The "furfural reductase" (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD(+) as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power.

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD(+) to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K ( d ) of 0.043μM for the FurX-NADH complex) and relatively low affinities for NAD(+) (e.g., K ( d ) of 87μM for FurX-NAD(+)). The kinetic data suggest that the four enzymes are efficient "furfural reductases" with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°') for ethanol-dependent reduction of furfural was determined to be -1.1kJmol(-1). The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.

Strict and direct transcriptional repression of the pobA gene by benzoate avoids 4‐hydroxybenzoate degradation in the pollutant degrader bacterium Cupriavidus necator JMP134

As other environmental bacteria, Cupriavidus necator JMP134 uses benzoate as preferred substrate in mixtures with 4-hydroxybenzoate, strongly inhibiting its degradation. The mechanism underlying this hierarchical use was studied. A C. necator benA mutant, defective in the first step of benzoate degradation, is unable to metabolize 4-hydroxybenzoate when benzoate is also included in the medium, indicating that this substrate and not one of its catabolic intermediates is directly triggering repression. Reverse transcription polymerase chain reaction analysis revealed that 4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and a fusion of pobA promoter to lacZ reporter confirmed that benzoate drastically decreases the transcription of this gene. Expression of pobA driven by a heterologous promoter in C. necator benA mutant, allows growth on 4-hydroxybenzoate in presence of benzoate, overcoming its repressive effect. In contrast with other bacteria, regulators of benzoate catabolism do not participate in repression of 4-hydroxybenzoate degradation. Moreover, the effect of benzoate on pobA promoter can be observed in heterologous strains with the sole presence of PobR, the transcriptional activator of pobA gene, indicating that PobR is enough to fully reproduce the phenomenon. This novel mechanism for benzoate repression is probably mediated by direct action of benzoate over PobR.

Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as a sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that the mnp cluster might have played important roles in MNP degradation. In order to experimentally confirm the prediction, we have now characterized mnpA-encoded meta-nitrophenol nitroreductase involved in the initial reaction of MNP degradation. Real-time PCR analysis indicated that mnpA played an essential role in MNP degradation. MnpA was purified to homogeneity as His-tagged proteins and was considered to be a dimer as determined by gel filtration. MnpA was an MNP nitroreductase with a tightly bound flavin mononucleotide (FMN), catalyzing the partial reduction of MNP to meta-hydroxylaminophenol via meta-nitrosophenol in the presence of NADPH and oxygen. The accumulation of meta-nitrosophenol was confirmed with the results of liquid chromatography-diode array detection and time-of-flight mass spectrometry for the first time. The low K (m) and high k (cat) of MnpA as well as MNP-inducible transcription of mnpA suggested that MNP was the physiological substrate for this nitroreductase. In addition, the phylogenetic analysis revealed that nitroreductases of known physiological function including MnpA constituted a new clade in the nitro-FMN-reductase superfamily.

Characterization of MnpC, a Hydroquinone Dioxygenase Likely Involved in the meta-Nitrophenol Degradation by Cupriavidus necator JMP134

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as the sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that MnpC might have played an important role in MNP degradation. In order to experimentally confirm the prediction, we have now characterized the mnpC-encoded (amino)hydroquinone dioxygenase involved in the ring-cleavage reaction of MNP degradation. Real-time PCR analysis indicated that mnpC played an essential role in MNP degradation. MnpC was purified to homogeneity as an N-terminal six-His-tagged fusion protein, and it was proved to be a dimer as demonstrated by gel filtration. MnpC was a Fe(2+)- and Mn(2+)-dependent dioxygenase, catalyzing the ring-cleavage of hydroquinone to 4-hydroxymuconic semialdehyde in vitro and proposed as an aminohydroquinone dioxygenase involved in MNP degradation in vivo. Phylogenetic analysis suggested that MnpC diverged from the other (chloro)hydroquinone dioxygenases at an earlier point, which might result in the preference for its physiological substrate.

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 FindingsIts 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/SignificanceThe 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.

Genuine genetic redundancy in maleylacetate-reductase-encoding genes involved in degradation of haloaromatic compounds by Cupriavidus necator JMP134

Maleylacetate reductases (MAR) are required for biodegradation of several substituted aromatic compounds. To date, the functionality of two MAR-encoding genes (tfdF(I) and tfdF(II)) has been reported in Cupriavidus necator JMP134(pJP4), a known degrader of aromatic compounds. These two genes are located in tfd gene clusters involved in the turnover of 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-CB). The C. necator JMP134 genome comprises at least three other genes that putatively encode MAR (tcpD, hqoD and hxqD), but confirmation of their functionality and their role in the catabolism of haloaromatic compounds has not been assessed. RT-PCR expression analyses of C. necator JMP134 cells exposed to 2,4-D, 3-CB, 2,4,6-trichlorophenol (2,4,6-TCP) or 4-fluorobenzoate (4-FB) showed that tfdF(I) and tfdF(II) are induced by haloaromatics channelled to halocatechols as intermediates. In contrast, 2,4,6-TCP only induces tcpD, and any haloaromatic compounds tested did not induce hxqD and hqoD. However, the tcpD, hxqD and hqoD gene products showed MAR activity in cell extracts and provided the MAR function for 2,4-D catabolism when heterologously expressed in MAR-lacking strains. Growth tests for mutants of the five MAR-encoding genes in strain JMP134 showed that none of these genes is essential for degradation of the tested compounds. However, the role of tfdF(I)/tfdF(II) and tcpD genes in the expression of MAR activity during catabolism of 2,4-D and 2,4,6-TCP, respectively, was confirmed by enzyme activity tests in mutants. These results reveal a striking example of genetic redundancy in the degradation of aromatic compounds.

3-Chlorobenzoate is taken up by a chromosomally encoded transport system in Cupriavidus necator JMP134

Cupriavidus necator JMP134(pJP4) is able to grow on 3-chlorobenzoate (3-CB), a model chloroaromatic pollutant. Catabolism of 3-CB is achieved via the expression of the chromosomally encoded benABCD genes and the tfd genes from plasmid pJP4. Since passive diffusion of benzoic acid derivatives at physiological pH is negligible, the uptake of this compound should be facilitated by a transport system. However, no transporter has so far been described to perform this function, and identification of chloroaromatic compound transporters has been limited. In this work, uptake experiments using 3-[ring-UL-(14)C]CB showed an inducible transport system in strain JMP134, whose expression is activated by 3-CB and benzoate. A similar level of 3-CB uptake was found for a mutant strain of JMP134, defective in chlorobenzoate degradation, indicating that metabolic drag is not an important component of the measured uptake rate. Competitive inhibitor assays showed that uptake of 3-CB was inhibited by benzoate and, to a lesser degree, by 3-CB and 3,5-dichlorobenzoate, but not by any of 12 other substituted benzoates tested. The expression of several gene candidates for this transport function was analysed by RT-PCR, including both permease-type and ABC-type ATP-dependent transporters. Induction of a chromosomally encoded putative permease transporter (benP gene) was found specifically in the presence of 3-CB or benzoate. A benP knockout mutant of strain JMP134 displayed an almost complete loss of 3-CB transport activity. This is to our knowledge the first report of a 3-CB transporter.

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacteriumCupriavidus necatorJMP134

Cupriavidus necator JMP134 is a model for chloroaromatics biodegradation, capable of mineralizing 2,4-D, halobenzoates, chlorophenols and nitrophenols, among other aromatic compounds. We performed the metabolic reconstruction of aromatics degradation, linking the catabolic abilities predicted in silico from the complete genome sequence with the range of compounds that support growth of this bacterium. Of the 140 aromatic compounds tested, 60 serve as a sole carbon and energy source for this strain, strongly correlating with those catabolic abilities predicted from genomic data. Almost all the main ring-cleavage pathways for aromatic compounds are found in C. necator: the beta-ketoadipate pathway, with its catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage branches; the (methyl)catechol meta ring-cleavage pathway; the gentisate pathway; the homogentisate pathway; the 2,3-dihydroxyphenylpropionate pathway; the (chloro)hydroxyquinol pathway; the (amino)hydroquinone pathway; the phenylacetyl-CoA pathway; the 2-aminobenzoyl-CoA pathway; the benzoyl-CoA pathway and the 3-hydroxyanthranilate pathway. A broad spectrum of peripheral reactions channel substituted aromatics into these ring cleavage pathways. Gene redundancy seems to play a significant role in the catabolic potential of this bacterium. The literature on the biochemistry and genetics of aromatic compounds degradation is reviewed based on the genomic data. The findings on aromatic compounds biodegradation in C. necator reviewed here can easily be extrapolated to other environmentally relevant bacteria, whose genomes also possess a significant proportion of catabolic genes.