Genome-wide investigation and functional characterization of the beta-ketoadipate pathway in the nitrogen-fixing and root-associated bacterium Pseudomonas stutzeri A1501

Abstract

Background: 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 beta-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 beta-ketoadipate pathway in root-associated diazotrophs.

Results: 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 beta-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 beta-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.

Conclusions: The expression of genes encoding proteins involved in the beta-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.

Figure 1

The catechol and protocatechuate branches of the β-ketoadipate pathway and its regulation in P. stutzeri A1501. (A) Localization of the gene clusters involved in degradation of benzoate and 4-hydroxybenzoate on a linear map of the chromosome. (B) Predicted biochemical steps for the catechol and protocatechuate pathways in P. stutzeri A1501. The question mark indicates an unknown mechanism that may be involved in the regulation of cat genes. Inactivation of pcaD is shown by "× " and accumulations of the intermediates catechol and cis, cis-muconate in the supernatants of the pcaD mutant are shown by red vertical arrows. Genes whose expression is under catabolite repression control (Crc) are indicated by "⊥".

Figure 2

Organization of benzoate (A) or 4-hydroxybenzoate (B) degradation gene clusters of A1501 and comparison with equivalent clusters from other bacteria. Two vertical lines indicate that the genes are not adjacent in the genome. Numbers beneath the arrows indicate the percentage of amino acid sequence identity between the encoded gene product and the equivalent product from A1501.

Figure 3

Bacterial growth of A1501 cultured in minimal medium containing 4 mM benzoate (black triangle), 8 mM benzoate (clear triangle), 0.4 mM 4-hydroxybenzoate (black dot) or 0.8 mM 4-hydroxybenzoate (clear dot).

Figure 4

Conversion of benzoate (BEN) to catechol (CAT) and cis, cis-muconate (CCM) by the pcaD mutant A1603. Cells were grown for 48 h in minimal medium supplemented with 4 mM benzoate. The elution profile of compounds separated by HPLC is shown. Accumulations of the intermediates catechol and cis, cis-muconate are indicated by red vertical arrows.

Figure 5

Transcriptional organization of the chromosomal ben-cat region. (A) The number of nucleotides in noncoding regions is shown in parentheses. Transcriptional units and directions are denoted by horizontal arrows in the upper panel. The designation and location of primers used for RT-PCR are in the lower panel. A pair of oligonucleotide primers is marked with a convergent arrow. (B) RT-PCR analysis of mRNA transcripts using gel electrophoresis of amplified cDNA fragments. The first and last lanes were loaded with molecular size markers. +, in the presence of inducer benzoate; -, in the absence of inducer benzoate.

Figure 6

Induction of the benA or catB promoters in culture media with several different inducers. The putative binding site for BenR or CatR is boxed. The putative -10/-35 promoter consensus sequences are indicated by asterisks. The predicted transcriptional start site (+1) and ribosome-binding site (RBS) are underlined. (A) Nucleotide sequence of the benR-benA intergenic region of strain A1501. (B) Quantitative real-time RT-PCR analysis of relative benA expression level of the wild type (black bars) and benR mutant (gray bars) in the presence or absence of benzoate (BEN), catechol (CAT), cis, cis-muconate (CCM), and lactate (LAC). (C) Comparison of the catB promoter of strain A1501 with those of P. putida PRS2000, P. aeruginosa PAO1 and P. fluorescens pf-5. Dashes indicate gaps to obtain maximal homology. (D) Quantitative real-time RT-PCR analysis of relative catB expression level of the wild type (black bars) and benR mutant (gray bars) in the presence or absence of benzoate (BEN), catechol (CAT), cis, cis-muconate (CCM), and lactate (LAC). Relative levels of transcripts are presented as the mean values ± SD, calculated from three sets of independent experiments.

Figure 7

Catabolite repression control in expression of the benA, catB or pcaD genes in the presence of 4 mM benzoate. Cells were harvested and transferred into minimal medium supplemented with succinate, lactate, acetate or glucose. To induce the catabolic promoter, benzoate was added to logarithmically growing cultures. Cultures were incubated at 30°C for 2 h, and samples were collected for quantitative real-time RT-PCR analysis.

Figure 8

The enhanced ability of A1501 to degrade benzoate by 4-hydroxybenzoate. (A) Time course of bacterial growth in the presence of 4 mM benzoate (black triangle) or a mixture of 4 mM benzoate and 0.4 mM (clear triangle) or 0.8 mM (clear dot) 4-hydroxybenzoate. (B) The benzoate consumption when A1501 was cultured in minimal medium containing 4 mM benzoate (black dot) or a mixture of 4 mM benzoate and 0.4 mM 4-hydroxybenzoate (clear dot), and changes in 4-hydroxybenzoate concentrations (clear diamond) were detected by HPLC. (C) The formation of catechol derived from benzoate (black square) or a mixture of benzoate and 4-hydroxybenzoate (clear square). Samples were collected at different times and the amount of the aromatic compound in the culture supernatant was determined by HPLC.