Burkholderia Xenovorans LB400 Research Articles

Micro-synteny conservation analysis revealed the evolutionary history of bacterial biphenyl degradation pathway.

Phenolic compounds have been enlisted by the United States Environmental Protection Agency (USEPA) and the European Union (EU) as pollutants of priority concern. The biphenyl degradation pathway plays an essential role in prokaryote polychlorinated biphenyls degradation. Our understanding of prokaryotic pathways and their evolution has dramatically increased in recent years with the advancements in prokaryotic genome sequencing and analysis tools. In this work, we applied bioinformatics tools to study the evolution of the biphenyl degradation pathway focusing on the phylogeny and initiation of four representative species (Burkholderia xenovorans LB400, Polaromonas naphthalenivorans CJ2, Pseudomonas putida F1 and Rhodococcus jostii RHA1). These species contained partial or full concatenated genes from bph gene cluster (i.e. bphRbphA1A2A3A4BCKHJID). The aim was to establish this pathway's origin and development mode in the prokaryotic world. Genomic screening revealed that many bacterial species possess genes for the biphenyl degradation pathway. However, the micro-synteny conservation analysis indicated that massive gene recruitment events might have occurred during the evolution of the biphenyl degradation pathway. Combining with the phylogenetic positions, this work points to the evolutionary process of acquiring the biphenyl degradation pathway by different fragments through horizontal gene transfer in these bacterial groups. This study reports the first-ever evidence of the birth of this pathway in the represented species.

Augmentation of an Engineered Bacterial Strain Potentially Improves the Cleanup of PCB Water Pollution.

ABSTRACTPolychlorinated biphenyls (PCBs) are recalcitrant organohalide pollutants, consisting of 209 congeners. PCB cleanup in natural landscapes is expected to be achieved by the metabolic activity of microorganisms, but aerobic PCB-degrading bacteria that inhabit sites polluted by PCBs cannot degrade all PCB congeners due to the specificity of their enzymes. In this study, we investigated the degradability of PCBs when a genetically modified PCB-degrading bacterium was compounded with wild-type PCB-degrading bacteria. We used two bacterial strains, Comamonas testosteroni YAZ2 isolated from a PCB-uncontaminated natural landscape and Escherichia coli BL21(DE3) transformed with a biphenyl dioxygenase (BphA) gene from a well-known PCB degrader, Burkholderia xenovorans LB400. The enzymatic specificities of BphA were 2,3-dioxygenation in the YAZ2 and 2,3- and 3,4-dioxygenations in the recombinant E. coli. For the PCB-degrading experiment, a dedicated bioreactor capable of generating oxygen microbubbles was prototyped and used. The combined cells of the recombinant and the wild-type strains with an appropriate composite ratio degraded 40 mg/L of Kaneclor KC-300 to 0.3 ± 0.1 mg/L within 24 h. All of the health-toxic coplanar PCB congeners in KC-300 were degraded. This study suggested that the augmentation of an engineered bacterial strain could improve the cleanup of PCB water pollution. It also revealed the importance of the ratio of the strains with different PCB-degrading profiles to efficient degradation and that the application of oxygen microbubbles could rapidly accelerate the cleanup.IMPORTANCE PCB cleanup technique in a natural environment relies on the use of enzymes from microorganisms, primarily biphenyl dioxygenase and dehalogenase. Herein, we focused on biphenyl dioxygenase and created a recombinant PCB-degrading E. coli strain. Despite the development of environments for the field use of transgenic microbial strains around the world, verification of the applicability of transgenic microbial strains for PCB cleanup in the field has not yet been reported. We tentatively verified the extent to which degradability could be obtained by an augmentation model of a transgenic strain, the enzyme expression of which is easily regulated in rivers and lakes with PCB pollution. Our experiments used a dedicated bioreactor to model the natural landscape and produced results superior to those of bioremediation or biostimulation methods. The application of micro-nano bubbles, which has recently been discussed, to the cleanup of environmental pollution was also found to be useful in this study.

Remediation of Organically Contaminated Soil Through the Combination of Assisted Phytoremediation and Bioaugmentation

Here, we aimed to bioremediate organically contaminated soil with Brassica napus and a bacterial consortium. The bioaugmentation consortium consisted of four endophyte strains that showed plant growth-promoting traits (three Pseudomonas and one Microbacterium) plus three strains with the capacity to degrade organic compounds (Burkholderia xenovorans LB400, Paenibacillus sp. and Lysinibacillus sp.). The organically contaminated soil was supplemented with rhamnolipid biosurfactant and sodium dodecyl benzenesulfonate to increase the degradability of the sorbed contaminants. Soils were treated with organic amendments (composted horse manure vs. dried cow slurry) to promote plant growth and stimulate soil microbial activity. Apart from quantification of the expected decrease in contaminant concentrations (total petroleum hydrocarbons, polycyclic aromatic hydrocarbons), the effectiveness of our approach was assessed in terms of the recovery of soil health, as reflected by the values of different microbial indicators of soil health. Although the applied treatments did not achieve a significant decrease in contaminant concentrations, a significant improvement of soil health was observed in our amended soils (especially in soils amended with dried cow slurry), pointing out a not-so-uncommon situation in which remediation efforts fail from the point of view of the reduction in contaminant concentrations while succeeding to recover soil health.

Analysis of the ars gene cluster from highly arsenic-resistant Burkholderia xenovorans LB400.

The Burkholderia xenovorans LB400 multireplicon genome displays a relatively high proportion of redundant genes, including several genes predicted to be related to arsenic resistance. These comprise an ars gene cluster, composed of the arsR3, acr3, arsC1 and arsH genes, as well as two arsB, arsC2, and seven individual arsR genes. The objective of this work was to elucidate the involvement of the ars gene cluster in arsenic resistance by the LB400 strain. Susceptibility tests showed that B. xenovorans LB400 is highly resistant to arsenate and arsenite. Arsenic resistance was induced by prior exposure of LB400 to arsenate or arsenite. reverse transcription-polymerase chain reaction assays using total RNA from LB400 showed arsenite-induced transcription of the arsR3 gene, suggesting that the ars gene cluster constitutes an arsenite-responsive operon. Transfer of cloned LB400 ars genes to heterologous Escherichia coli or Pseudomonas aeruginosa strains demonstrated that the ArsR3 transcriptional repressor, ArsC1 arsenate reductase, and the Acr3 arsenite efflux pump encoded in the LB400 ars gene cluster, are all associated to the arsenic resistance phenotype of this strain. The ars gene cluster from Burkholderia xenovorans LB400 is responsible for the inducible arsenic-resistance phenotype of the bacterium.

Linkage of the Nit1C gene cluster to bacterial cyanide assimilation as a nitrogen source.

A genetic linkage between a conserved gene cluster (Nit1C) and the ability of bacteria to utilize cyanide as the sole nitrogen source was demonstrated for nine different bacterial species. These included three strains whose cyanide nutritional ability has formerly been documented (Pseudomonas fluorescens Pf11764, Pseudomonas putida BCN3 and Klebsiella pneumoniae BCN33), and six not previously known to have this ability [Burkholderia (Paraburkholderia) xenovorans LB400, Paraburkholderia phymatum STM815, Paraburkholderia phytofirmans PsJN, Cupriavidus (Ralstonia) eutropha H16, Gluconoacetobacter diazotrophicus PA1 5 and Methylobacterium extorquens AM1]. For all bacteria, growth on or exposure to cyanide led to the induction of the canonical nitrilase (NitC) linked to the gene cluster, and in the case of Pf11764 in particular, transcript levels of cluster genes (nitBCDEFGH) were raised, and a nitC knock-out mutant failed to grow. Further studies demonstrated that the highly conserved nitB gene product was also significantly elevated. Collectively, these findings provide strong evidence for a genetic linkage between Nit1C and bacterial growth on cyanide, supporting use of the term cyanotrophy in describing what may represent a new nutritional paradigm in microbiology. A broader search of Nit1C genes in presently available genomes revealed its presence in 270 different bacteria, all contained within the domain Bacteria, including Gram-positive Firmicutes and Actinobacteria, and Gram-negative Proteobacteria and Cyanobacteria. Absence of the cluster in the Archaea is congruent with events that may have led to the inception of Nit1C occurring coincidentally with the first appearance of cyanogenic species on Earth, dating back 400-500 million years.

Burkholderia xenovorans LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the pha genes and synthesizes poly(3-hydroxybutyrate) undernitrogen-limiting conditions.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located atchromosome 1 of strain LB400.During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40 ± 0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.

Nano/bio treatment of polychlorinated biphenyls with evaluation of comparative toxicity

The persistence of polychlorinated biphenyl (PCB) Aroclor 1248 in soils and sediments is a major concern because of its toxicity and presence at high concentrations. In this study, we developed an integrated remediation system for PCBs using chemical catalysis and biodegradation. The dechlorination of Aroclor 1248 was achieved by treatment with bimetallic nanoparticles Pd/nFe under anoxic conditions. Among the 32 PCB congeners of Aroclor 1248 examined, our process dechlorinated 99%, 92%, 84%, and 28% of tri-, tetra-, penta-, and hexachlorinated biphenyls, respectively. The resulting biphenyl was biodegraded rapidly by Burkholderia xenovorans LB400. Benzoic acid was detected as an intermediate during the biodegradation process. The toxicity of the residual PCBs after nano-bio treatment was evaluated in terms of toxic equivalent values which decreased from 33.8×10−5μgg−1 to 9.5×10−5μgg−1. The residual PCBs also had low cytotoxicity toward Escherichia coli as demonstrated by lower reactive oxygen species levels, lower glutathione peroxidase activity, and a reduced number of dead bacteria.

Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7(T) (AcdDPN7) during degradation of 3,3'-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). AcdDPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. AcdS110, AcdB4, AcdH16, and AcdKT2440 were able to dehydrogenate isobutyryl-CoA. AcdKT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas AcdDSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only AcdTBEA6, AcdN-1, and AcdLB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for AcdBL21 and AcdWT001. Spectrophotometric assays provided apparent Km and Vmax values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of uncharacterized acyl-CoA dehydrogenases. Furthermore, C. necator N-1 was found to utilize 3SP as the sole source of carbon and energy.

Genomic and Functional Analyses of the 2-Aminophenol Catabolic Pathway and Partial Conversion of Its Substrate into Picolinic Acid in Burkholderia xenovorans LB400

2-aminophenol (2-AP) is a toxic nitrogen-containing aromatic pollutant. Burkholderia xenovorans LB400 possess an amn gene cluster that encodes the 2-AP catabolic pathway. In this report, the functionality of the 2-aminophenol pathway of B. xenovorans strain LB400 was analyzed. The amnRJBACDFEHG cluster located at chromosome 1 encodes the enzymes for the degradation of 2-aminophenol. The absence of habA and habB genes in LB400 genome correlates with its no growth on nitrobenzene. RT-PCR analyses in strain LB400 showed the co-expression of amnJB, amnBAC, amnACD, amnDFE and amnEHG genes, suggesting that the amn cluster is an operon. RT-qPCR showed that the amnB gene expression was highly induced by 2-AP, whereas a basal constitutive expression was observed in glucose, indicating that these amn genes are regulated. We propose that the predicted MarR-type transcriptional regulator encoded by the amnR gene acts as repressor of the amn gene cluster using a MarR-type regulatory binding sequence. This report showed that LB400 resting cells degrade completely 2-AP. The amn gene cluster from strain LB400 is highly identical to the amn gene cluster from P. knackmussi strain B13, which could not grow on 2-AP. However, we demonstrate that B. xenovorans LB400 is able to grow using 2-AP as sole nitrogen source and glucose as sole carbon source. An amnBA − mutant of strain LB400 was unable to grow with 2-AP as nitrogen source and glucose as carbon source and to degrade 2-AP. This study showed that during LB400 growth on 2-AP this substrate was partially converted into picolinic acid (PA), a well-known antibiotic. The addition of PA at lag or mid-exponential phase inhibited LB400 growth. The MIC of PA for strain LB400 is 2 mM. Overall, these results demonstrate that B. xenovorans strain LB400 posses a functional 2-AP catabolic central pathway, which could lead to the production of picolinic acid.

Effects of growth substrate on triclosan biodegradation potential of oxygenase-expressing bacteria

Triclosan is an antimicrobial agent, an endocrine disrupting compound, and an emerging contaminant in the environment. This is the first study investigating triclosan biodegradation potential of four oxygenase-expressing bacteria: Rhodococcus jostii RHA1, Mycobacterium vaccae JOB5, Rhodococcus ruber ENV425, and Burkholderia xenovorans LB400. B. xenovorans LB400 and R. ruber ENV425 were unable to degrade triclosan. Propane-grown M. vaccae JOB5 can completely degrade triclosan (5mgL−1). R. jostii RHA1 grown on biphenyl, propane, and LB medium with dicyclopropylketone (DCPK), an alkane monooxygenase inducer, was able to degrade the added triclosan (5mgL−1) to different extents. Incomplete degradation of triclosan by RHA1 is probably due to triclosan product toxicity. The highest triclosan transformation capacity (Tc, defined as the amount of triclosan degraded/the number of cells inactivated; 5.63×10−3ng triclosan/16S rRNA gene copies) was observed for biphenyl-grown RHA1 and the lowest Tc (0.20×10−3ng-triclosan/16S rRNA gene copies) was observed for propane-grown RHA1. No triclosan degradation metabolites were detected during triclosan degradation by propane- and LB+DCPK-grown RHA1. When using biphenyl-grown RHA1 for degradation, four chlorinated metabolites (2,4-dichlorophenol, monohydroxy-triclosan, dihydroxy-triclosan, and 2-chlorohydroquinone (a new triclosan metabolite)) were detected. Based on the detected metabolites, a meta-cleavage pathway was proposed for triclosan degradation.

Identification and In-vivo Characterization of a Novel OhrR Transcriptional Regulator in Burkholderia xenovorans LB400

Transcriptional regulators (TRs) are an important and versatile group of proteins, yet very little progress has been achieved towards the discovery and annotation of their biological functions. We have characterized a previously unknown organic hydroperoxide resistance regulator from Burkholderia xenovoransLB400, Bxe_B2842, which is homologous to E. coli’s OhrR. Bxe_B2842 regulates the expression of an organic hydroperoxide resistance protein (OsmC). We utilized frontal affinity chromatography coupled with mass spectrometry (FAC-MS) and electrophoretic mobility gel shift assays (EMSA) to identify and characterize the possible effectors of the regulation by Bxe_B2842. Without an effector, Bxe_B2842 binds a DNA operator sequence (DOS) upstream of osmC. FAC-MS results suggest that 2-aminophenol binds to the protein and is potentially an effector molecule. EMSA analysis shows that 2-aminophenol attenuates the Bxe_B2842’s affinity for its DOS. EMSA analysis also shows that organic peroxides attenuate Bxe_B2842/DOS affinity, suggesting that binding of the TR to its DOS is regulated by the two-cysteine mechanism, common to TRs in this family. Bxe_B2842 is the first OhrR TR to have both oxidative and effector-binding mechanisms of regulation. This paper reveals further mechanistic diversity TR mediated gene regulation and provides insights into methods for function discovery of TRs.

Enhanced degradation of haloacid by heterologous expression in related Burkholderia species

Haloacids are environmental pollutant and can be transformed to non-toxic alkanoic acids by microbial dehalogenase. Bacterium Burkholderia species MBA4 was enriched from soil for its ability to bioremediate haloacids such as mono-chloroacetate (MCA), mono-bromoacetate (MBA), 2-mono-chloropropionate, and 2-mono-bromopropionate. MBA4 produces an inducible dehalogenase Deh4a that catalyzes the dehalogenation process. The growth of MBA4 on haloacid also relies on the presence of a haloacid-uptake system. Similar dehalogenase genes can be found in the genome of many related species. However, wildtype Burkholderia caribensis MWAP64, Burkholderia phymatum STM815, and Burkholderia xenovorans LB400 were not able to grow on MCA. When a plasmid containing the regulatory and structural gene of Deh4a was transformed to these species, they were able to grow on haloacid. The specific enzyme activities in these recombinants ranges from 2- to 30-fold that of MBA4 in similar condition. Reverse transcription-quantitative real-time PCR showed that the relative transcript levels in these recombinant strains ranges from 9 to over 1,600 times that of MBA4 in similar condition. A recombinant has produced nearly five times of dehalogenase that MBA4 could ever achieve. While the expressions of Deh4a were more relaxed in these phylogenetically related species, an MCA-uptake activity was found to be inducible. These metabolically engineered strains are better degraders than the haloacid-enriched MBA4.

Transformation of hydroxylated derivatives of 2,5-dichlorobiphenyl and 2,4,6-trichlorobiphenyl by Burkholderia xenovorans LB400

The polychlorinated biphenyl (PCB)-degrading bacterium, Burkholderia xenovorans LB400, was capable of transforming three hydroxylated derivatives of 2,5-dichlorobiphenyl (2,5-DCB) (2'-hydroxy- (2'-OH-), 3'-OH-, and 4'-OH-2,5-DCB) when biphenyl was used as the carbon source (i.e., biphenyl pathway-inducing condition), although only 2'-OH-2,5-DCB was transformed when the bacterium was growing on succinate (i.e., condition non-inductive of the biphenyl pathway). On the contrary, hydroyxlated derivatives of 2,4,6-trichlorobiphenyl (2,4,6-TCB) (2'-OH-, 3'-OH-, and 4'-OH-2,4,6-TCB) were not significantly transformed by B. xenovorans LB400, regardless of the carbon source used. Gene expression analyses showed a clear correlation between the transformation of OH-2,5-DCBs and expression of genes of the biphenyl pathway. The PCB metabolite, 2,5-dichlorobenzoic acid (2,5-DCBA), was produced following the transformation of OH-2,5-DCBs. 2,5-DCBA was not further transformed by B. xenovorans LB400. The present study is significant because it provides evidence that PCB-degrading bacteria are capable of transforming hydroxylated derivatives of PCBs, which are increasingly considered as a new class of environmental contaminants.

Remediation of Polychlorinated Biphenyl Impacted Sediment by Concurrent Bioaugmentation with Anaerobic Halorespiring and Aerobic Degrading Bacteria

Bioremediation of sediments contaminated with commercial polychlorinated biphenyls (PCBs) is potentially achievable by the sequential activity of anaerobic halorespiration to convert higher chlorinated congeners to less chlorinated congeners that are susceptible to aerobic respiratory degradation. The efficacy of bioaugmentation with anaerobic halorespiring Dehalobium chlorocoercia DF1 and aerobic Burkholderia xenovorans LB400 added concurrently with granulated activated carbon (GAC) as a delivery system was determined in 2 L laboratory mesocosms containing weathered Aroclor-contaminated sediment from Baltimore Harbor, MD, USA. The greatest effect was seen in the mesocosm bioaugmented with both DF1 and LB400 together, which resulted in an 80% decrease by mass of PCBs, from 8 to <2 mg/kg after 120 days. There was no significant increase in lesser-chlorinated congeners, indicating that both anaerobic dechlorination by DF1 and aerobic degradation by LB400 occurred. In contrast, nonbioaugmented controls containing filtered culture supernatant showed only a 25% decrease in total levels of PCBs after 365 days, which was likely due to biostimulation of the indigenous population by the medium. Direct colony counts and molecular analysis targeting a putative reductive dehalogenase gene of D. chlorocoercia or the bphA gene of LB400 showed the presence of viable DF1 and LB400 in bioaugmented mesocosms after 365 days, indicating that both nonindigenous strains were sustainable within the indigenous microbial community. These results suggest that an in situ treatment employing the simultaneous application of anaerobic and aerobic microorganisms could be an effective and environmentally sustainable strategy to reduce PCBs levels in contaminated sediment.

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.

Triclosan susceptibility and co-metabolism – A comparison for three aerobic pollutant-degrading bacteria

The antimicrobial agent triclosan is an emerging and persistent environmental pollutant. This study evaluated the susceptibility and biodegradation potential of triclosan by three bacterial strains (Sphingomonas wittichii RW1, Burkholderia xenovorans LB400 and Sphingomonas sp. PH-07) that are able to degrade aromatic pollutants (dibenzofuran, biphenyl and diphenyl ether, respectively) with structural similarities to triclosan. These strains showed less susceptibility to triclosan when grown in complex and mineral salts media. Biodegradation experiments revealed that only strain PH-07 was able to catabolize triclosan to intermediates that included hydroxylated compounds (monohydroxy-triclosan, and dihydroxy-triclosan) and the ether bond cleavage products (4-chlorophenol and 2,4-dichlorophenol), indicating that the initial dihydroxylation occurred on both aromatic rings of triclosan. Additional growth inhibition tests demonstrated that the main intermediate, 2,4-dichlorophenol, was less toxic to strain PH-07 than was triclosan. Our results indicate that ether bond cleavage might be the primary mechanism of avoiding triclosan toxicity by this strain.

Biochemical and Structural Characterization of the Paralogous Benzoate CoA Ligases from Burkholderia xenovorans LB400: Defining the Entry Point into the Novel Benzoate Oxidation ( box) Pathway

Xenobiotic aromatic compounds represent one of the most significant classes of environmental pollutants. A novel benzoate oxidation ( box) pathway has been identified recently in Burkholderia xenovorans LB400 (referred to simply as LB400) that is capable of assimilating benzoate and intimately tied to the degradation of polychlorinated biphenyls (PCBs). The box pathway in LB400 is present in two paralogous copies ( box M and box C) and encodes eight enzymes with the first committed step catalyzed by benzoate CoA ligase (BCL). As a first step towards delineating the biochemical role of the box pathway in LB400, we have carried out functional studies of the paralogous BCL enzymes (BCL M and BCL C) with 20 different putative substrates. We have established a structural rationale for the observed substrate specificities on the basis of a 1.84 Å crystal structure of BCL M in complex with benzoate. These data show that, while BCL M and BCL C display similar overall substrate specificities, BCL M is significantly more active towards benzoate and 2-aminobenzoate with tighter binding ( K m) and a faster reaction rate ( V max). Despite these clear functional differences, the residues that define the substrate-binding site in BCL M are completely conserved in BCL C, suggesting that second shell residues may play a significant role in substrate recognition and catalysis. Furthermore, comparison of the active site of BCL M with the recently solved structures of 4-chlorobenzoate CoA ligase and 2, 3-dihydroxybenzoate CoA ligase offers additional insight into the molecular features that mediate substrate binding in adenylate-forming enzymes. This study provides the first biochemical characterization of a Box enzyme from LB400 and the first structural characterization of a Box enzyme from any organism, and further substantiates the concept of distinct roles for the two paralogous box pathways in LB400.

A large gene cluster in Burkholderia xenovorans encoding abietane diterpenoid catabolism.

Abietane diterpenoids are defense compounds synthesized by trees that are abundant in natural environments and occur as significant pollutants from pulp and paper production. Burkholderia xenovorans LB400 has diverse catabolic capabilities and represents an important group of heterotrophic bacteria in soil environments. The genome sequence of LB400 revealed homologs of the dit genes of Pseudomonas abietaniphila BKME-9, which encode abietane diterpenoid degradation. LB400 grew on abietic acid (AbA), dehydroabietic acid (DhA), palustric acid (PaA), and 7-oxo-DhA. A Xeotron microarray set, with probes for 8450 of the estimated 9000 LB400 genes, was used to compare the transcriptomes of LB400 growing on DhA versus on succinate. On DhA, 97 genes were upregulated, 43 of which were within an 80-kb cluster located on the 1.47-Mbp megaplasmid of LB400. Upregulated genes in this cluster encode a permease, a ring-hydroxylating dioxygenase system (DitA), a ring-cleavage dioxygenase (DitC), a P450 monooxygenase (DitQ), and enzymes catalyzing beta-oxidation-type reactions. Disruption of the ditA1 gene, encoding the alpha-subunit of DitA, abolished growth on these abietanes. Analyses of the metabolism of abietanes by cell suspensions of wild-type LB400 and the ditA1 mutant indicate a convergent pathway, with 7-oxo-DhA as a common intermediate for ring hydroxylation by DitA. Also, 7-oxo-PaA was identified as a metabolite of both AbA and PaA. Sequence analysis indicates that genes encoding this pathway have been horizontally transferred among Betaproteobacteria and Gammaproteobacteria.

Chlorobenzoate inhibits growth and induces stress proteins in the PCB-degrading bacterium Burkholderia xenovorans 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.

Synthesis of highly hydroxylated aromatics by evolved biphenyl dioxygenase and subsequent dihydrodiol dehydrogenase

The evolved bphA1 (2049) gene, in which nine amino acids from the Pseudomonas pseudoalcaligenes KF707 BphA1 were changed to those from the Burkholderia xenovorans LB400 BphA1 (M247I, H255Q, V258I, G268A, D303E, -313G, S324T, V325I, and T376N), was expressed in Escherichia coli along with the bphA2A3A4 and bphB genes derived from strain KF707. This recombinant E. coli cells converted biphenyl and several heterocyclic aromatic compounds into the highly hydroxylated products such as biphenyl-2,3,2',3'-tetraol (from biphenyl), 2-(2,3-dihydroxyphenyl)benzoxazole-4,5-diol (from 2-phenylbenzoxazole), and 2-(2,5-dihydroxyphenyl)benzoxazole-4,5-diol [from 2-(2-hydroxyphenyl)benzoxazole]. The antioxidative activity of these generated compounds was markedly higher than that of the original substrate used.