INTRODUCTION

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A general strategy used by microbes to degrade diverse aromatic compounds anaerobically is to first convert them to benzoyl-coenzyme A (benzoyl-CoA), a compound that is the starting point for a central pathway of aromatic ring reduction and cleavage (18). Peripheral reactions leading to benzoyl-CoA formation include modification and removal of benzene ring substituents, often via 4-hydroxybenzoate (4-HBA) or 4-hydroxybenzoyl-CoA as an intermediate. The conversion of 4-HBA to benzoyl-CoA is a relatively well-studied reaction sequence (Fig. 1). First, CoA is added to 4-HBA by 4-hydroxybenzoate-CoA ligase. Enzymes catalyzing this reaction have been purified from the purple nonsulfur bacterium Rhodopseudomonas palustris (16) and from the denitrifying species Thauera aromatica (4). The R. palustris gene encoding this enzyme, hbaA, has been cloned and sequenced (16). 4-Hydroxybenzoyl-CoA (4-HBA-CoA) is then dehydroxylated by 4-HBA-CoA reductase to yield benzoyl-CoA (17). The genes encoding the dehydroxylating reductase have been cloned from both R. palustris and T. aromatica, and sequenced, and this enzyme has been purified from T. aromatica (5).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   The hba gene cluster for 4-HBA degradation. These genes are part of a 26-kb cluster of genes whose products are involved in anaerobic degradation of aromatic compounds by R. palustris (12). The roles of the hba gene products in the conversion of 4-HBA to benzoyl-CoA are also shown (16, 17).

In previous work on anaerobic 4-HBA degradation, we identified a transcriptional activator, AadR, that is required for anaerobic growth of R. palustris on 4-HBA and for expression of hbaA (9). AadR is a member of the FNR family of transcriptional regulators, and based on the presence of the conserved cysteine residues shown to be essential for sensing anaerobiosis by FNR (23), we have proposed that AadR functions in oxygen sensing (9). In addition to being involved in expression of hbaA, AadR is required for expression of the benzoyl-CoA reductase genes, badDEFG (11). Here we describe HbaR, a second transcriptional activator and member of the FNR-CRP superfamily that is involved in regulating the anaerobic degradation of 4-HBA in response to 4-HBA as an effector molecule.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. The bacterial strains and plasmids used are described in Table 1. R. palustris cultures were grown anaerobically in defined mineral medium (21) prepared as described previously (19). Carbon sources were added at the time of inoculation to a final concentration of 3 mM except for succinate, which was added to 10 mM. Cultures were incubated at 30°C and illuminated with a 40-W incandescent light bulb for phototrophic growth or in the dark with shaking for aerobic growth. Escherichia coli cultures were routinely grown in Luria broth (LB) (7) at 37°C. For -galactosidase activity assays, E. coli cultures were grown in LB containing 0.2% glucose, either aerobically with shaking or anaerobically in butyl-rubber-stoppered tubes incubated statically. Pseudomonas aeruginosa cultures were grown at 37°C in LB for routine cultivation and in defined minimal medium (25) with 10 mM succinate for cultures to be assayed for -galactosidase activity. Antibiotics were used at the following concentrations (in micrograms per milliliter): for R. palustris, gentamicin (100) and kanamycin (100); for P. aeruginosa, kanamycin (100) and gentamicin (20); and for E. coli, ampicillin (100), gentamicin (10), kanamycin (100), and spectinomycin (100).

Cloning and DNA manipulation. Standard protocols were used for cloning and transformations (30). Plasmid DNA was purified by using the QIAprep Spin Miniprep kit (Qiagen Inc., Chatsworth, Calif.). DNA fragments were purified from agarose gels by using the GeneClean spin kit from Bio 101 (La Jolla, Calif.). Chromosomal DNA was purified by using a variation on the method of Saito and Miura (29) as described previously (10).

Expression of HbaR in E. coli. HbaR was expressed by using the T7 promoter system (3). DNA containing hbaR was PCR amplified and cloned into the NdeI and SmaI sites of pT7-7 (35) to generate pPE905. E. coli BL21(DE3) cells containing pPE905 were grown in LB medium to an A₆₆₀ of approximately 0.25. Cells (1 ml) were harvested, washed, and resuspended in 1.0 ml of basal medium supplemented with 0.02% concentrations of each of 18 amino acids (no methionine or cysteine). After a 30-min incubation at 30°C, isopropyl--D-thiogalactopyranoside (IPTG) was added to 1 mM, and incubation was continued for 30 min. Rifampin was added to a final concentration of 0.5 mg/ml, and cells were incubated at 42°C for 10 min to allow rifampin to enter the cells. After an additional 30 min at 30°C, cells were incubated with 10 µCi of [³⁵S]methionine for 2 min. Proteins were separated on sodium dodecyl sulfate-15% polyacrylamide gels.

Gel mobility shift assays. Gel mobility shift assays were attempted by using several different protocols (15, 27). Binding buffers containing various salts at a range of concentrations were used. Target DNA fragments of different lengths were also tried.

Primer extension and reverse transcriptase PCR analysis. Primer extension analysis was used to determine the transcriptional start sites of hbaR and hbaA. The avian myeloblastosis virus reverse transcriptase primer extension system was used according to the protocol supplied by the manufacturer (Promega Corp., Madison, Wis.). The primer used to map the hbaR start site was complementary to nucleotides 13 to 33 of hbaR. The primer used to map the hbaA start site was complementary to bases 147 to 164 of hbaA. Primer extension products were analyzed on a 6% polyacrylamide gel next to a sequence ladder generated by using the same primers. Sequencing reactions were performed with the fmol DNA sequencing system from Promega.

DNA sequencing and analysis. DNA sequences were determined at the University of Iowa DNA Facility by using dye terminator cycle sequencing. The reactions were run on and analyzed with an Applied Biosystems model 373A stretch fluorescent automated sequencer. DNA sequences were analyzed with GENE Inspector, version 1.0.1 (Textco Inc., West Lebanon, N.H.). Similar sequences were identified from the SWISS-PROT 26 and GENPEP 78.0 databases by using the BLAST network service at the National Center for Biotechnology Information (Bethesda, Md.). The GAP program from the University of Wisconsin Genetics Computer Group software package, version 9.0, was used to make sequence comparisons and alignments. The AllAll program from the Computational Biochemistry Research Group (cbrg.inf.ethz.ch./section3_1.html) was used for phylogenetic analysis.

Enzyme assays. 4-HBA-CoA ligase activity was measured by using a spectrophotometric assay described previously (16). Briefly, reaction mixtures containing 50 mM Tris (pH 9.2), 5 mM MgCl₂, 0.5 mM ATP, 0.8 mM reduced CoA, 0.25 mM 4-HBA, and cell extract were monitored for increase in absorbance at 330 nm due to formation of 4-HBA-CoA. Activity was calculated by using a millimolar extinction coefficient of 24.

Nucleotide sequence accession number. The DNA sequence of hbaR has been assigned GenBank accession no. AF172325.

	RESULTS

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Characteristics of hbaR. A 726-bp open reading frame designated hbaR was found next to the R. palustris genes encoding enzymes responsible for the conversion of 4-HBA to benzoyl-CoA (Fig. 1) (16, 17). HbaR was similar in its deduced amino acid sequence to members of the FNR-CRP superfamily of transcriptional regulators. It was most similar (52% similar, 42% identical) to NNR, a positive regulator of nitrite and nitric oxide reductase gene expression in Paracoccus denitrificans (37). The other proteins with the highest levels of amino acid sequence identity to HbaR were members of the DNR group of the FNR-CRP superfamily of transcriptional regulators, as described by Vollack et al. (39) (Fig. 2). HbaR had a low level of amino acid sequence similarity (36%) to the R. palustris protein AadR, an FNR-like regulator that contains the conserved cysteine residues shown to be involved in redox sensing by FNR (9, 23). HbaR does not contain these cysteine residues.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Phylogenetic tree of selected members of the FNR-CRP superfamily of transcriptional regulators constructed with the AllAll program from the Computational Biochemistry Research Group. Subdivisions shown are those proposed by Fischer (13) and recently modified by Vollack et al. (39). The proteins and their sources and accession numbers, respectively, are as follows: AadR, R. palustris, M92426; ANR, P. aeruginosa, P23926; CLP, Xanthomonas campestris, M58745; CRP, E. coli, U18997; CysR, Synechococcus sp., AAA73046; DNR, P. aeruginosa, D50019; DnrD, DnrE, and DnrS, P. stutzeri, AJ131715, AJ131716, and AJ131717, respectively; FixK (A. caul.), Azorhizobium caulinodans, P26488; FixK (B. jap.) and FixK2 (B. jap.), Bradyrhizobium japonicum, M86805 and CAA06287, respectively; FixK (R. mel.), Rhizobium meliloti, X15079; FNR, E. coli, P03019; FnrA, P. stutzeri, Z26044; FnrL, Rhodobacter capsulatus, U78309; FnrP, Paracoccus denitrificans, U34353; HbaR, R. palustris, AF172325; NNR, Paracoccus denitrificans, U17435; NtcA, Anabaena variabilis, Q05061.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   (A) Mapping the hbaR start site by primer extension. Lane 1 contains the primer extension product from RNA isolated from cells grown anaerobically on succinate. A sequencing ladder generated with the same primer is shown. The arrow indicates the start site of transcription. (B) Nucleotide sequence of the hbaR promoter region showing the start site of transcription (+1), the putative 10 region (underlined), and the inverted repeat matching the consensus FNR binding site (inverted arrows). (C) Alignment of FNR boxes from R. palustris promoter regions (9, 11) and the consensus FNR binding sequence (33).

Characterization of an hbaR mutant. An hbaR mutant (strain CGA612) was unable to grow on 4-HBA under anaerobic conditions, but it grew at wild-type rates on benzoate. The hbaR mutant also grew at wild-type rates on succinate and, like the wild-type parent strain, could grow aerobically on 4-HBA. The defect in anaerobic growth on 4-HBA was complemented in trans by a plasmid-borne copy of hbaR supplied on pPE901. The growth phenotype of this mutant suggested that hbaR is involved in regulating the enzymes that convert 4-HBA to benzoyl-CoA. The first step in this process is the addition of CoA to 4-HBA by 4-HBA-CoA ligase. The activity of 4-HBA-CoA ligase in extracts from wild-type cells grown anaerobically with 4-HBA (0.3 mM) plus succinate (10 mM) was 10.2 nmol/min/mg of protein, while the hbaR mutant had levels of 4-HBA-CoA ligase activity below the level of detection for the assay used (<0.25 nmol/min/mg of protein).

HbaR activates expression of 4-HBA-CoA ligase in response to 4-HBA. To examine a possible regulatory role of HbaR, the hbaR mutation was introduced into an R. palustris strain containing a chromosomal hbaA::'lacZ fusion (CGA507). Levels of expression of hbaA, the gene encoding 4-HBA-CoA ligase, in wild-type and hbaR (CGA614) backgrounds were then compared. Cells with an intact hbaR gene had 20-fold-higher levels of hbaA expression when 4-HBA was present in anaerobic growth medium than when they were grown on succinate only. In contrast, expression of the hbaA::'lacZ fusion was not induced by 4-HBA in hbaR mutant cells (Table 2). This 20-fold activation by HbaR is consistent with the difference in 4-HBA-CoA ligase activity between the wild type and hbaR mutant.

View this table: [in this window] [in a new window]   TABLE 2.   Effects of hbaR on -galactosidase activity expressed from a chromosomally encoded hbaA::'lacZ fusion in R. palustris

HbaR is active under aerobic conditions. Primer extension analysis was used to identify the promoter region of hbaA (Fig. 4). The possibility that HbaR is active in the presence of oxygen was investigated by expressing hbaR in aerobically growing P. aeruginosa cells containing a reporter plasmid (pMD505) that had the hbaA promoter fused to a promoterless lacZ gene. Such cells exhibited a fivefold increase in -galactosidase expression over the levels seen in the absence of hbaR (Fig. 5). When 4-HBA was present in the growth medium, levels of -galactosidase activity increased twofold more over levels in cells grown on succinate only, for a net 10-fold induction. This suggests that HbaR acts directly at the hbaA promoter and activates expression in response to 4-HBA. In addition, this shows that, although hbaA is not expressed in aerobically growing R. palustris cells (Table 2), HbaR is able to activate transcription from the hbaA promoter in the presence of oxygen.

View larger version (43K): [in this window] [in a new window]   FIG. 4.   (A) Mapping the hbaA start site by primer extension. Lane 1 contains the primer extension product (arrow) from RNA isolated from cells grown anaerobically on 4-HBA. A sequencing ladder generated with the same primer is shown. (B) Nucleotide sequence of the hbaA promoter region showing the start site of transcription (+1) and a putative 10 region that matches the E. coli 70 consensus (TATAAT) at four of six positions.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Expression of -galactosidase activity from P. aeruginosa cells containing either the PhbaA-'lacZ reporter plasmid pMD505 (filled bars) or a promoterless negative control (pHRP311) (open bar) and either the HbaR-expressing plasmid pPE901 (+HbaR) or the vector pBBR1MCS-2 (HbaR). Cells were grown aerobically on succinate in the presence or absence of 0.5 mM 4-HBA. -Galactosidase activity is in Miller units. Error bars represent standard deviations.

Expression of HbaR in E. coli. HbaR was expressed in E. coli cells from the plasmid pPE905 as described in Materials and Methods. The apparent molecular mass of the HbaR peptide (25 kDa) was close to that predicted based on the deduced amino acid sequence of HbaR (28.5 kDa). E. coli cell extracts containing overexpressed HbaR were tested to see if they would cause a shift in gel mobility of DNA fragments containing the hbaA promoter region. No change in the mobility of a fragment containing the hbaA promoter region was seen with these extracts or with extracts from cultures of R. palustris or P. aeruginosa expressing HbaR (data not shown). The addition of 4-HBA to the gel shift assay mixtures had no apparent effect on binding. An HbaR-His fusion peptide purified to near homogeneity was also tested in gel mobility shift assays. The purified peptide was mostly insoluble, precipitated out of solution easily, and bound nonspecifically to all DNA fragments tested including vector DNA (data not shown).

E. coli FNR can activate hbaR expression. The hbaR promoter region has an inverted repeat centered at 42.5 bp from the start site of transcription that matches the FNR consensus binding site (Fig. 3B). To determine whether hbaR expression might be activated by an FNR-type regulator, a 226-bp fragment containing the hbaR promoter region was cloned in front of the promoterless lacZ gene in pHRP309 by using a two-step cloning system described previously (26). -Galactosidase expression from the PhbaR-'lacZ plasmid (pPE906) was measured in wild-type and fnr mutant E. coli cells. In wild-type cells, the levels of PhbaR-'lacZ fusion expression were threefold higher when cells were grown anaerobically than when cells were grown in the presence of oxygen (Table 3). PhbaR-'lacZ activity was not induced in response to anaerobiosis in the fnr mutant strain of E. coli. This suggests that hbaR could also be regulated by an FNR-like protein in R. palustris. Similar experiments were done to examine the possible effect of FNR on hbaA expression. We found that -galactosidase expression from a PhbaA-'lacZ plasmid present in E. coli was not influenced by the fnr mutation.

View this table: [in this window] [in a new window]   TABLE 3.   Effects of fnr on -galactosidase activity expressed from a plasmid-borne PhbaR-'lacZ fusion in E. coli

	DISCUSSION

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Data presented here indicate that HbaR is a transcriptional activator that senses 4-HBA as an effector molecule and induces expression of hbaA, the gene encoding 4-HBA-CoA ligase and first enzyme of 4-HBA degradation. Although we were unable to demonstrate binding of HbaR to the hbaA promoter in vitro with gel mobility shift assays, experiments with HbaR expressed in P. aeruginosa indicate that HbaR activates expression of the hbaA promoter. These experiments also show that when expressed in P. aeruginosa, HbaR can activate expression from the hbaA promoter in the presence of oxygen (Fig. 5). In contrast, hbaA expression was not activated in aerobically grown R. palustris cells. This suggests that a second regulator is required for activation of hbaA expression in response to anaerobiosis. Our results showing that E. coli fnr influences hbaR expression and the fact that hbaR has an FNR binding site in its promoter region suggest that hbaR expression is activated in response to anaerobiosis in R. palustris by an FNR homologue. HbaR, in turn, activates hbaA expression in response to 4-HBA. The R. palustris FNR homologue in question is, presumably, AadR, since AadR is required for HbaA expression in this organism (9). This restriction of HbaR expression in R. palustris to anaerobically growing cells indicates that this organism must have a separate system for detecting 4-HBA under aerobic conditions and for activating the genes encoding enzymes of the aerobic 4-HBA degradation pathway.

HbaR is a member of the FNR-CRP superfamily of transcriptional regulators. This protein family has been divided into three classes based on sequence similarity to the reference proteins FNR, which activates gene expression in response to anaerobiosis, CRP, a regulator of catabolic functions, and NtcA, a regulator of genes involved in nitrogen and sulfur metabolism (13) (Fig. 2). The FNR class (which includes AadR of R. palustris) has been further subdivided into four groups based on the presence and spacing of the conserved cysteine residues required for assembly of the redox-sensitive iron-sulfur center. The sequence of HbaR places it in a group (DNR) of the FNR class that is comprised of proteins that lack the cysteine residues required for iron-sulfur center coordination and oxygen sensing (39). With the exception of HbaR, all the proteins within the DNR group are global regulators that control expression of genes involved in denitrification (1, 36-38).

A recently recognized theme among members of the FNR-CRP superfamily is the involvement of two members of the protein family in regulation of the same system. Transcriptional regulation of genes involved in denitrification in P. aeruginosa and Paracoccus denitrificans also involves control by two members of the FNR-CRP superfamily (2, 37). The involvement of multiple family members has also been demonstrated in Pseudomonas stutzeri, which has at least four FNR family members involved in regulating metabolic processes (39). In some cases, including the system we have described here, this multiplicity of regulators involves a regulatory cascade, with one protein acting as an oxygen sensor and, in turn, activating expression of a second regulator (2, 39). Hierarchical expression of two regulators that are members of the greater FNR family would be an effective strategy to prevent cross talk and provide regulation of relatively specialized target genes, as in the case of 4-HBA degradation, under conditions of oxygen deprivation. This study expands the range of functions regulated by the greater FNR-CRP superfamily to include aromatic carbon source utilization and expands the range of effectors sensed by members of this superfamily to include aromatic acids.