Benzoate Catabolite Repression of the Phthalate Degradation Pathway in Rhodococcus sp. Strain DK17

Abstract

Rhodococcus sp. strain DK17 exhibits a catabolite repression-like response when provided simultaneously with benzoate and phthalate as carbon and energy sources. Benzoate in the medium is depleted to detection limits before the utilization of phthalate begins. The transcription of the genes encoding benzoate and phthalate dioxygenase paralleled the substrate utilization profile. Two mutant strains with defective benzoate dioxygenases were unable to utilize phthalate in the presence of benzoate, although they grew normally on phthalate in the absence of benzoate.

Aromatic hydrocarbons are found ubiquitously in the environment; indeed, second only to glucosyl residues, the benzene ring is among the most widely distributed units of chemical structure (4). Bacteria in the natural environment are thus often presented with mixtures of aromatic compounds, which can be utilized as growth substrates. Successful growth under these conditions depends on the bacterium's ability to quickly choose the best available carbon source and adapt gene expression for efficient metabolism. During the past three decades, much research has centered on elucidating the metabolic pathways for degradation of various aromatic hydrocarbons, and the details of these metabolic pathways at the biochemical and molecular levels have been relatively well documented (5, 13, 26, 27, 28). In contrast, very little work has been reported on the degradation of mixtures of aromatic compounds or on the degradation of an individual aromatic hydrocarbon when present in a mixture of structurally similar compounds. One interesting observation was the preferred metabolism of benzoate over 4-hydroxybenzoate (4-HBA) in some gram-negative soil bacteria, such as Acinetobacter and Pseudomonas spp. (8, 21).

The metabolically versatile Rhodococcus sp. strain DK17 was originally isolated for the ability to grow on o-xylene and has the capability to utilize such aromatic compounds as benzene, alkylbenzenes (toluene, ethylbenzene, isopropylbenzene, and n-propyl- to n-hexylbenzenes), phenol, and phthalates as sole carbon and energy sources (2, 14, 15, 16, 17). This catabolic versatility led us to investigate the adaptability of DK17 to the presence of multiple aromatic hydrocarbons as carbon and energy sources.

As an initial step to elucidate the response of DK17 to simultaneously available aromatic hydrocarbons, the effects of benzoate and phthalate on each other's metabolism in DK17 were examined. These two aromatic acids were selected mainly due to their structural similarities. DK17 was grown overnight in 50 ml of mineral salt basal (MSB) liquid medium (25) containing 20 mM glucose. The overnight-grown cells were harvested, washed, resuspended in 100 ml of fresh MSB medium containing 5 mM benzoate plus 5 mM phthalate at an initial optical density at 600 nm (OD₆₀₀) of approximately 0.1, and incubated at 30°C with shaking (180 rpm). The numbers of CFU were determined by plating the appropriate dilutions of the culture. Also, a half milliliter of the culture was harvested at the same time intervals, and the concentrations of benzoate and phthalate were determined by using high-pressure liquid chromatography (HPLC). HPLC analysis was performed with a Hewlett-Packard model 1100 HPLC apparatus equipped with a 5-μm ZORBAX column (4.6 by 250 mm). The mobile phase used was a 45-min linear gradient of methanol-water (from 5% to 95%) containing 1% acetic acid at a flow rate of 1.0 ml/min. As shown in Fig. 1, DK17 exhibited diauxic growth when cultivated on a mixture of benzoate and phthalate. The benzoate concentration in the culture supernatant began to decrease sharply as cells entered the first exponential growth phase, and it reached almost zero in the subsequent 10 h (4.83 ± 0.07 mM benzoate at hour 4 to 0.0 mM benzoate at hour 14). The concentration of phthalate began to drop, however, only after the concentration of benzoate reached almost zero. In contrast, in a parallel experiment where only phthalate was added to the DK17 culture, the disappearance of phthalate was observed starting as early as two hours after the addition of substrate (data not shown).

FIG. 1.

Preferential utilization of benzoate over phthalate by Rhodococcus sp. strain DK17. A glucose-grown culture was used as the inoculum. Numbers of CFU (hatched circles) and concentrations of benzoate (open circles) and phthalate (closed circles) are shown. Error bars around each symbol represent the standard deviation of the measurements taken at that time point over three assay replications. The upper panels show results of an agarose gel electrophoresis of RT-PCR products for oxygenase component large subunits of benzoate (benA1, 1,377 bp) and phthalate (ophA1, 1,446 bp) dioxygenases and for a 1,466-bp 16S rRNA gene fragment as an internal RT-PCR control. The first lanes were loaded with molecular size markers (1,018, 1,636, and 2,036 bp). Numbers above the lanes indicate cell-harvest time points. Proposed pathways for early steps in benzoate and phthalate degradation by Rhodococcus sp. strain DK17 are shown in the lower panel.

The above results suggest that the presence of benzoate inhibits DK17 phthalate metabolism. In order to further examine this possibility, reverse transcription (RT)-PCR experiments were performed. DK17 cells exposed to 5 mM benzoate plus 5 mM phthalate were collected at various time points. Cells were broken with glass beads in a FastPrep FP120 system (BIO 101), and total RNA extraction was carried out according to the method of Chomczynski and Sacchi (3). The extracted total RNA was further purified by spin column and DNase I treatments according to the manufacturer's instructions (QIAGEN, Germany). RT-PCRs were performed by using a 20-μl solution with 100 ng of total RNA and 10 pmol of each primer with a ONE-STEP RT-PCR PreMix kit (iNtRON, Korea). Primer sequences for benzoate dioxygenase were 5′-ATGACTGACACCCTGTAC-3′ (benA1 forward) and 5′-TCAGCGGTTGTTCGCGGC-3′ (benA1 reverse) and were based on the gene sequence of the benzoate dioxygenase large subunit from Rhodococcus sp. RHA1 (18). Indeed, the application of these primers amplified an approximately 1.4-kb fragment, as expected from the benA1 gene target. Subsequent cloning and sequencing of the PCR product revealed 99% identity with the nucleotide sequence of benA1 of RHA1 (18). Primer sequences for phthalate dioxygenase were 5′-ATGATCCCGGCGCACATC-3′ (ophA1 forward) and 5′-TCATGCCAGCACCGCCCC-3′ (ophA1 reverse) and were based on our previous work on the induction of the DK17 phthalate operon (2). The thermocycler program used for the RT-PCRs was as follows: 45°C for 30 min; 94°C for 5 min; 30 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min; and 72°C for 5 min. As displayed in the two uppermost panels of Fig. 1, benA1 transcripts had appeared already at hour 2, continued to be expressed until hour 13, and became undetectable by hour 14. In contrast, ophA1 transcripts began to appear only at hour 12. Furthermore, RT-PCR experiments clearly show that a shift in gene expression from benA1 to ophA1 occurs between hours 12 and 14. Also, by using the 27F and 1492R universal primers (12), the 16S rRNA was amplified by RT-PCR as an internal control, which showed no significant variation throughout the 22-hour incubation period (Fig. 1, third panel from top). Taken together with the data on the preferential consumption of benzoate, these RT-PCR results strongly suggest that benzoate mediates a certain form of transcriptional repression over the utilization of phthalate by transcriptional inhibition of the oph operon in DK17.

In order to better address the issue of benzoate repression on phthalate utilization by DK17, attempts were made to generate mutant strains defective in the metabolism of benzoate. UV mutagenesis was performed according to the method of Carlton et al. (1) with slight modification as described previously (17). After approximately 1,000 colonies were screened, one mutant strain, designated KC710, was isolated for the inability to grow on benzoate as well as the inability to utilize benzoate. Although KC710 is unable to grow on benzoate, it is still able to grow normally on other aromatic acids, such as phthalate, terephthalate, and vanillate. Both DK17 and KC710 took four, three, or four days to form a 1.0-mm-diameter colony on MSB plates containing 5 mM of phthalate, terephthalate, or vanillate, respectively, as the sole carbon source. The mutant strain KC710 was grown overnight in 50 ml of MSB liquid medium containing 20 mM glucose. The overnight-grown cells were harvested, washed, and resuspended in 100 ml of fresh MSB medium containing 5 mM benzoate plus 5 mM phthalate or containing 5 mM phthalate. As clearly shown by the data presented in Fig. 2, benzoate completely inhibits the ability of KC710 to grow on phthalate, despite the fact that KC710 grows normally on phthalate in the absence of benzoate. Using the nucleotide sequence as a guide, we determined that the mutant strain KC710 has a nonsense mutation in the 78th codon of the benA1 gene (a CAG Gln codon changed to a TAG stop codon). An amino acid sequence alignment of iron sulfur protein large subunits of different benzoate dioxygenases (lower panel in Fig. 2) clearly shows the conservation of two cysteine-histidine pairs and an amino acid triplet (two histidines plus one aspartate), which coordinate the Rieske iron-sulfur center and the mononuclear iron, respectively (7). This mutation in KC710 is thus predicted to result in the production of a truncated BenA1 protein (less than 20% of its normal size of 457 amino acid residues) without two functionally important domains: a Rieske domain and a catalytic domain. Through a further screening of 1,000 additional UV-treated colonies, we identified a second mutant strain, KC720, which is unable to grow on benzoate. KC720 also retained the ability to grow on phthalate, terephthalate, and vanillate as fast as DK17. When growing in MSB medium containing 5 mM benzoate plus 5 mM phthalate, KC720 showed the same phenotype as the mutant strain KC710, and its growth on phthalate was completely inhibited by the presence of benzoate. It was also determined that KC720 has a missense mutation in the 234th codon of the benA1 gene (a TAC Tyr codon changed to a CAC His codon) (Fig. 2, lower panel).

FIG. 2.

Growth of the benzoate-negative mutant Rhodococcus sp. strain KC710 with phthalate alone or a mixture of benzoate and phthalate. A glucose-grown culture was used as the inoculum, and each substrate was provided at a final concentration of 5 mM. Error bars around each symbol represent the standard deviation of the measurements taken at that time point over three assay replications. The lower panel shows the alignment of iron sulfur protein large subunits of different benzoate dioxygenases, revealing the conservation of the Rieske iron-sulfur center and the mononuclear iron-binding site. Conserved amino acids are in boldface type. The cysteine-histidine pairs of the Rieske iron-sulfur center are marked by asterisks. The inverted triangles indicate the 2-His-1-carboxylate motif for the coordination of the mononuclear iron. An arrow indicates the Tyr234 residue, which is changed to histidine in the mutant strain KC720. DK17, RHA1, 19070, ADP1, YS-314, and 2CBS designate iron sulfur protein large subunits of benzoate dioxygenases from Rhodococcus sp. strain DK17, Rhodococcus sp. strain RHA1 (18), Rhodococcus sp. strain 19070 (10), Acinetobacter calcoaceticus ADP1 (20), Corynebacterium efficiens YS-314 (22), and Burkholderia cepacia 2CBS (9), respectively.

The present work provides strong evidence that benzoate completely inhibits the ability of Rhodococcus sp. strain DK17 to utilize phthalate. This is something of a surprise, because we previously observed that the gene encoding protocatechuate 3,4-dioxygenase was expressed following growth on either benzoate or phthalate (24). Recently, Patrauchan et al. (23) also reported catabolic redundancy in the downstream pathway for benzoate and phthalate degradation in Rhodococcus sp. strain RHA1, which is similar to DK17 in its metabolic capability to degrade monocyclic aromatic hydrocarbons. In Pseudomonas putida PRS2000, on the other hand, benzoate represses 4-HBA degradation, allowing the bacterial cells to utilize benzoate in preference to 4-HBA (21). The authors suggested an explanation for this preference, in terms of the energetic demands of the early steps of 4-HBA and benzoate. Namely, the conversion of 4-HBA to protocatechuate by a monooxygenase reaction requires the oxidation of NADPH (6), while the dioxygenase-catalyzed oxidation of benzoate to catechol consumes no net reducing equivalents, because the NADH oxidized in the initial oxidation of benzoate to 2-hydro-1,2-dihydroxybenzoate (cis-benzoate dihydrodiol) is recovered during the next dehydrogenation to catechol by an NAD⁺-dependent dehydrogenase (19). However, this explanation cannot apply to Rhodococcus sp. strain DK17, since the initial steps in the metabolism of both benzoate and phthalate are performed by the combination of the corresponding dioxygenase and dehydrogenase (2), resulting in a zero net utilization of NAD(P)H. Also, from the growth curve in Fig. 1, the doubling times of DK17 on benzoate and phthalate were determined to be 4.2 ± 0.11 and 2.8 ± 0.04 h, respectively. The much faster growth rate of DK17 on phthalate is seemingly contradictory to the preferential utilization of benzoate. However, as pointed out by Higgins and Mendelstam (11), it is impossible to decide whether growth rate was the factor determining the evolution of the corresponding control system without complete knowledge of the natural habitat of the organism. Their assertion was also based on the observation that benzoate was used in preference to mandelate by a Pseudomonas putida strain, although the latter supports a faster growth rate. Even though more extensive studies are needed to completely understand the catabolite repression of phthalate degradation by benzoate in DK17, the finding obtained from this study provides new insights into bacterial degradation of aromatic hydrocarbons when present in a mixture of analogous compounds.