Table 1.
Primer sets designed and used in this study.
Figure 1.
Predicted mhb genes encoding the gentisate central pathway and related genes in B. xenovorans LB400.
The mhb genes located at the major chromosome (C1) and the minor chromosome (C2). The genes encoding gentisate 1,2-dioxygenase are indicated with black arrows. The orientations of ORFs are represented by open arrows. The promoter regions for mhbR1, mhbR2, mhbT1, mhbT2, BxeA2626 and BxeA4527 are denoted with small black arrows, bent in the directions of transcription. The sizes of the genes and the intergenic regions are on scale.
Figure 2.
Relationship between bacterial transcriptional regulators for gentisate and protocatechuate catabolic pathways.
LysR, MarR and IclR transcriptional regulators from B. xenovorans LB400 and other bacteria were depicted. The dendogram was obtained by the neighbor-joining method using MEGA 5.0 based on sequence alignment calculated by CLUSTAL W using the default options. Circles denote proteins encoded in the genome from B. xenovorans LB400. The sequence and their accession number are: CatM, Acinetobacter baylyi ADP1 (P07774); BenM, A. baylyi ADP1 (AAC46441); TfdT, Cupriavidus pinatubonensis JMP134 (AAC44724); CdoR, Comamonas sp. JS765 (AAC79916); MhbR, Klebsiella pneumoniae M5a1 (Q5EXK6); PcaQ, Cupriavidus metallidurans CH34 (ABF10579); PcaQ, Agrobacterium tumefaciens C58/ATCC3397 (P0A4T6); PcaQ, B. xenovorans LB400 (Q13RR8); SalR, A. baylyi ADP1 (AAF04311); NagR, Ralstonia sp. U2 (Q9EXL7); PcaR, B. xenovorans LB400 (ABE35311); MhbR, B. xenovorans LB400 (ABE30329); BadR, Rhodopseudomonas palustris CGA009 (AAC23923); CbaR, Comamonas testosteroni TA441 (AAG00065); HpaR, Escherichia coli W (Z37980); MhpR, C. testosteroni TA441 (P77569); PcaR, Pseudomonas putida PRS2000 (Q52154); CatR, Rhodococcus opacus (Q33539); PcaR, P. putida KT2440 (Q88N41); PcaR, A. tumefaciens C58 (Q7CV82); PcaU, A. baylyi ADP1 (AAC37157); PobR, A. baylyi ADP1 (A36893).
Table 2.
Predicted genes encoding the gentisate and protocatechuate pathways and peripheral reactions from B. xenovorans LB400.
Figure 3.
Organization of the gene clusters involved in the catabolism of gentisate in B. xenovorans LB400 and other bacteria.
The genes encoding gentisate 1,2-dioxygenase are indicated with black arrows. The sizes of the genes and the intergenic regions are on scale.
Figure 4.
Genetic organization of the pca genes involved in protocatechuate catabolism in Burkholderia xenovorans LB400.
The pca genes are located in the minor chromosome (C2), major chromosome (C1) and megaplasmid (MP). The genes encoding the alpha and beta subunits of the protocatechuate 3,4-dioxygenase are indicated with black arrows. The orientations of ORFs are represented by open arrows. The promoter regions for pcaH, pcaQ, pcaI and pcaR are denoted with small black arrows, bent in the directions of transcription. The sizes of the genes and the intergenic regions are on scale.
Figure 5.
Organization of the pca genes involved in the catabolism of protocatechuate in B. xenovorans LB400 and other bacteria.
The genes encoding the alpha and beta subunits of the protocatechuate 3,4-dioxygenase are indicated with black arrows. The sizes of the genes and the intergenic regions are on scale.
Figure 6.
Growth of B. xenovorans LB400 on hydroxybenzoates, gentisate and protocatechuate.
LB400 cells were grown in M9 minimal medium using gentisate (5 mM), protocatechuate (5 mM), 3-HBA (10 mM) or 4-HBA (5 mM) as sole carbon and energy source. Each point is an average ± SDs of results from, at least, three independent assays.
Figure 7.
Transcriptional analysis of the mhb and pca genes during LB400 growth on hydroxybenzoates, gentisate and protocatechuate.
LB400 cells were grown on 3-HBA (lane 1), 4-HBA (lane 2), gentisate (lane 3), protocatechuate (lane 4) and glucose (lane 5) as sole carbon source. (A) Transcription of mhbD and mhbR genes; (B) Transcription of pcaG, pcaQ and pcaR genes. (C) Transcription of mhbM and pobA genes. RT-PCR (15, 20 and 25 amplification cycles) assays were performed using RNA extracted from LB400 cells collected at mid exponential growth phase. Transcription of 16S rRNA gene was used as control (not shown).
Figure 8.
Gentisate and protocatechuate dioxygenase activities of B. xenovorans LB400.
Cells were grown on 3-HBA (black square), 4-HBA (empty square) and glucose (empty circles) as sole carbon source. (A) Gentisate 1,2-dioxygenase activity measured by maleylpyruvate formation. (B) Protocatechuate 3,4-dioxygenase activity measured by protocatechuate disappearance. Each point is an average ± SDs of values from three independent assays.
Figure 9.
Models of 3-HBA and 4-HBA catabolic pathways in B. xenovorans LB400.
(A) 3-HBA and 4-HBA catabolism via the gentisate central pathway (continuous line). The substrates and products are: 3-hydroxybenzoate (1); gentisate (2); maleylpyruvate (3); fumarylpyruvate (4); pyruvate (5); fumarate (6) and 4-hydroxybenzoate (7). The enzymes are MhbM (3-HBA 6-hydroxylase), MhbD (gentisate 1,2-dioxygenase), MhbI (maleylpyruvate isomerase) and MhbH (fumarylpyruvate hydrolase). The gene encoding the 4-HBA 1-hydroxylase is unknown. (B) 4-HBA catabolism via protocatechuate (dotted line). The substrates and products are: 4-hydroxybenzoate (7); protocatechuate (8); 3-carboxy-cis,cis,-muconate (9); 4-carboxymucolactone (10); β-ketoadipate enol-lactone (11); β-ketoadipate (12); β-ketoadipyl-CoA (13); Acetyl-CoA (14); Succinyl-CoA (15).The enzymes are: PobA (4-hydroxybenzoate 3-monooxygenase), PcaGH (protocatechuate 3,4-dioxygenase), PcaB (3-carboxymuconate cycloisomerase), PcaC (4-carboxymuconolactone decarboxylase), PcaD (β-ketoadipate enol-lactone hydrolase), PcaJI (β-ketoadipate:succinyl-CoA tranferase) and PcaF (β-ketoadipyl-CoA thiolase).