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.


Introduction
Aerobic bacterial degradation of aromatic compound proceeds generally in two phases. In the first phase, the aromatic compound is prepared for ring cleavage by a variety of ring modification reactions. The second phase of aerobic degradation includes ring fission of the aromatic compound and subsequent reactions leading to the formation of Krebs cycle intermediates [1][2][3][4]. Betaproteobacteria of the Burkholderiales order possess an amazing metabolic versatility to degrade a large number of aromatic compounds [1,3]. Burkholderia xenovorans LB400 is a bacterium able to degrade polychlorobiphenyls (PCBs) and diverse aromatic compounds [3][4][5][6][7][8]. LB400 genome has a size of 9.73 Mbp distributed in three replicons: the major chromosome (C1; 4.90 Mbp), the minor chromosome (C2; 3.36 Mbp) and the megaplasmid (MP; 1.47 Mbp) [3]. Genomic analysis of B. xenovorans strain LB400 revealed the presence of genes encoding an unusual high number of central and peripheral pathways for the degradation of aromatic compounds [3]. A recent study confirmed functionality of some predicted aromatic pathways [4]. However, the function of genes encoding diverse aromatic catabolic routes including the gentisate and protocatechuate pathways of B. xenovorans strain LB400 remains to be elucidated.
The aims of this study were to characterize the gentisate and protocatechuate central pathways in B. xenovorans LB400 using genomic and functional approaches. In addition, the role of these ring-cleavage pathways during 3-HBA and 4-HBA degradation in strain LB400 was studied. This report describes for the first time the metabolism of 3-HBA and 4-HBA in B. xenovorans LB400. This study demonstrates functional gentisate and protocatechuate catabolic pathways in B. xenovorans LB400 that are involved in the degradation of 3-HBA and 4-HBA.

Bacterial Strain and Culture Conditions
B. xenovorans strain LB400 was cultivated in M9 mineral medium with trace solution and glucose (5 mM) [23], 3-HBA (5 and 10 mM), 4-HBA (5 mM), gentisate (5 mM) or protocatechuate (5 mM) as sole carbon and energy source at 30uC [3,23]. Growth was determined by measuring turbidity at 525 nm and by counting colony-forming units (CFU). Aliquots taken from bacterial cultures were diluted and plated on Luria-Bertani (LB) medium. CFU mL 21 values were calculated as the mean 6 SD of values from at least three independent experiments.
Isolation of RNA and RT-PCR RNA was isolated from LB400 cells grown until mid-exponential growth phase (turbidity 525 nm 0.4-0.6) on 3-HBA, 4-HBA and glucose. RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany). DNase I treatment was performed using the RNase-Free DNase Set (Qiagen, Hilden, Germany). The RNA was quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). In this study, specific primers for mhbD (BxeA2627, BxeA4526), mhbT (BxeA2625, BxeA4528), mhbR (BxeA2624, BxeA4529), mhbM (BxeB2526), pcaG (BxeB2776), pcaQ (BxeB2772), pcaR (BxeB0642) and pobA (BxeA2040) genes were designed and used (Table 1). Reverse transcription-PCR (RT-PCR) was carried out with 40 mg of total RNA and the sequence-specific primers using SuperScript One-step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA, USA). Amplification of the 16S rRNA gene was performed as control for DNA contamination using the primers 27f (59-AGAGTTTGATCMTGGCTCAG-39) and 1492r (59-TACGGYTACCTTGTTACGACTT-39) as reported [4]. Negative and positive controls were included in each RT-PCR assay. The expression of the constitutively expressed 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.

Preparation of Bacterial Cell Extracts
Cells grown until exponential phase with 3-HBA, 4-HBA or glucose were harvested by centrifugation (10,733 g for 10 min) at 4uC. Bacterial cells washed with sodium phosphate buffer (50 mM, pH 7.0) and concentrated were disrupted using ultrasonic cells disruptor Microson at 4uC. Cell lysates were clarified by centrifugation (19,300 g for 15 min) at 4uC. The protease inhibitor cocktail Tablet Complete mini (Roche, Indianapolis, USA) was added to cell extracts to reach a final concentration of 0.8 mg/ml. Protein concentration was measured using the Quantit-iT Protein Assay kit (Invitrogen, Carlsbad, CA, USA) that uses a fluorescent dye whose quantum yields are enhanced significantly when binding at the detergent-protein interface and measuring fluorescence at room temperature with the Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) [24].

Dioxygenase Activity Assays
Gentisate 1,2-dioxygenase activity was determined spectrophotometrically by measuring the formation of maleylpyruvate at 330 nm [13]. The assay volume (10 ml) contained sodium phosphate buffer (50 mM, pH 7.0), crude extract (1000 mg of protein) and gentisate (0.6 mM). The reactions were carried out at 30uC and initiated by the addition of gentisate. Gentisate 1,2dioxygenase activity was calculated using the molar extinction coefficient of maleylpyruvate 10,300 M 21 cm 21 [13,25]. Protocatechuate 3,4-dioxygenase activity was determined spectrophotometrically by the disappearance of protocatechuate measuring the absorbance at 290 nm (A 290 nm ) [26]. The assay volume (10 ml) contained sodium phosphate buffer (50 mM, pH 7.0), crude extract (1000 mg of protein) and protocatechuate (0.6 mM). The reactions were carried out at 30uC and initiated by the addition of protocatechuate. Protocatechuate 3,4-dioxygenase activity was calculated using the difference of the molar extinction coefficient of protocatechuate and its product under these conditions that is 2,280 M 21 cm 21 [27]. One enzyme unit corresponds to the transformation under the conditions described above of 1 mmol of substrate to product per minute per milligram of protein at 30uC. The enzymatic activity assays were done in triplicate.

Genomic Analysis of the Gentisate Pathway and Related Peripheral Routes
The mhb genes encoding the gentisate catabolic pathway were analyzed in the genome of B. xenovorans LB400. Table 2 describes the predicted genes encoding the gentisate central pathway and related peripheral routes in B. xenovorans LB400. Two identical mhbRTDHI gene clusters encoding the gentisate pathway were identified at C1 (Fig. 1). Both mhb gene clusters are located adjacent in two identical chromosomal regions comprising thirty genes. Sequence analyses suggest that mhbD, mhbH and mhbI genes of strain LB400 encode the gentisate 1,2-dioxygenase, maleylpyruvate isomerase and fumarylpyruvate hydratase, respectively. The mhbD, mhbH and mhbI gene products of strain LB400 possess .52% sequence identity with the respective MhbD, MhbH and MhbI proteins from K. pneumoniae M5a1 [13,30]. Sequence analyses of both mhbDHI gene clusters neighborhoods in strain LB400, revealed that the BxeA2625 and BxeA4528 (hereafter, mhbT1 and mhbT2) genes encode major facilitator superfamily (MFS) transporters that belong to the aromatic acid:H + symporter (AAHS) family. Genomic context suggests that the mhbT1 and mhbT2 genes encode a MFS transporters probably involved in gentisate transport in B. xenovorans LB400. The BxeA2624 and BxeA4529 genes (hereafter, mhbR1 and mhbR2) were located upstream and adjacent to the mhbT genes, and divergently transcribed. The mhbR gene product shares 30% sequence identity with MarR-type transcriptional regulator related to the mhb gene cluster from Polaromonas naphthalenivorans CJ2 (Table 2). Sequence alignment of transcriptional regulators associated to central aromatic pathways showed that the mhbR gene product from B. xenovorans LB400 is a MarR-type transcriptional regulator (Fig. 2), which probably regulates the expression of the mhbT gene. The MhbR regulator in K. pneumoniae M5a1 is a LysR-type activator of the mhbTDHI gene cluster [39]. Genome analyses indicated that two divergent s 70 -type promoters were present between mhbR and mhbT. A regulatory binding site with a palindromic sequence for a MarR-type transcriptional regulator (59-ATTGTTTCA-CAAACTAAT-39) was identified upstream from the mhbT gene promoter, which is similar to the consensus MarR-type regulator binding site Genome analysis indicated that an independent s 70 -type promoter is located upstream of the BxeA2626 (BxeA4527) and the mhbDHI genes. Upstream from the BxeA2626 (BxeA4527) gene promoter is located a LysR-type regulator binding sequence (59-ATTCACTTCGAGAAT-39) that has the conserved (59-T-N 11 -A-39) motif critical for binding of LysR-type transcriptional regulators [41]. The specific LysR-type transcriptional regulator gene of the mhbDHI gene cluster of strain LB400 has not been identified in this study.
Gene organization of the mhb cluster in B. xenovorans LB400 is similar to the mhb cluster from K. pneumoniae M5a1, except for the presence of a coding sequence of unknown function between mhbT and mhbD genes in strain LB400, the presence of the mhbM gene encoding 3-HBA 6-hydroxylase located downstream to mhbI in strain M5a1 and that the mhbR genes encode different transcriptional regulators (Fig. 3). The mhb gene clusters from other Burkholderiales strains including Ralstonia solanacearum GMI100, B. multivorans CGD1, P. naphthalenivorans CJ2 and Acidovorax avenae subsp. avenae ATCC19860 showed different mhb gene organizations (Fig. 3).

Genomic Analysis of Protocatechuate Pathway and Related Peripheral Routes
Predicted pca genes encoding the protocatechuate central pathway are distributed within the B. xenovorans LB400 genome (Fig. 4). Predicted pca genes encoding the protocatechuate pathway and related peripheral routes are listed in Table 2. Sequence analyses suggest that pcaG, pcaH, pcaQ, pcaF (BxeB2167; hereafter pcaF1), pcaC, pcaD (BxeB0646; hereafter pcaD1), pcaB (BxeB0645; hereafter pcaB1), pcaJ (BxeB0644; hereafter pcaJ1), pcaI (BxeB0643; hereafter pcaI1), pcaQ and pcaR genes encoding the protocatechuate catabolic pathway are located at C2. Copies of pcaB (BxeB1906; hereafter pcaB2) and pcaD genes (BxeB0595; hereafter pcaD2) were identified at C2. Two pcaF gene copies (hereafter pcaF2 and pcaF3, respectively), and a pcaJ gene copy (hereafter pcaJ3) were identified by sequence analyses at C1. In addition, pcaJ and pcaI genes copies (hereafter pcaJ2 and pcaI2, respectively) were identified by sequence analyses at MP. The pcaG and pcaH genes encoding the alpha and beta subunits from a protocatechuate 3,4dioxygenase were located 2.3 Mbp distant region from pcaIJBDC gene cluster. The pcaG and pcaH gene products of strain LB400 showed $90% identity with the PcaG and PcaH proteins from Acinetobacter lwoffii K24 [31]. The spread in the organization of pca genes and genes encoding related peripheral pathways in B. xenovorans LB400 is also observed in Cupriadus metallidurans CH34   5). Predicted pcaQ gene located next to the pcaHG genes and divergently transcribed encodes a LysR-type transcriptional regulator (Fig. 2) that might regulate the expression of the pcaHG gene cluster. The PcaQ protein shared a 48% sequence identity with the LysR-type transcriptional regulator PcaQ from Agrobacterium tumefaciens A348 (Table 2) [43]. Two divergent s 70 -type promoters are present between the pcaQ gene and the pcaGH gene cluster. A PcaQ LysR-type regulatory binding sequence (59-ATAACGGCAGGTTAT-39) was identified upstream of the pcaH gene promoter. This regulatory sequence has the consensus sequence for the PcaQ regulator recognition site sequence of Burkholderiales (59-ATAACN 5 GTTAT-39) [43]. Predicted pcaR gene (BxeB0642) located adjacent to the pcaIJBDC genes and divergently transcribed encodes a LysR-type transcriptional regulator (Fig. 2). The pcaR gene product shared a 95% sequence identity with a LysR-type transcriptional regulator adjacently located to the pcaIJBDC gene cluster from B. phytofirmans PsJN ( Table 2) [38]. Genomic analysis of the pcaCD1B1J1I1R gene cluster showed that two divergent s 70 -type promoters are present between the pcaR gene and the pcaCD1B1J1I1 gene cluster. A putative LysR-type regulatory binding sequence (59-GTTCGCTAAACGAAT-39) was identified upstream to the pcaR gene promoter. This sequence has the conserved (59-T-N 11 -A-39) motif critical for binding of LysR-type autoregulatory transcriptional regulators [41].
The genes encoding the enzymes of the 3-HBA and 4-HBA peripheral reactions that funnel into the protocatechuate pathway were searched in the LB400 genome. The pobA gene (BxeA2040) encoding for a 4-HBA 3-monooxygenase that converts 4-HBA into protocatechuate was identified by sequence analyses at C1 (Fig. 4). The pobA gene protein from strain LB400 shares 51% sequence identity with the PobA enzyme from P. fluorescens strain Pf-5 [21]. In addition, protocatechuate has been described as a central route for 3-HBA degradation by a 3-HBA 4-hydroxylase encoded by the mobA gene in bacteria. The mobA gene was not found in the LB400 genome.

Growth of Strain LB400 on Gentisate, Protocatechuate and HBAs
Sequence analyses of B. xenovorans LB400 genome revealed the presence of genes encoding the gentisate and the protocatechuate ring-cleavage pathways and 3-HBA and 4-HBA peripheral pathways. To investigate whether these catabolic pathways are functional, in a first approach growth assays were performed. The growth of B. xenovorans strain LB400 on the central metabolites gentisate and protocatechuate was studied. B. xenovorans LB400 was able to grow on gentisate (5 mM) and on protocatechuate (5 mM) as sole carbon and energy source (Fig. 6). In addition, the growth of B. xenovorans strain LB400 on 3-HBA or 4-HBA was studied. Strain LB400 was able to grow on 3-HBA (10 mM) and 4-HBA (5 mM) (Fig. 6). B. xenovorans LB400 attained higher cell concentration at stationary phase on protocatechuate (1.1 610 9 CFU mL 21 ) and 4-HBA (1.0610 9 CFU mL 21 ). The growth of strain LB400 on gentisate or 3-HBA yielded 7.0610 8 CFU mL 21 (Fig. 6). These results indicate functional 3-HBA and 4-HBA peripheral pathways and gentisate and protocatechuate central pathways in B. xenovorans LB400.

Transcriptional Analyses of the mhb and pca Genes
In order to determine the role of predicted genes encoding the gentisate and protocatechuate ring-cleavage routes and 3-HBA and 4-HBA peripheral pathways in strain LB400, transcriptional analyses of key catabolic genes were performed. In addition, the expression of genes encoding potential transcriptional regulators was analyzed. The expression of the mhbD gene encoding the enzyme gentisate 1,2-dioxygenase during exponential growth phase of LB400 cells grown on gentisate, protocatechuate, 3-HBA, 4-HBA, and glucose was analyzed. The mhbD gene was transcribed during growth of strain LB400 in gentisate, 3-HBA and 4-HBA (Fig. 7). In contrast, expression of the mhbD gene was not observed during growth on protocatechuate or glucose (Fig. 7). The mhbR gene encoding a MarR-type transcriptional regulator was transcribed during LB400 growth on 3-HBA, 4-HBA, gentisate, protocatechuate and glucose (Fig. 7). These results suggest that gentisate, 3-HBA and 4-HBA or its metabolites induce transcription of the mhbD gene in B. xenovorans LB400. On the other side, the mhbR gene has probably a basal constitutive expression. Transcription analysis of the mhbM gene encoding the 3-HBA 6-hydroxylase was performed. The mhbM gene of strain LB400 was expressed was during growth on 3-HBA, 4-HBA, gentisate, protocatechuate and glucose (Fig. 7). However, higher expression of the mhbM gene on HBAs than on glucose was observed.
Transcription of the pcaG gene encoding the key protocatechuate 3,4-dioxygenase alpha subunit during growth of strain LB400 with different aromatic substrates was studied. Predicted pcaG gene was expressed during growth of strain LB400 on protocatechuate and 4-HBA (Fig. 7). However, pcaG transcripts were not observed during growth of strain LB400 on gentisate, 3-HBA and glucose. These results suggest that by 4-HBA, protocatechuate or its metabolites induce the transcription of the pcaG gene. Both pcaQ and pcaR genes encoding the transcriptional regulators of the pcaGH genes and the pcaIJBDC gene cluster were transcribed during LB400 growth on HBAs, gentisate, protocatechuate and glucose (Fig. 7). Therefore, the pcaQ and pcaR genes have probably a basal constitutive expression.
Transcription of the pobA gene encoding the 4-HBA 3monooxygenase of strain LB400 was analyzed. The pobA gene was expressed during LB400 growth only on 3-HBA, 4-HBA and glucose (Fig. 7). The pobA gene expression was higher during growth on HBAs than on glucose.
These results indicate that expression of the mhb and pca genes encoding catabolic enzymes is regulated in LB400 cells. These regulations are probably exerted by MarR-type and LysR-type transcriptional regulators. This study also suggests a regulated expression of the mhbM gene encoding the peripheral 3-HBA 6hydroxylase and of the pobA gene encoding the peripheral 4-HBA 3-monooxygenase but with a basal constitutive expression of the mhbM gene. Overall, the transcriptional analyses suggest that 3-HBA is catabolized and funneled exclusively through the gentisate central pathway, whereas 4-HBA can be degraded through the protocatechuate and gentisate central pathways in strain LB400.

Gentisate and Protocatechuate Ring-cleavage Activities
To further characterize the gentisate and protocatechuate central pathways in B. xenovorans strain LB400, the activities of the key enzymes gentisate 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were measured. Both dioxygenase activities were determined in crude extract of cells cultured until mid exponential phase in 3-HBA, 4-HBA and glucose. Higher gentisate 1,2dioxygenase activity was observed in cells grown on 3-HBA and 4-HBA (0.04960.004 U mg 21 protein), whereas the gentisate 1,2-dioxygenase activity was not detected in glucose-growth cells (Fig. 8A). LB400 cells grown on 4-HBA showed high protocatechuate 3,4dioxygenase activity (0.1560.02 U mg 21 protein), whereas the protocatechuate dioxygenase activity was not observed in LB400 cells grown on 3-HBA and glucose (Fig. 8B). Enzyme activity assays suggest an active protocatechuate pathway in 4-HBA-grown cells, whereas the gentisate central pathway was found to be active in both 3-HBA-and 4-HBA-grown cells.

Discussion
This study has focused on a genomic analysis on the gentisate and protocatechuate catabolic pathways and its related peripheral pathways in B. xenovorans LB400. Experimental evidence was provided on the functionality of these ring-cleavage pathways, and their roles during 3-HBA and 4-HBA catabolism were proposed. Strain LB400 is able to grow on gentisate as sole carbon source, indicating a functional gentisate catabolic pathway. The gentisate catabolic pathway is present only in few Burkholderia strains [1]. Both mhbRTDHI gene clusters were found in two identical and adjacently located segments of 30 genes in LB400 major chromosome, indicating a gene cluster duplication process. Gene duplication is frequently observed in aromatic catabolic pathways and suggests functional redundancies, which could potentially increase the bacterial fitness and its adaptation to adverse environmental conditions. The mhbDHI gene cluster organization  is similar in strains from Burkholderiales order including B. xenovorans LB400. However, the complete mhbRTDHI gene cluster with a coding sequence of unknown function between mhbT and mhbD was found in strain LB400, which has not been observed in other bacteria and its function remains to be elucidated.
Growth studies indicated that a 3-HBA peripheral pathway is present in B. xenovorans LB400. Gentisate has been described as the main ring-cleavage pathway for 3-HBA assimilation in bacteria, such as K. pneumoniae M5a1 [11], Corynebacterium glutamicum RES167 [44] and Rhodococcus sp. strain NCIMB 12038 [45]. In B. xenovorans strain LB400 the mhbM gene encoding the 3-HBA 6-monooxygenase is located at C2, whereas the mhbRTDHI gene cluster is located at C1 (Fig. 1). In contrast, in strains M5a1, RES167, NCIMB 12038 and in Comamonas and other Burkholderia strains, a 3-HBA 6 hydroxylase-encoding mhbM gene is usually found within the mhbDHI gene cluster. An increased transcription of mhbM gene  in strain LB400 was observed during growth on 3-HBA. Therefore, we propose that the predicted mhbM gene product of strain LB400 is involved in the hydroxylation of 3-HBA to gentisate.
It is worth noting that B. xenovorans LB400 was able to grow using 10 mM 3-HBA, whereas no growth of LB400 cells was achieved on 5 mM 3-HBA. We propose that a 3-HBA (5 mM) did not support the growth of B. xenovorans strain LB400, probably due to an insufficient transport of 3-HBA across the membrane. These results suggest that strain LB400 lacks an active transport for 3-HBA. 4-HBA catabolism through the gentisate pathway has been described in bacterial strains from Bacillus genus [46] and the archaeal isolate Haloarcula sp. strain D1 [42]. B. xenovorans LB400 was able to grow using 4-HBA (5 mM) as sole carbon source. The LB400 growth on 4-HBA (5 mM) but not on 3-HBA (5 mM) suggests an efficient transport of 4-HBA rather than 3-HBA through the membrane by an active 4-HBA transport system. This is supported by our bioinformatics analyses that indicated that the mhbT gene product of strain LB400 has 33% sequence identity with the PcaK protein of P. putida PRS2000, which is an active transporter system for 4-HBA [29,33]. The mhbT gene of B. xenovorans LB400 encodes a MFS transporter of the AAHS family that may be involved in the active transport of gentisate and 4-HBA.
Genomic analyses suggest that the mhbR gene, which is upstream and divergently transcribed to the mhbT gene, encodes for a transcriptional regulator of the mhbT gene in strain LB400. The mhbR gene of strain LB400 was transcribed during growth on 3-HBA, 4-HBA, gentisate, protocatechuate and glucose, suggesting a basal constitutive expression. The mhbR gene product from B. xenovorans LB400 shares 30% sequence identity with MarR-type transcriptional regulators from the mhb gene cluster from P. naphthalenivorans CJ2 [28] and C. testosteroni CNB-2 [47]. MarR regulators are winged helix-turn-helix DNA-binding proteins that respond to specific ligands and act as dimmers. In absence of the ligand, MarR regulators bind to palindromic sequences within the promoter, generally causing transcriptional repression [40]. We propose that the MarR-type regulator encoded by the mhbR gene from B. xenovorans LB400 is a transcriptional repressor that regulates the expression of the mhbT gene. Transcriptional analysis showed that the mhbD gene encoding a gentisate 1,2-dioxygenase was expressed during growth on gentisate, 3-HBA and 4-HBA, but not on glucose. Transcriptional analyses suggest that either 3-HBA, 4-HBA, gentisate or its metabolic intermediates induced the transcription of the mhb gene cluster. The expression of mhbD gene correlates with an increased gentisate 1,2-dioxygenase activity observed in LB400 cells grown on 3-HBA and 4-HBA. Genomics analysis showed the presence of a conserved sequence (59-T-N 11 -A-39) critical for LysR-type transcriptional regulator binding, located upstream of the -35 box of BxeA2626 and BxeA4527 genes. However, a LysR-type transcriptional regulator gene was not found in the neighborhoods of both mhb gene clusters. Although further studies are needed to survey the regulation mechanisms of 3-HBA and 4-HBA degradation pathways via gentisate in strain LB400, these genomic analyses suggest that mhbDHI cluster is regulated by a LysR-type transcriptional regulator in strain LB400. Overall, transcriptomic analyses and dioxygenase activity assays indicate that the gentisate pathway in strain LB400 is active during 3-HBA and 4-HBA degradation, suggesting that both substrates may be funneled into the gentisate central pathway (Fig. 9). We propose that a 4-HBA 1-hydroxylase could be involved in the conversion of 4-HBA into gentisate in B. xenovorans LB400. Experimental data with deuterated substrates in the archaeal strain Haloarcula sp. D1 supported the mechanism in which 4-HBA is converted into gentisate by hydroxylation of 4-HBA at C-1, with a concomitant 1,2-carboxyl group migration. The transformation is called NIH shift and is catalysed by a 4-HBA 1-hydroxylase [42]. Although no gene sequence information of the 4-HBA 1-hydroxylase enzyme is available to perform a genomic search, an active gentisate pathway during 4-HBA assimilation suggests a 4-HBA 1-hydroxylase activity in B. xenovorans strain LB400. Recently, we reported that B. xenovorans strain LB400 degrades 4-hydroxyphenylacetate via homogentisate, in which also a migration of the acetate group by a NIH shift reaction may also be involved [4]. Hydroxylation of monosubstituted aromatic rings at C-1, followed by a migration of the carboxymethyl side chain to C-2 has been described in P. acidovorans and Nitrosomonas europaea 19718 [48,49]. Future studies are needed to characterize the genes encoding the 4-HBA 1hydroxylase that converts 4-HBA into gentisate in bacteria, including B. xenovorans strain LB400.
Most bacteria metabolize 4-HBA by hydroxylation at C-3 to yield protocatechuate as central intermediate [1,2]. The 4-HBA 3monooxygenase encoded by the pobA gene catalyzes 4-HBA conversion into protocatechuate in Pseudomonas strains [21,22]. Genomic analyses revealed the presence of the pca genes encoding the complete protocatechuate central pathway are located at C2 and pca gene copies are located at C1 and MP, while the pobA gene is separately located at C1 in strain LB400 genome. Spreading of pca genes and genes encoding its related peripheral reactions is also observed in Betaproteobacteria, such as Cupriavidus (Fig. 4), Burkholderia and Ralstonia strains [1]. Diverse pca gene organizations have been reported in bacteria [1,2,20]. The pcaGH genes are clustered with other pca genes of the protocatechuate route and related peripheral pathways in the Gammaproteobacteria A. baylyi ADP1 and P. putida KT2440, the Alphaproteobacteria strains Agrobacterium tumefaciens C58 and Sinorhizobium meliloti 1021, and the Betaproteobacteria from the Cupriavidus genus such as C. metallidurans CH34, C. pinatubonensis JMP143 and C. necator H16 [1]. In contrast, in B. xenovorans LB400 and other bacterial strains from the Burkholderia genus, the pcaGH genes are not clustered with other pca genes. B. xenovorans strain LB400 possesses the pcaQHG and the pcaRIJBDC gene clusters and separately pcaF gene copies. In addition, LB400 genome contains a pcaIJ gene cluster and pcaB, pcaD and pcaJ gene single-copies, indicating genetic redundancy. B. xenovorans LB400 was able to grow on 4-HBA and protocatechuate, indicating functional 4-HBA peripheral and protocatechuate ring-cleavage pathways. Transcriptional analyses showed that pcaG gene encoding the protocatechuate 3,4dioxygenase alpha subunit was expressed during protocatechuate and 4-HBA degradation by strain LB400. The expression of the pcaG gene during 4-HBA degradation correlates with an increased protocatechuate 3,4-dioxygenase activity observed in LB400 cells extracts grown on 4-HBA. In contrast, neither pcaG gene expression nor protocatechuate 3,4-dioxygenase activity was observed in 3-HBA and glucose-grown LB400 cells. These results suggest that 4-HBA is exclusively degraded via protocatechuate, in which a hydroxylation of 4-HBA at C-3 occurred for protocatechuate formation. The pobA gene of strain LB400 was transcribed during growth on 4-HBA, suggesting that the pobA gene product is involved in 4-HBA hydroxylation to produce protocatechuate in B. xenovorans strain LB400 (Fig. 9). B. xenovorans LB400 as well as most strains belonging to Burkholderia and Ralstonia genera that possess the pcaGH genes have a 4-HBA 3hydroxylase-encoding gene, indicating that protocatechuate is the preferred catabolic pathway for 4-HBA degradation. In addition, these results suggest that either 4-HBA, protocatechuate or its metabolic intermediates induced the transcription of protocatech-uate 3,4-dioxygenase-encoding genes. The pcaQ gene adjacently located to pcaGH genes encodes a LysR-type transcriptional regulator and was transcribed during growth on 4-HBA, protocatechuate, 3-HBA, gentisate and glucose, suggesting a constitutive expression of the pcaQ gene. Thus, the PcaQ LysR-type transcriptional regulator of strain LB400 probably controls the expression of the pcaGH genes during 4-HBA and protocatechuate degradation in strain LB400. LysR-type transcriptional regulators act as activators during aromatic compounds catabolism. In general, the inducers are metabolic intermediates of the catabolic pathway [2,20]. The PcaQ regulator from strain LB400 probably acts as a transcriptional activator of the expression of the divergently transcribed pcaGH genes, like other PcaQ proteins that are transcriptional activators of the pca genes in Agrobacterium tumefaciens and Sinorhizobium meliloti [32,43]. The pcaR gene adjacently located upstream of the pcaIJBDC gene cluster encodes a LysR-type transcriptional regulator and was transcribed during growth on 4-HBA, protocatechuate, 3-HBA, gentisate and glucose. In P. putida strains PRS2000 and KT2440, IclR-type transcriptional regulators regulate the pca gene cluster encoding enzymes for the degradation of 3-carboxy-cis,cis-muconate into Krebs cycle intermediates [2,20]. We propose that the PcaR protein may regulate the expression of the pcaIJBDC gene cluster in strain LB400. The pcaRIJBDC gene organization with a LysRtype regulator with high sequence identity (95%) was observed in the closely relative strain B. phytofirmans PsJN. In accordance with this, it has been reported that all strains of the 'B. cepacia complex' as well as B. phymatum STM815 and B. glumae BGR1 possess a LysR-type transcriptional regulator gene clustered with the pcaIJBDC genes [1]. It is likely that the pcaR gene product of strain LB400 acts as a transcriptional activator of the expression of the divergently transcribed pcaIJBDC gene cluster. Further studies are required to confirm that PcaQ and PcaR proteins regulate the expression of the pcaHG and pcaIJBDC gene clusters in B. xenovorans LB400. The protocatechuate pathway is widespread among pathogenic and environmental bacteria, and only a small number of strains lack this pathway. Therefore, the protocatechuate catabolic pathway can be an important catabolic feature and play a key role to improve bacterial fitness in the environment and in pathogenesis.  (7); protocatechuate (8); 3-carboxy-cis,cis,-muconate (9); 4carboxymucolactone (10); b-ketoadipate enol-lactone (11); b-ketoadipate (12); b-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 (4carboxymuconolactone decarboxylase), PcaD (b-ketoadipate enol-lactone hydrolase), PcaJI (b-ketoadipate:succinyl-CoA tranferase) and PcaF (bketoadipyl-CoA thiolase). doi:10.1371/journal.pone.0056038.g009 In this report, we described functional gentisate and protocatechuate ring-cleavage pathways in B. xenovorans LB400, revealing inducible expression of key genes encoding gentisate and protocatechuate dioxygenases. Additionally, in this study 3-HBA and 4-HBA peripheral reactions in strain LB400 were reported. Based on genomic analyses, growth assays, gene expression analyses and key dioxygenase activities, we propose that 3-HBA degradation is channeled exclusively through the gentisate central pathway in B. xenovorans strain LB400, whereas 4-HBA can be degraded and funneled actively into the protocatechuate and gentisate central pathways (Fig. 9). This report reveals novel catabolic capabilities of B. xenovorans LB400, which can use two different ring-cleavage pathways for the metabolism of 3-HBA and 4-HBA. This study confirms that B. xenovorans LB400 possesses a high metabolic versatility, which is reflected on its extensive gene and functional redundancy for the degradation of aromatic compounds.