p-Cymene Promotes Its Catabolism through the p-Cymene and the p-Cumate Pathways, Activates a Stress Response and Reduces the Biofilm Formation in Burkholderia xenovorans LB400.

p-Cymene is an aromatic terpene that is present in diverse plant species. The aims of this study were to study the p-cymene metabolism in the model aromatic-degrading bacterium Burkholderia xenovorans LB400, and its response to p-cymene. The catabolic p-cymene (cym) and p-cumate (cmt) genes are clustered on the LB400 major chromosome. B. xenovorans LB400 was able to grow on p-cymene as well as on p-cumate as a sole carbon and energy sources. LB400 growth attained higher cell concentration at stationary phase on p-cumate than on p-cymene. The transcription of the key cymAb and cmtAb genes, and p-cumate dioxygenase activity were observed in LB400 cells grown on p-cymene and on p-cumate, but not in glucose-grown cells. Diverse changes on LB400 proteome were observed in p-cymene-grown cells compared to glucose-grown cells. An increase of the molecular chaperones DnaK, GroEL and ClpB, the organic hydroperoxide resistance protein Ohr, the alkyl hydroperoxide reductase AhpC and the copper oxidase CopA during growth on p-cymene strongly suggests that the exposure to p-cymene constitutes a stress condition for strain LB400. Diverse proteins of the energy metabolism such as enolase, pyruvate kinase, aconitase AcnA, succinyl-CoA synthetase beta subunit and ATP synthase beta subunit were induced by p-cymene. Electron microscopy showed that p-cymene-grown cells exhibited fuzzy outer and inner membranes and an increased periplasm. p-Cymene induced diverse membrane and transport proteins including the p-cymene transporter CymD. Biofilm formation was reduced during growth in p-cymene in strain LB400 compared to glucose-grown cells that may be associated with a decrease of diguanylate cyclase protein levels. Overall, these results indicate active p-cymene and p-cumate catabolic pathways in B. xenovorans LB400. In addition, this study showed that p-cymene activated a stress response in strain LB400 and reduced its biofilm formation.

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.

Genomic and functional analyses of the gentisate and protocatechuate ring-cleavage pathways and related 3-hydroxybenzoate and 4-hydroxybenzoate peripheral pathways in Burkholderia xenovorans LB400.

In this study, the gentisate and protocatechuate pathways in Burkholderia xenovorans LB400 were analyzed by genomic and functional approaches, and their role in 3-hydroxybenzoate (3-HBA) and 4-hydroxybenzoate (4-HBA) degradation was proposed. The LB400 genome possesses two identical mhbRTDHI gene clusters encoding the gentisate pathway and one mhbM gene encoding a 3-HBA 6-hydroxylase that converts 3-HBA into gentisate. The pca genes encoding the protocatechuate pathway and the pobA gene encoding the 4-HBA 3-monooxygenase that oxidizes 4-HBA into protocatechuate are arranged in gene clusters and single genes mainly at the minor chromosome, but also at the major chromosome and the megaplasmid. Strain LB400 was able to grow on gentisate, protocatechuate, 3-HBA and 4-HBA. Transcriptional analyses showed that the mhbD gene encoding the gentisate 1,2-dioxygenase was expressed during growth on 3-HBA, 4-HBA and gentisate, whereas the pcaG gene encoding the protocatechuate 3,4-dioxygenase was expressed only during growth on 4-HBA and protocatechuate. The mhbM gene encoding the 3-HBA 6-hydroxylase was transcribed in strain LB400 during growth on HBAs, gentisate, protocatechuate and glucose. The pobA gene encoding the 4-HBA 3-monooxygenase was expressed during growth on HBAs and glucose. 3-HBA- and 4-HBA-grown LB400 cells showed gentisate 1,2-dioxygenase activity, whereas protocatechuate 3,4-dioxygenase activity was observed only in 4-HBA-grown cells. The mhbR gene encoding a MarR-type transcriptional regulator that probably regulates the expression of the MhbT transporter, and the pcaQ and pcaR genes encoding LysR-type transcriptional regulators that regulate pcaHG and pcaIJBDC genes, respectively, were transcribed during growth on both HBAs, gentisate, protocatechuate and glucose, suggesting a basal constitutive expression. The results indicate active gentisate, protocatechuate, 3-HBA and 4-HBA catabolic pathways in B. xenovorans LB400 and suggest that 3-HBA is channeled exclusively through the gentisate route, whereas 4-HBA is funneled into the protocatechuate central pathway and potentially into the gentisate pathway.

Inference system using softcomputing and mixed data applied in metabolic pathway datamining.

This paper describes the development of an inference system used for the identification of genes that encode enzymes of metabolic pathways. Input sequence alignment values are used to classify the best candidate genes for inclusion in a metabolic pathway map. The system workflow allows the user to provide feedback, which is stored in conjunction with analysed sequences for periodic retraining. The construction of the system involved the study of several different classifiers with various topologies, data sets and parameter normalisation data models. Experimental results show an excellent prediction capability with the classifiers trained with mixed data providing the best results.

Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales.

The relevance of the ß-proteobacterial Burkholderiales order in the degradation of a vast array of aromatic compounds, including several priority pollutants, has been largely assumed. In this review, the presence and organization of genes encoding oxygenases involved in aromatics biodegradation in 80 Burkholderiales genomes is analysed. This genomic analysis underscores the impressive catabolic potential of this bacterial lineage, comprising nearly all of the central ring-cleavage pathways reported so far in bacteria and most of the peripheral pathways involved in channelling of a broad diversity of aromatic compounds. The more widespread pathways in Burkholderiales include protocatechuate ortho ring-cleavage, catechol ortho ring-cleavage, homogentisate ring-cleavage and phenylacetyl-CoA ring-cleavage pathways found in at least 60% of genomes analysed. In general, a genus-specific pattern of positional ordering of biodegradative genes is observed in the catabolic clusters of these pathways indicating recent events in its evolutionary history. In addition, a significant bias towards secondary chromosomes, now termed chromids, is observed in the distribution of catabolic genes across multipartite genomes, which is consistent with a genus-specific character. Strains isolated from environmental sources such as soil, rhizosphere, sediment or sludge show a higher content of catabolic genes in their genomes compared with strains isolated from human, animal or plant hosts, but no significant difference is found among Alcaligenaceae, Burkholderiaceae and Comamonadaceae families, indicating that habitat is more of a determinant than phylogenetic origin in shaping aromatic catabolic versatility.

The homogentisate and homoprotocatechuate central pathways are involved in 3- and 4-hydroxyphenylacetate degradation by Burkholderia xenovorans LB400.

BACKGROUND: Genome characterization of the model PCB-degrading bacterium Burkholderia xenovorans LB400 revealed the presence of eleven central pathways for aromatic compounds degradation, among them, the homogentisate and the homoprotocatechuate pathways. However, the functionality of these central pathways in strain LB400 has not been assessed and related peripheral pathways has not been described. METHODOLOGY/PRINCIPAL FINDINGS: The aims of this study were to determine the functionality of the homogentisate and homoprotocatechuate central pathways in B. xenovorans LB400 and to establish their role in 3-hydroxyphenylacetate (3-HPA) and 4-hydroxyphenylacetate (4-HPA) catabolism. Strain LB400 was able to grow using 3-HPA and 4-HPA as sole carbon source. A genomic search in LB400 suggested the presence of mhaAB and hpaBC genes clusters encoding proteins of the 3-hydroxyphenylacetate and 4-hydroxyphenylacetate peripheral pathways. LB400 cells grown with 3-HPA and 4-HPA degraded homogentisate and homoprotocatechuate and showed homogentisate 1,2-dioxygenase and homoprotocatechuate 2,3-dioxygenase activities. Transcriptional analyses by RT-PCR showed the expression of two chromosomally-encoded homogentisate dioxygenases (BxeA2725 and BxeA3900) and the hpaD gene encoding the homoprotocatechuate 2,3-dioxygenase during 3-HPA and 4-HPA degradation. The proteome analyses by two-dimensional polyacrilamide gel electrophoresis of B. xenovorans LB400 grown in 3-HPA and 4-HPA showed the induction of fumarylacetoacetate hydrolase HmgB (BxeA3899). CONCLUSIONS/SIGNIFICANCE: This study revealed that strain LB400 used both homogentisate and homoprotocatechuate ring-cleavage pathways for 3- hydroxyphenylacetate and 4-hydroxyphenylacetate catabolism and that these four catabolic routes are functional, confirming the metabolic versatility of B. xenovorans LB400.

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.

Response to (chloro)biphenyls of the polychlorobiphenyl-degrader Burkholderia xenovorans LB400 involves stress proteins also induced by heat shock and oxidative stress.

We report the effects of 4-chlorobiphenyl and biphenyl on the physiology, morphology and proteome of the polychlorobiphenyl-degrader Burkholderia xenovorans LB400. The exposure to 4-chlorobiphenyl decreases the growth of LB400 on glucose, and cells exhibit irregular outer membranes, a larger periplasmic space and electron-dense granules in the cytoplasm. Additionally, lysis of cells was observed during incubation with 4-chlorobiphenyl or biphenyl. Proteome of B. xenovorans LB400 exposed to biphenyl and 4-chlorobiphenyl were analysed by two-dimensional gel electrophoresis. Besides induction of the Bph enzymes of biphenyl catabolic pathways, incubation with 4-chlorobiphenyl or biphenyl results in the induction of the molecular chaperones DnaK and GroEL. Induction of these chaperones, which were also induced during heat shock, strongly suggests that exposure to (chloro)biphenyls constitutes stress conditions for LB400. During growth of LB400 on biphenyl, oxidative stress was evidenced by the induction of alkyl hydroperoxide reductase AhpC, which was also induced during exposure to H(2)O(2). 4-chlorobiphenyl and biphenyl induced catechol 1,2-dioxygenase, as well as polypeptides involved in energy production, amino acid metabolism and transport.

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility.

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven "central aromatic" and twenty "peripheral aromatic" pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.