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 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.