Genome-wide investigation and functional characterization of the β-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeriA1501
- Danhua Li†1, 2,
- Yongliang Yan†2,
- Shuzhen Ping2,
- Ming Chen2,
- Wei Zhang2, 3,
- Liang Li2,
- Wenna Lin2,
- Lizhao Geng2,
- Wei Liu2,
- Wei Lu2, 3Email author and
- Min Lin1, 2Email author
© Li et al; licensee BioMed Central Ltd. 2010
Received: 28 September 2009
Accepted: 8 February 2010
Published: 8 February 2010
Soil microorganisms are mainly responsible for the complete mineralization of aromatic compounds that usually originate from plant products or environmental pollutants. In many cases, structurally diverse aromatic compounds can be converted to a small number of structurally simpler intermediates, which are metabolized to tricarboxylic acid intermediates via the β-ketoadipate pathway. This strategy provides great metabolic flexibility and contributes to increased adaptation of bacteria to their environment. However, little is known about the evolution and regulation of the β-ketoadipate pathway in root-associated diazotrophs.
In this report, we performed a genome-wide analysis of the benzoate and 4-hydroxybenzoate catabolic pathways of Pseudomonas stutzeri A1501, with a focus on the functional characterization of the β-ketoadipate pathway. The P. stutzeri A1501 genome contains sets of catabolic genes involved in the peripheral pathways for catabolism of benzoate (ben) and 4-hydroxybenzoate (pob), and in the catechol (cat) and protocatechuate (pca) branches of the β-ketoadipate pathway. A particular feature of the catabolic gene organization in A1501 is the absence of the catR and pcaK genes encoding a LysR family regulator and 4-hydroxybenzoate permease, respectively. Furthermore, the BenR protein functions as a transcriptional activator of the ben operon, while transcription from the catBC promoter can be activated in response to benzoate. Benzoate degradation is subject to carbon catabolite repression induced by glucose and acetate in A1501. The HPLC analysis of intracellular metabolites indicated that low concentrations of 4-hydroxybenzoate significantly enhance the ability of A1501 to degrade benzoate.
The expression of genes encoding proteins involved in the β-ketoadipate pathway is tightly modulated by both pathway-specific and catabolite repression controls in A1501. This strain provides an ideal model system for further study of the evolution and regulation of aromatic catabolic pathways.
Aromatic compounds, one of the most abundant classes of natural carbon compounds, accumulate primarily due to the degradation of plant-derived molecules (e.g., lignin). These structurally diverse compounds are independently converted to a small number of structurally simpler common intermediates, such as catechol and protocatechuate, which are subsequently metabolized to tricarboxylic acid intermediates via the β-ketoadipate pathway [1–3]. Therefore, many soil bacteria are characterized by considerable metabolic flexibility and physiological adaptability with a minimum number of functional proteins.
The β-ketoadipate pathway for degradation of aromatic compounds is widely distributed among bacteria. In addition, the microbial degradation of aromatic compounds has tremendous environmental significance. Therefore, the metabolic and genomic characteristics of the aromatic catabolic pathways from Acinetobacter, Pseudomonas, Geobacterter and Dechloromonas have been studied extensively [2, 4–6]. For example, A. baylyi ADP1 (formerly known as Acinetobacter sp. ADP1) and P. putida KT2440 have long been used as a model for studying aromatic compound biodegradation and have contributed greatly to the elucidation of gene regulation of the β-ketoadipate pathway. In A. baylyi ADP1, the β-ketoadipate pathway consists of two parallel branches for the conversion of catechol and protocatechuate, which are derived from benzoate and 4-hydroxybenzoate, respectively . At least 19 genes involved in the peripheral pathways for the catabolism of benzoate (ben) and 4-hydroxybenzoate (pob) and in the catechol (cat) and protocatechuate (pca) branches of the β-ketoadipate pathway have been identified in A. baylyi ADP1 . P. putida KT2440 is another well-characterized bacterium capable of utilizing benzoate and 4-hydroxybenzoate [2, 7–9]. Genome sequence analysis of strain KT2440 predicts the existence of the protocatechuate (pca genes) and catechol (cat genes) branches of the β-ketoadipate pathway . Further enzymatic studies and amino acid sequence data revealed that the pob, pca, ben and cat gene products are highly conserved in Acinetobacter and Pseudomonas strains. These products are usually synthesized in the presence of their respective substrates. Two different regulatory proteins, an XylS-type BenR in P. putida  and a LysR-type BenM in A. baylyi , are known to be involved in activating the ben gene expression in response to benzoate. In most cases, BenR/BenM is necessary for the ben expression but not for the expression of the cat genes, which can be regulated by CatR/CatM [11, 12]. For example, BenR and CatR jointly activate more than a dozen chromosomal ben and cat genes responsible for benzoate catabolism in P. putida [9, 13]. Thus, BenR-CatR or BenM-CatM regulation may serve as a practical model for complex regulatory circuits involved in the biodegradation of benzoate.
Aromatic compounds are not preferred as growth substrates. In most cases, synthesis of the catabolic enzymes is reduced when certain rapidly metabolizable carbon sources are simultaneously present . One such control mechanism is called catabolite repression, which can integrate different signals, thus increasing the complexity of the system . Although the molecular mechanism responsible for global control is not yet well understood, available data suggest that catabolite repression control (Crc) is a component of a signal transduction pathway that modulates carbon metabolism in some soil bacteria. In addition, Crc has also been observed in several Pseudomonas species . Very recently, A. baylyi Crc was proposed to be involved in determining the transcript stability of the pca-qui operon, thereby mediating catabolite repression .
The β-ketoadipate pathway is found almost exclusively in soil microorganisms, especially in Pseudomonas species, emphasizing the importance of aromatic compound catabolism in this family [18, 19]. Establishment of the complete genome sequence of Pseudomonas strains enabled mapping of the entire catabolic gene cluster in their chromosomes [2, 20, 21]. Despite the current extensive knowledge about the aerobic catabolism of aromatic compounds in Pseudomonas strains, there remains much more to understand. For instance, the large information gap between sequence information and function for genes responsible for aromatic catabolism is a major challenge to the field of functional genomics. In particular, the evolutionary and regulatory mechanisms of aromatic catabolic pathways in the nitrogen-fixing and root-associated bacteria have been poorly documented. P. stutzeri A1501 was isolated from paddy soil in South China in the early 1980s for its ability to fix nitrogen under microaerobic conditions in the free-living state and to colonize rice endophytically [22–24]. As previously mentioned, aromatic compounds are highly abundant in the soil, so they can serve as a normal carbon source for A1501 when this bacterium colonizes on root surfaces of host plants. In this study, genomic analysis showed that A1501 contains sets of genes encoding enzymes and regulators involved in the biodegradation of benzoate and 4-hydroxybenzoate. Herein, we present evidence that benzoate degradation is subject to catabolite repression control. We also describe, for the first time, that low concentrations of 4-hydroxybenzoate significantly enhance the ability of A1501 to degrade benzoate.
Genome-wide analysis of the aromatic catabolism pathways
Functional characterization of the β-ketoadipate pathway
A1501 grew well on 4 mM benzoate and reached an OD600 of 0.5 after 24 h of incubation, whereas no growth was observed in the presence of 8 mM benzoate. A1501 grow poorly on 0.4 mM 4-hydroxybenzoate, while 4-hydroxybenzoate at concentrations above 0.8 mM completely inhibited bacterial growth (Figure 3). Further investigation of the β-ketoadipate pathway was made by constructing and characterizing three mutants: benR mutant A1601, pcaR mutant A1602 and pcaD mutant A1603 (Table 1). When the wild type and mutants were cultured in media containing lactate, their growth rates were not affected (data not shown). As expected, the benR mutant failed to grow on benzoate, and the pcaR and pcaD mutants failed to grow on 4-hydroxybenzoate as the sole carbon source. Furthermore, both the pcaR and pcaD mutants lost their ability to utilize benzoate as a carbon source. We constructed three complementary plasmids containing the entire pcaD, pcaR and benR genes for further growth complementation assays. Complementation of the three mutants with the corresponding complementary plasmids restored the catabolic activity, and the three corresponding complementary strains grew on benzoate as the sole carbon source (data not shown). Results from gene disruption analyses and genetic complementation tests demonstrate that the three genes are required for the growth of A1501 on benzoate.
Strains and plasmids used in this study
Strains or plasmids
Source or reference
Wild type, Chinese culture CGMCC 0351, Ben++, Cat++, 4HBA+
A1501 benR::pK18mob ΔbenR, Ben-, Cat++, 4HBA+
A1501 pcaR::pK18mob ΔpcaR, Ben-, Cat-, 4HBA-
A1501 pcaD::pK18mob ΔpcaD, Ben-, Cat-, 4HBA-
A1601 harboring complement plasmid pLbenR
A1602 harboring complement plasmid pLpcaR
A1603 harboring complement plasmid pLpcaD
Kmr; oriColE1 Mob+ lacZα+, used for directed insertional disruption
Kmr; Tra+, oriColE1
Tcr; Tra-, Mob+, cos, RK2 replicon
Kmr; 293 bp EcoR I-Hind III fragment containing part of benR in pK18mob
Kmr; 299 bp EcoR I-Hind III fragment containing part of pcaR in pK18mob
Kmr; 361 bp EcoR I-Hind III fragment containing part of pcaD in pK18mob
Tcr; 1041 bp EcoR I-Hind III fragment containing benR with its native promoter in pLAFR3
Tcr; 1457 bp EcoR I-Hind III fragment containing pcaR with its native promoter in pLAFR3
Tcr; 820 bp EcoR I-Hind III fragment containing pcaD with the lac promoter in pLAFR3
Primers for RT-PCR and Quantitative Real Time RT-PCR
782 bp benRA intergenic region
456 bp benAB intergenic region
503 bp benBC intergenic region
546 bp benCD intergenic region
610 bp benDK intergenic region
1074 bp benK and catB intergenic region
309 bp catBC intergenic region
1030 bp catCA intergenic region
186 bp internal fragment
159 bp internal fragment
173 bp internal fragment
BenR activates expression of the benABCDoperon in responseto benzoate
Benzoate-mediated induction of the catBCoperon in A1501
In P. putida, the catBC operon encodes cis,cis-muconate lactonizing enzyme I (CatB) and muconolactone isomerase (CatC), which catalyze the second and third steps of the catechol branch of the β-ketoadipate pathway, respectively . The transcription of this operon requires CatR and cis,cis-muconate . Additionally, the translational starts of catR and catBC are separated by 136 bp of intervening DNA containing -35/-10-type promoters and the CatR-binding sites in P. putida [13, 33]. However, we found that only 29 nucleotides are present in the noncoding regions between benK and catB in A1501, suggesting that the promoter region of the catBC operon overlaps with the coding region of the benK gene. The promoter region of the catBC operon from A1501 shows very low similarity to those of the three other Pseudomonas strains, notably the lack of the typical binding site for CatR present in the catB promoter region of other Pseudomonas strains (Figure 6C). Although a catR orthologue could not be identified in A1501, quantitative real-time PCR experiments indicated that benzoate has the strongest induction effect on expression of the catBC operon (Figure 6D). Since benzoate induces expression of catB in the benR mutant background and this mutant is unable to metabolize benzoate, we proposed that induction of the catBC expression is not due to the production of benzoate metabolites, such as cis,cis-muconate. As reported in P. putida, induction of the catBC operon requires cis,cis-muconate, an intermediate of benzoate degradation, and CatR, a well-studied activator in the β-ketoadipate pathway . However, benzoate itself has a significant induction effect on expression of the catBC operon in A1501, strongly suggesting the existence of an uncharacterized regulatory mechanism.
Benzoate degradation in A1501 is subject to carbon catabolite repression
In Pseudomonas and Acinetobacter strains, the Crc global regulator controls the expression of genes involved in benzoate degradation when other preferred carbon sources are present in the culture medium [16, 17]. Based on sequence comparison, we found a Crc-like protein in the A1501 genome (Figure 1A). The A1501 Crc-like protein shows highest amino acid identity with P. aeruginosa Crc (86%), whereas relatively low amino acid identity (only 38%) is observed between A1501 and A. baylyi Crc proteins.
4-hydroxybenzoate enhances the ability of A1501 to degrade benzoate
A study reported that high concentrations of aromatic hydrocarbons are harmful to cells because they disrupt membrane components . In the plate assay, A1501 grew extremely poorly on 4-hydroxybenzoate as the sole carbon source with colonies of less than 1.0 mm in diameter after 3 days, whereas it produced normal-sized colonies (> 5 mm) on benzoate alone in the same period. These results indicate that 4-hydroxybenzoate itself directly inhibits A1501 growth, which is likely caused by the toxicity of 4-hydroxybenzoate. It is unclear whether the lack of pcaK results in the loss of 4-hydroxybenzoate transport, leaving A1501 unable to metabolize 4-hydroxybenzoate efficiently. In subsequent experiments, growth of A1501 was examined in a mixture of 4 mM benzoate and 0.4 mM 4-hydroxybenzoate. A1501 showed a shorter lag phase and a higher growth rate when cells were grown on the mixture than when benzoate was supplied alone (Figure 8A). Furthermore, under the latter growth conditions, the culture gradually became dark brown in color because of autoxidation of the accumulated catechol (data not shown). However, when the 4-hydroxybenzoate concentration increased to 0.8 mM, growth of A1501 was completely inhibited (Figure 8A). These results indicate that 4-hydroxybenzoate at low concentrations can enhance the ability of A1501 to grow on benzoate.
We then evaluated the effect of 4-hydroxybenzoate on the metabolism of benzoate using HPLC. When 4 mM benzoate alone was provided to the culture, it was completely consumed within 26 h, and metabolic intermediates were present. When 4 mM benzoate and 0.4 mM 4-hydroxybenzoate were provided together as growth substrates, benzoate was completely consumed within 18 h, while no discernible loss of 4-hydroxybenzoate was detected (Figure 8B). Additionally, analysis of the intracellular metabolites by HPLC revealed accumulation of catechol derived from benzoate both in the presence and absence of 4-hydroxybenzoate in the growth medium. The concentration of catechol reached 0.28 mM when A1501 grew on benzoate alone, whereas the concentration of catechol reached approximately 0.12 mM when both benzoate and 4-hydroxybenzoate were in the growth medium (Figure 8C). Collectively, these results suggest that 4-hydroxybenzoate can significantly enhance the ability of A1501 not only to degrade benzoate, but also to remove the catechol accumulated from benzoate.
The data presented here reveal that the sequence and organization of the ben, pob, cat, and pca genes in A1501 are very similar to those within other well-studied Pseudomonas strains, raising the question of whether these genes have common origins. Increasing evidence indicates that horizontal gene transfer is an efficient mechanism for introducing catabolic pathways into different bacterial genomes . In general, recently acquired transferable genomic regions are associated with insertion sequence elements and mobility-related genes, whereas anciently acquired genomic regions may lose these genetic elements . Furthermore, horizontally acquired DNA regions are usually chromosomally inserted in the vicinity of tRNA or rRNA genes . We also discovered that an rRNA operon is located directly downstream of the ben gene cluster and that a tRNA-Gly gene is located downstream of the pca gene cluster. Although insertion sequence elements and mobility-related genes are absent, the packing of the catabolic pathway genes as well as the difference in the G+C % content from the rest of the genome favor the hypothesis that the β-ketoadipate pathway in A1501 is acquired from horizontal gene transfer, which contributes to an increased adaptability in the soil environment.
As shown in Figure 2, the gene arrangement of the ben, cat, and pca clusters differs between different bacteria. Apparently, various DNA rearrangements have occurred during its evolution in each particular host. Furthermore, we observed the lack of the catR and pcaK genes, a distinguishing feature of the catabolic gene organization in A1501, suggesting that gene deletion events responsible for the loss of the two genes have occurred over a long period of evolution. In most cases, the complex regulatory circuits involving the two sets of transcriptional regulators, BenR/BenM and CatR/CatM, have evolved to allow optimal expression of catabolic genes [39, 40]. Unlike P. putida in which the transcription of the catBC operon requires CatR and cis,cis-muconate , we could not identify a catR orthologue or a consensus sequence typical of CatR-dependent promoters in A1501. In particular, benzoate, but not cis,cis-muconate, has a significant induction effect on the expression of the catBC operon in A1501. Therefore, we propose that an uncharacterized regulatory mechanism might be involved in the regulation of the β-ketoadipate pathway in A1501, but this hypothesis requires further investigation.
A1501 contains all of the enzymes involved in the 4-hydroxybenzoate degradation pathway. However, this strain shows extremely poor growth on 4-hydroxybenzoate as the sole carbon source. A plausible explanation for this observation is due to the lack of PcaK, a 4-hydroxybenzoate transporter, thereby leaving A1501 unable to metabolize 4-hydroxybenzoate efficiently. In most cases, the pcaK mutation had a negative effect on bacterial 4-hydroxybenzoate uptake and growth. For example, mutants blocked in 4-hydroxybenzoate transport have been identified in two biovars of Rhizobium leguminosarum . Growth of these mutants was completely blocked when cultured on 4-hydroxybenzoate. By contrast, growth of the P. putida pcaK mutant was not significantly impaired on 4-hydroxybenzoate at neutral pH . Furthermore, repression of 4-hydroxybenzoate transport and degradation by benzoate has been reported in P. putida . Unexpectedly, our results indicate that low concentrations of 4-hydroxybenzoate significantly enhance the ability of A1501 to degrade benzoate, potentially due to 4-hydroxybenzoate-mediated induction of enzymes, such as PcaD, required for dissimilation of benzoate by the β-ketoadipate pathway. Pesticides and industrial wastes often contain aromatic constituents, including many that are toxic to living organisms. The degradation of aromatic compound mixtures has recently received a great deal of attention. To our knowledge, this is the first report of enhanced benzoate degradation by 4-hydroxybenzoate, highlighting its potential physiological significance. The metabolic capacity for utilizing different aromatic compounds as carbon or energy sources confers a selective advantage, notably for exposure to a mixture of aromatic compounds. The findings obtained from this study will help to investigate novel regulatory mechanisms and catabolic activities that can be of great biotechnological interest for improving the microbial degradation of aromatic environmental pollutants.
We have shown that A1501 contains sets of genes encoding enzymes and regulators responsible for the entire benzoate or 4-hydroxybenzoate-degrading pathways. The unique features found in the A1501 catabolic pathway are not just rearrangements of structural genes but represent the existence of an uncharacterized regulatory mechanism and the lack of CatR, a well-studied activator in other benzoate-degrading bacteria. We also described for the first time that low concentrations of 4-hydroxybenzoate significantly enhance the ability of A1501 to degrade benzoate. More extensive studies are needed to fully understand mechanisms involved in the regulation of cat genes and to further improve the ability of A1501 to degrade aromatic environmental pollutants.
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this work are listed in Table 1. Bacterial strains were grown in Luria-Bertani (LB) and minimal lactate-containing medium (medium K), as previously described . When required, carbon sources were supplemented at the following final concentrations: 4 mM glucose, 4 mM succinate, 4 mM lactate, 4 mM acetate, 4 mM benzoate, 0.4 mM catechol and 0.4 mM 4-hydroxybenzoate. The following antibiotics were added as required at the indicated final concentrations: 10 μg/ml tetracycline (Tc) and 50 μg/ml kanamycin (Km).
Construction of nonpolar mutants
We constructed a nonpolar insertion into the benR, pcaR, and pcaD genes, respectively, by homologous suicide plasmid integration, as described previously , using pK18mob as the vector . DNA fragments (~300 bp) were amplified using the total DNA of A1501 as the template and appropriate oligonucleotide primers. Oligonucleotide primers were designed to generate amplicons for the creation of nonpolar mutations enabling transcription of downstream genes. The amplicons were ligated into the vector pK18mob and the resulting plasmids were introduced into P. stutzeri A1501 from Escherichia coli JM109 by triparental conjugation using pRK2013  as the helper plasmid. The nonpolar mutant strains A1601, A1602, and A1603 were generated in which benR, pcaR, and pcaD, respectively, were disrupted without blocking the transcription of downstream genes. Correct recombination was confirmed by PCR analysis. For further growth complementation assays, we used the broad host vector pLAFR3 to construct three complementary plasmids, pLbenR, pLpcaD and pLpcaR, as described previously . Three complementary plasmids and the corresponding complementary strains are listed in Table 1.
RT-PCR and Quantitative real-time PCR
Total RNA was isolated with an SV Total RNA Isolation System (Promega, Madison, WI, USA) and treated with RNase-free DNase I (Promega). The integrity of RNA was analyzed by agarose gel electrophoresis. To check for DNA contamination, samples were analyzed with PCR using primers for benA. First-strand cDNAs were synthesized from 1 μg of total RNA in a 20 μl reaction volume using the Protoscript First-Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA).
For quantitative real-time PCR (Q-PCR) experiments, primer pairs, as shown in Table 2, were designed based on the published reference genome sequence of P. stutzeri A1501 using the Primer 4 server. Amplicons (100 to 200 bp) and reaction specificity were confirmed by agarose gel electrophoresis and product dissociation curves. Q-PCR reactions contained 1 μl of cDNA, 10 μl of 2× QuantiTect SYBR Green PCR Master Mix (Qiagen, Hilden, Germany), 0.5 μl of each primer (20 μM stock), and 8 μl of RNase-free water. Amplifications were conducted on an ABI PRISM 7000 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 31 s at 55°C, and 31 s at 72°C, followed by a melting-curve program (55°C to 99°C, with a 5-s hold at each temperature). Q-PCR data were analyzed using the ABI PRISM 7000 Sequence Detection System Software (Applied Biosystems). All cDNA samples were run in triplicate. The expression of l6S rRNA was used as an internal control and the signal was used to normalize variations due to different reverse transcription efficiencies. The comparative CT (threshold cycle) method was used to determine the average fold induction of mRNA by comparing the CT of the target gene to that of the reference gene, as described previously . The average fold change and standard deviation from three independent RNA samples are reported for each point tested.
High-performance liquid chromatography (HPLC) analysis
To monitor metabolism, the pcaD mutant and wild-type strains were grown in minimal medium supplemented with benzoate or a mixture of benzoate and 4-hydroxybenzoate. One-milliliter culture samples were centrifuged to pellet cells. Any cells remaining in the supernatant were removed by passage through a low-protein-binding, 0.22 μm pore size, syringe filter (MSI, Westborough, MA, USA). HPLC analysis was performed using an Agilent Technologies (Santa Clara, CA, USA) 1200 series chromatography system. A 20-μl sample of the filtrate was analyzed on a C18 reverse-phase HPLC column (Agilent Technologies). Elution at a rate of 0.8 ml/min was carried out with 30% acetonitrile and 0.1% phosphoric acid, and the eluant was detected at 254 nm. Under these conditions, the retention times for benzoate, catechol, cis, cis-muconate, and 4-hydroxybenzoate standards were 6.071, 2.388, 3.358, and 2.770 min, respectively. Peak areas corresponding to standard and experimental samples were integrated using the manufacturer's software package (Agilent Technologies).
We would like to thank Dr. Russell Nicholson and Dr. Haiyang Wang for continuous support and critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (No. 30925002, 30970093 and 30800022) and the National Basic Research (973) Program of China (No. 2010CB126504).
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