Fig 1.
Organization of the genes encoding the p-cymene peripheral and the 2,3-dihydroxy-p-cumate central pathways in B. xenovorans LB400 and comparison with related bacterial gene clusters.
Organization of genes encoding the p-cymene, p-cumate and the 2,3-dihydroxy-p-cumate pathways in Burkholderia xenovorans LB400 (A), Pseudomonas putida F1 (B) and Rhodococcus sp. T104 (C). The cym genes encode proteins from the p-cymene catabolic pathway, and the cmt genes encode proteins from the p-cumate and 2,3-dihydroxy-p-cumate catabolic pathways.
Table 1.
Predicted genes encoding the p-cymene peripheral and the 2,3-dihydroxy-p-cumate central pathways from B. xenovorans LB400.
Fig 2.
Model of p-cymene and 2,3-dihydroxy-p-cumate catabolic pathways in B. xenovorans LB400.
The box with dotted border indicates the p-cymene peripheral pathway, which converts p-cymene into 2,3-dihydroxy-p-cumate. The substrate is p-cymene and the products are p-cumic alcohol, p-cumic aldehyde, p-cumate, 2,3-dihydroxy-2,3dihydro-p-cumate and 2,3-dihydroxy-p-cumate. The enzymes are CymA (p-cymene monooxygenase), CymB (p-cumic alcohol dehydrogenase) CymC (p-cumic aldehyde dehydrogenase), CmtA (p-cumate dioxygenase) and CmtB (2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase). The reactions of the 2,3-dihydroxy-p-cumate central pathway are presented in a box with continuous border. The substrate is 2,3-dihydroxy-p-cumate and the final products are isobutyrate, pyruvate and acetyl-CoA. The enzymes are CmtC (2,3-dihydroxy-p-cumate-3,4-dioxygenase), CmtD (2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate decarboxylase), CmtE (2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase), CmtF (2-hydroxypenta-2,4-dienoate hydratase), CmtG (4-hydroxy-2-pentanoate aldolase) and CmtH (acetaldehyde dehydrogenase).
Fig 3.
Growth of B. xenovorans LB400 on p-cymene and p-cumate.
LB400 cells were grown in M9 medium using p-cymene (vapor phase) or p-cumate (5 mM) as the sole carbon and energy source. Cells without carbon source were used as control. CFU/mL values were calculated as the mean ± SD of at least three independent experiments.
Fig 4.
Expression of the cymAb and cmtAb genes during LB400 growth on p-cymene and p-cumate, and activity of p-cumate dioxygenase.
(A) Expression of cymAb and cmtAb genes during LB400 growth on p-cymene and p-cumate. Expression of cymAb (BxeA3559) and cmtAb (BxeA3556) genes in LB400 cells grown in glucose (lane 1), succinate (lane 2), p-cymene (lane 3), p-cumate (lane 4) and p-cymene plus succinate (lane 5). RT-PCR assays were performed using RNA from LB400 cells collected at exponential growth phase. The expression of 16S rRNA was used as a control to normalize across samples. At least three independent RNA samples were collected at each condition and two independent RT-PCR reactions for each sample were done to assess reproducibility. (B) Activity of the p-cumate dioxygenase CmtA in LB400 cells. LB400 cells grown overnight in LB medium and washed with M9 medium were incubated with 2.5 mM p-cumate and 0.3% lactate. Absorbance values are the mean ± SD of at least three independent experiments.
Fig 5.
Effects of growth on p-cymene and p-cumate on the cell morphology of B. xenovorans LB400.
Cells were grown in M9 minimal medium using succinate (A), p-cumate (B), p-cymene (C), and succinate plus p-cymene (D) as sole carbon and energy sources. Arrows and arrowheads indicate the outer and the inner membrane, respectively. Some internal granules are indicated within boxes. The micrographs shown were representative and selected from, at least, 10 fields.
Fig 6.
Effects of p-cymene on the proteome of B. xenovorans LB400.
Total protein pattern of LB400 cells grown in M9 medium using glucose (A1) and p-cymene (B1) as sole carbon and energy sources. Induced proteins in p-cymene-grown cells on the total protein pattern of strain LB400 grown in glucose (A2) and p-cymene (B2) are boxed. Proteins repressed in p-cymene-grown cells on the total protein pattern of strain LB400 grown in glucose (A3) and p-cymene (B3) are boxed. The gels depicted are representative of at least three biological replicates and at least six 2-DE gels.
Table 2.
Identification of LB400 proteins induced during growth on p-cymene.
Table 3.
Identification of LB400 proteins repressed during growth on p-cymene.
Fig 7.
Effect of p-cymene on biofilm formation by B. xenovorans LB400.
Cells were grown in M9 medium using glucose or p-cymene as sole carbon source until exponential phase (Turbidity600nm = 0.6) and washed were resuspended in M9 medium supplemented with glucose (5 mM) or p-cymene (vapor phase). Microscopic characterization of B. xenovorans LB400 biofilms grown in M9 medium supplemented with glucose (5 mM) or p-cymene (vapor phase) during 48 h. Cells in the biofilms were stained with the BacLight kit showing viable (green fluorescence) and non-viable (red fluorescence) bacteria (C and F). The viable and non-viable cells were marked with green fluorescence (A and D). The dead cells were stained red (B and E). Images were horizontal and vertical three-dimensional reconstructed in the x–y plane and x–y plane, respectively. In all images the scale bar is 25 μm.
Fig 8.
Schematic metabolic network activated by p-cymene in B. xenovorans LB400.
Capital and small letters indicate protein and genes, respectively. Red arrow indicates reduced protein synthesis or gene expression. Blue arrow indicates an increase in enzymatic activity, protein level or gene expression. Main p-cymene induced metabolic and physiological changes are highlighted by yellow boxes. OM, outer membrane; PS, periplasmic space; IM, inner membrane. BphA2, biphenyl 2,3-dioxygenase beta subunit; BphB, biphenyl dihydrodiol dehydrogenase; CmtAb, p-cumate dioxygenase large subunit; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; PK, pyruvate kinase; AcnA, aconitate hydratase; SCoAS, Succinyl-CoA synthetase; NADHDH, NADH dehydrogenase; SDH, Succinate dehydrogenase (Complex II); UQ, ubiquinone; Cytb, ubiquinol-cytochrome C (Complex III); CytC, cytochrome C; Cyo, cytochrome C oxidase (Complex IV); ATPase, ATP synthase; PPiase, inorganic pyrophosphatase; BxeA4132, 4-diphosphocytidyl-2-methyl-D-erythritol kinase.