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Positive and Negative Transcriptional Regulators of Glutathione-Dependent Formaldehyde Metabolism

Jason W. Hickman,^1, Vernon C. Witthuhn Jr.,¹ Miguel Dominguez,² and Timothy J. Donohue^1*

Departments of Bacteriology,¹ Genetics, University of Wisconsin—Madison, Madison, Wisconsin²

Received 23 April 2004/ Accepted 1 September 2004

	ABSTRACT

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A glutathione (GSH)-dependent pathway is used for formaldehyde metabolism by a wide variety of prokaryotes and eukaryotes. In this pathway, S-hydroxymethylglutathione, produced by the reaction of formaldehyde with the thiolate moiety of glutathione, is the substrate for a GSH-dependent formaldehyde dehydrogenase (GSH-FDH). While expression of GSH-FDH often increases in the presence of metabolic or exogenous sources of formaldehyde, little is known about the factors that regulate this response. Here, we identify two signal transduction pathways that regulate expression of adhI, the gene encoding GSH-FDH, in Rhodobacter sphaeroides. The loss of the histidine kinase response regulator pair RfdRS or the histidine kinase RfdS increases adhI transcription in the absence of metabolic sources of formaldehyde. Cells lacking RfdRS further increase adhI expression in the presence of metabolic sources of formaldehyde (methanol), suggesting that this negative regulator of GSH-FDH expression does not respond to this compound. In contrast, mutants lacking the histidine kinase response regulator pair AfdRS or the histidine kinase AfdS cannot induce adhI expression in the presence of either formaldehyde or metabolic sources of this compound. AfdR stimulates activity of the adhI promoter in vitro, indicating that this protein is a direct activator of GSH-FDH expression. Activation by AfdR is detectable only after incubation of the protein with acetyl phosphate, suggesting that phosphorylation is necessary for transcription activation. Activation of adhI transcription by acetyl-phosphate-treated AfdR in vitro is inhibited by a truncated RfdR protein, suggesting that this protein is a direct repressor of GSH-FDH expression. Together, the data indicate that AfdRS and RfdRS positively and negatively regulate adhI transcription in response to different signals.

	INTRODUCTION

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Formaldehyde is a cytogenic compound that is produced by environmental, industrial, and metabolic processes including the oxidative demethylation of amino acids and osmoprotectants or oxidation of one-carbon compounds like methanol, methyl halides, and methane (10, 11, 16, 20-22, 24, 35, 37, 38). The toxicity of formaldehyde stems from its reactivity with amino and sulfhydryl groups of biological molecules, causing alkylation, mutations, and cross-links that destroy the function of membranes, proteins, and nucleic acids (18, 25). When the sources of formaldehyde, its toxicity, and the carbon skeletons and reducing power derived from its oxidation are considered, it is clear that cells benefit from metabolism of this compound.

We are studying formaldehyde metabolism by the facultative bacterium Rhodobacter sphaeroides. This -proteobacterium uses a glutathione (GSH)-dependent pathway for formaldehyde metabolism that is similar to those present in several prokaryotic and eukaryotic cells (5, 7). In this pathway, formaldehyde reacts with the thiolate moiety of GSH to form S-hydroxymethylglutathione, a substrate for a GSH-dependent formaldehyde dehydrogenase (GSH-FDH). GSH-FDH oxidizes S-hydroxymethylglutathione to S-formylglutathione with concomitant reduction of NAD. S-Formylglutathione is hydrolyzed to glutathione and formate before oxidation of formate to CO₂ by a NAD-dependent formate dehydrogenase. Evidence supporting the role of GSH-FDH in this pathway includes the inability of GSH-FDH mutants to grow in the presence of metabolic sources of formaldehyde and the defect in formaldehyde oxidation seen in cells that lack this enzyme (5, 19).

Given this role of GSH-FDH, it makes sense that expression of this enzyme is regulated. In Saccharomyces cerevisiae, GSH-FDH levels are increased by the presence of methylated compounds like methyl methanesulphonate or formaldehyde (36). In many eubacteria, including R. sphaeroides, expression of the GSH-FDH gene (adhI) is increased when either formaldehyde or metabolic sources of this compound, such as methanol or choline, are present (6, 15, 17). These and other results led to the proposal that a pathway intermediate, possibly formaldehyde itself, increases adhI transcription. Additional support for the regulated expression of R. sphaeroides GSH-FDH expression includes the existence of mutations, such as the spd-7 allele, that increase adhI transcription (5). Unfortunately, little molecular information is available on the proteins or signals that regulate expression of GSH-FDH homologs.

To increase our understanding of GSH-FDH expression, we have identified genes that control adhI transcription. In one set of experiments, we utilized a previously characterized mutant containing the spd-7 allele, which increases expression of the adhI-cycI operon (30). Increased adhI-cycI expression by the spd-7 allele restores photosynthetic growth to a mutant that lacks cytochrome c₂ by increasing levels of the cytochrome c₂ isoform, isocytochrome c₂, encoded by cycI (31). By screening for wild-type DNA sequences that abolished photosynthetic growth of cells containing the spd-7 allele, we identified a putative two-component signal transduction system that negatively regulates adhI-cycI transcription, which we termed RfdRS (repressor of formaldehyde dehydrogenase). Mutants lacking RfdRS still increase adhI transcription in response to metabolic sources of formaldehyde, suggesting the presence of additional regulators of adhI transcription. By searching the R. sphaeroides genome sequence, we identified a second related two-component regulatory system, AfdRS (activator of formaldehyde dehydrogenase), that stimulates adhI transcription in vitro and is necessary for formaldehyde-dependent induction of adhI expression in vivo. Both AfdRS and RfdRS display similarity with proteins in the methanol-oxidizing bacterium Paracoccus denitrificans (FlhRS) that have been implicated in controlling the expression of GSH-FDH in that organism. Based on these findings, we propose that R. sphaeroides GSH-FDH expression is positively and negatively regulated. Our data indicate that AfdRS is necessary for formaldehyde-dependent increases in adhI transcription, while negative regulation of adhI transcription by RfdRS responds to a different and unknown signal.

	MATERIALS AND METHODS

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Strains, plasmids, and growth conditions. Table 1 lists strains and plasmids. R. sphaeroides was grown in Sistrom's minimal medium A containing 35 mM succinate at 30°C (33). For photosynthetic growth on solid medium, plates were placed in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, Md.) with H₂-CO₂ generators. Escherichia coli strain DH5 was used as a plasmid host, S17-1 was used for conjugation of plasmids into R. sphaeroides (11), and ER2566 was used for expression of intein-chitin-binding domain fusions. E. coli strains were typically grown in Luria-Bertani medium at 37°C. When necessary, the medium was supplemented with spectinomycin (25 µg/ml), kanamycin (25 µg/ml), trimethoprim (30 µg/ml), or tetracycline (1 µg/ml) for R. sphaeroides and ampicillin (100 µg/ml), tetracycline (10 µg/ml), spectinomycin (25 µg/ml), or kanamycin (25 µg/ml) for E. coli. All primer sequences used are available from the authors upon request.

Cloning and inactivation of rfdRTS or afdRTS. To create a strain lacking RfdRTS, an Sp^r cartridge was inserted into codon 23 of the rfdR gene (rfd-1 allele). To do this, a 2.0-kb HindIII fragment containing the Sp^r cartridge was purified from pHP45, and the ends of this fragment were filled in by incubation with the Klenow fragment of DNA polymerase. This fragment was ligated into a filled-in XhoI site of pVW17-4, a plasmid which contains a BamHI fragment encoding rfdR from pUI8017. The resultant plasmid, pRfdR1, was digested with BamHI, and a 3.4-kb fragment carrying the interrupted rfdR gene was purified and its end was filled in. This fragment was ligated into a filled-in EcoRI site of pSUP202. This Tc^r Sp^r plasmid, pRfdR2, was mobilized into wild-type R. sphaeroides, and colonies exhibiting an Sp^r Tc^s phenotype were analyzed with genomic Southern blots to ensure that the rfd-1 allele was incorporated by an even number of crossovers (data not shown).

To create a strain lacking RfdS, a 2.2-kb EcoRI fragment containing the last 250 codons of rfdT and the first 459 codons of rfdS was cloned into EcoRI-digested pUC19, generating plasmid pVW17-35. This plasmid was digested with ClaI and StuI and filled in with a Klenow fragment of DNA polymerase, deleting codons 6 to 290 of rfdS. A 2.0-kb SmaI fragment containing the Sp^r cartridge was purified from pHP45 and inserted into this mutant rfdS gene by ligation into digested pVW17-35 (rfd-2 allele). This plasmid, pRfdS1, was digested with EcoRI, and the 4.2-kb fragment containing the R. sphaeroides DNA with the Sp^r cartridge was isolated and ligated into pSUP202 that had been digested with EcoRI. This suicide plasmid, pRfdS2, was introduced into wild-type R. sphaeroides, and mutants were verified as described above.

A 600-bp in-frame deletion in rfdT (codons 71 to 271) was generated by a two-step process. In the first step, PCRs were performed to create products surrounding the region of rfdT to be deleted, each of which contained a 10-bp complementary overlap at the 3' end of the upstream product and the 5'end of the downstream product. These products were mixed in a 1:1 ratio, and a second reaction was performed by using only the outside primers from the first two reactions, resulting in the formation of a 4-kb fragment containing the rfd-3 allele and XbaI sites to facilitate cloning. The product from this second reaction was digested with XbaI, cloned into XbaI-digested pUC19 to generate plasmid pDelT4, and sequenced to confirm the desired in-frame deletion in rfdT. This 4-kb XbaI fragment was cloned into XbaI-digested pCM62. The resultant plasmid, pRfdT1, was mobilized into R. sphaeroides RfdR1 to generate a strain lacking RfdT.

For complementation of the rfdRTS mutants, a 4.6-kb fragment from pUI8017 that contains wild-type rfdRTS was generated by PCR and cloned into XbaI-digested pUC19, generating plasmid pRfd3. This 4.6-kb XbaI fragment was then cloned into XbaI-digested pCM62, generating a plasmid, pRfd4, that was used for complementation analysis.

To clone the afdRTS locus, a 5-kb fragment containing the R. sphaeroides open reading frames RSP2591 (afdR), RSP2593 (afdS), and RSP2592 (afdT) was amplified from genomic DNA, and the PCR product was digested with KpnI, phosphorylated with T4 polynucleotide kinase, and ligated into KpnI-HincII-digested pUC19, resulting in pAfd1.

To generate a polar insertion in afdR (afd-1 allele), a 1.4-kb region containing this gene was amplified from genomic DNA, placing a KpnI site in the upstream primer. The product was purified, digested with KpnI, phosphorylated, and ligated into HincII-KpnI-digested pUC19, creating pAfdR1. A gene encoding trimethoprim resistance (Tp^r) was inserted into codon 84 of afdR by ligation into BtrI-digested pAfdR1 to generate pDelAfdR1. A 2-kb PCR product was generated from pDelAfdR1 by using vector-specific primers (1224 and 1233) and cloned into ScaI-digested pSup202 to create pDelAfdR3. pDelAfdR3 was conjugated into R. sphaeroides, and strains in which afd-1 had replaced the wild-type gene were verified as described above (strain AfdR7).

To generate a polar insertion in afdS (afd-2 allele), a 2.5-kb region was amplified from genomic DNA, and the product was phosphorylated and ligated into SmaI-digested pUC19 to generate pAfdS1. A BamHI fragment containing a gene encoding Tp^r was inserted into codon 142 of afdS by ligation into pAfdS1 that was digested by BglII, forming pDelAfdS1. Plasmid pDelAfdS1 was used to generate a 3.5-kb PCR product containing the afd-2 allele by using vector-specific primers (1224 and 1233). The product was phosphorylated and ligated into ScaI-digested pSUP202, generating pDelAfdS2. pDelAfdS2 was conjugated into R. sphaeroides, and strains in which afd-2 replaced the wild-type gene were verified as described above (strain AfdS1).

To generate an afdRTS-containing plasmid for complementation, a 5-kb PvuII fragment from pAfd1 was ligated into ScaI-digested pSUP202, resulting in pAfdRTS5. For single-copy complementation, pAfdRTS5 was conjugated into R. sphaeroides, and merodiploids were identified by screening for resistance to both trimethoprim (from the afd-1 or afd-2 allele) and tetracycline (from pSUP202) to generate strains AfdR7-5 and AfdS1-5. The presence of the wild-type afdRTS locus or the afd-1 or afd-2 allele was confirmed by PCR.

Enzyme assays. The levels of ß-galactosidase activity in aerobic cells containing an adhI::lacZ reporter gene were assayed (31) while cultures were at cell densities of 3.0 x 10⁸ to 7.0 x 10⁸ CFU/ml. Values reported are averages of data from at least three separate isolates grown in separate cultures. The error reported is the standard deviation between individual isolates (31). Values for GSH-dependent formaldehyde dehydrogenase activity (7) are the averages of three independent assays. Protein concentration was determined by the Bradford assay (8a).

Western blots. Western blot analysis with antiserum to isocytochrome c₂ (29) was performed on crude cell extracts prepared by sonication of cells from 500-ml cultures grown to a cell density of approximately 10⁹ cells per ml by aeration with a gas mixture of 69% N₂, 30% O₂, and 1% CO₂. Equal amounts of crude extract protein (12.5 µg) were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (4 to 12% gradient) (Invitrogen, Carlsbad, Calif.). Proteins were transferred to nitrocellulose membranes and probed for isocytochrome c₂ with a 1:500 dilution of rabbit antiserum (29). Secondary anti-rabbit immunoglobulin G conjugated to horseradish peroxidase was used for detection.

Purification of AfdR and RfdR. AfdR or a truncated version of RfdR lacking its presumed N-terminal receiver and oligomerization domains (RfdR-CTD) was purified by using an intein-chitin-binding domain fusion system (New England Biolabs, Beverly, Mass.) that had been previously used to obtain active R. sphaeroides response regulators (9).

To generate an intein fusion to AfdR, the afdR gene was amplified and cloned into HincII-digested pUC19, generating pAfdR-Int1. After pAfdR-Int1 was digested with NdeI and SmaI, a 750-bp fragment was ligated into pTYB2 that was digested with NdeI and SmaI, forming pAfd-Int2. Before use, pAfd-Int2 was sequenced to ensure that afdR was fused in frame with the intein-chitin-binding domain. To purify AfdR, 1 liter of ER2566 (pAfdR-Int2) cells were grown at 37°C to an optical density at 600 nm of 0.4 to 0.5. IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to 0.3 mM, and the culture was incubated at 30°C for 3 h. Cells were harvested by centrifugation, resuspended in 5 ml of column buffer (20 mM HEPES [pH 8.0], 250 mM KCl, 0.1 mM EDTA, 0.1% Triton X-100), and lysed by sonication. The extract was centrifuged at 15,000 x g for 30 min, and the supernatant was loaded onto a 6-ml chitin column equilibrated with 10 volumes of column buffer. The column was washed with 10 volumes of column buffer, flushed with column buffer containing 60 mM dithiothreitol, and stored for 36 h at 4°C to allow intein cleavage. Released protein was eluted in column buffer and collected in 2-ml fractions before analysis by SDS-PAGE. Fractions containing AfdR were pooled and dialyzed against storage buffer (20 mM HEPES, 250 mM KCl, 0.1 mM EDTA, 50% glycerol, pH 8.0). AfdR used for in vitro transcription experiments was estimated to be >95% pure by SDS-PAGE analysis of pooled fractions. Phosphorylated AfdR was prepared by mixing the protein with 25 mM acetyl phosphate for 1 h at 30°C (9).

To generate an intein fusion to RfdR-CTD, DNA encoding the C-terminal domain (amino acids 150 to 226) was amplified and cloned into SmaI-digested pUC19, generating pMAD1. After pMAD1 was digested with NdeI and SmaI, a 230-bp fragment was ligated into pTYB2 that was digested with NdeI and SmaI, forming pMAD47. Before use, pMAD47 was sequenced to ensure that the C-terminal region of rfdR was fused in frame with the intein-chitin-binding domain. To purify RfdR-CTD, 800 ml of ER2566 (pMAD47) cells were grown at 37°C to an optical density at 600 nm of 0.4 to 0.5. IPTG was added to 0.3 mM, and the culture was incubated at 30°C for 3 h. Cells were harvested by centrifugation, resuspended in 5 ml of column buffer (20 mM Tris-HCl [pH 7.9], 5 mM MgCl₂, 500 mM KCl, 0.1 mM EDTA, 0.1% Triton X-100), and lysed by sonication. The cell extract was centrifuged at 15,000 x g for 30 min, and the supernatant was loaded onto a 10-ml chitin column equilibrated with 10 column volumes of column buffer. The column was washed with 10 column volumes of column buffer, flushed with column buffer containing 50 mM dithiothreitol, and stored overnight at 4°C to allow intein cleavage. Released protein was eluted in column buffer and collected in 1-ml fractions before analysis by SDS-PAGE. Fractions containing RfdR-CTD were pooled and dialyzed against storage buffer (40 mM Tris-HCl [pH 7.9], 5 mM MgCl₂, 50 mM KCl, 1 mM dithiothreitol, 20% glycerol).

Gel shift assays. A 476-bp DNA fragment containing the adhI promoter that extends from –296 to +180 bp relative to the known transcription initiation site (5) was end labeled with [-³²P]ATP. The indicated concentrations of pure recombinant RfdR-CTD were incubated with 2 ng of the end-labeled adhI promoter DNA in a reaction mix containing 10 mM KPO₄ (pH 7.5), 100 mM potassium glutamate, 1 mM EDTA, 0.05 mM dithiothreitol, 0.5 mg of bovine serum albumin/ml, and 30 µg of salmon sperm DNA/ml. Binding reactions were incubated at 30°C for 40 min. After incubation, protein-DNA complexes were resolved on a 6% polyacrylamide gel (Invitrogen) by using 0.5x Tris-borate-EDTA as a running buffer. The gel was run at 4°C for 1.5 h at 100 V. The gel was dried and exposed to a PhosphorImager screen overnight and visualized by using a PhosphorImager and ImageQuant software (Molecular Dynamics).

In vitro transcription reactions. A typical 20-µl reaction included R. sphaeroides RNA polymerase (9), 20 nM plasmid pJWH20 (see below), and AfdR or RfdR-CTD in transcription buffer (40 mM Tris-Cl [pH 7.9], 50 mM KCl, 5 mM MgCl₂, 0.2 mM EDTA, 0.1 mM dithiothreitol, 0.1 mg of bovine serum albumin/ml). This mixture was incubated for 20 min at 30°C prior to initiating the assay. After transcription was initiated by the addition of nucleoside triphosphates (0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, and 0.05 mM UTP plus 10 µCi of [-³²P]UTP), the reaction mixture was incubated at 30°C for 20 min. Assays were stopped by adding 10 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). Samples were heated to 95°C for 5 min, and the products were separated on a 6% polyacrylamide-urea gel. The gel was dried and exposed to a PhosphorImager screen. Transcript levels were quantified by using ImageQuant software (Molecular Dynamics). Data shown for induction of adhI transcription were calculated relative to the background level and amount of the control RNA1 product that was present in each reaction.

Potential adhI promoter sequences (–296 to +14 relative to known transcriptional initiation site) were obtained by digestion of pEPS296 (6) with KpnI and HincII. This 310-bp fragment was cloned into pRKK96 digested with KpnI and StuI to generate pJWH20. The sequencing of pJWH20 with plasmid-specific primers was used to ensure the desired orientation of adhI promoter sequences relative to the SpoT40 transcription terminator on pRKK96.

	RESULTS

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A negative regulator of GSH-FDH expression. To search for negative regulators of GSH-FDH expression, we utilized an R. sphaeroides mutant, CYCA65R7, which contains the spd-7 allele (28). The spd-7 allele restores photosynthetic growth to cells lacking cytochrome c₂ by increasing transcription of the adhI-cycI operon, which encodes GSH-FDH (adhI) and isocytochrome c₂ (cycI) (7). Increased levels of isocytochrome c₂ are required for photosynthetic growth in the absence of cytochrome c₂, so we reasoned that wild-type DNA sequences reducing adhI-cycI expression would abolish photosynthetic growth of cells containing the spd-7 allele.

To search for DNA that reduced adhI-cycI expression, a wild-type cosmid library was mobilized into cells containing an spd-7 allele (CYCA65R7), and exconjugants were scored for photosynthetic growth. One cosmid that blocked photosynthetic growth, pUI8017, was identified. To test that the loss of photosynthetic growth of cells containing an spd-7 allele was due to reduced expression of the adhI-cycI operon, we measured the levels of these gene products. To measure levels of the cycI gene product, a Western blot with CycI-specific antibody was used to determine the relative abundance of isocytochrome c₂ in aerobically grown cells (Fig. 1). This analysis showed that there were low, but detectable, amounts of isocytochrome c₂ in both wild-type cells and cells lacking cytochrome c₂ (CYCA65). There is increased abundance of isocytochrome c₂ in cells containing the spd-7 allele, similar to results reported previously (29). The presence of pUI8017 in the spd-7 mutant decreased the abundance of isocytochrome c₂, providing an explanation for why the presence of this cosmid blocked photosynthetic growth of cells containing the spd-7 allele.

View larger version (36K): [in this window] [in a new window]   FIG. 1. Cosmid pUI8017 reduces adhI-cycI expression. (Top) A Western blot monitoring the relative abundance of isocytochrome c2 in extracts of aerobically grown cells is shown. The strains used in this analysis are indicated above the lanes. (Bottom) GSH-FDH specific activities (in nanomoles per minute per milligram of protein) measured in extracts of these strains. Numbers in parentheses indicate the standard deviations in GSH-FDH specific activity.

To identify genes responsible for this decrease in adhI-cycI expression, we tested a series of plasmids derived from this cosmid for their ability to prevent photosynthetic growth of cells containing the spd-7 allele. Sequence analysis of a smaller region that prevented photosynthetic growth of cells containing an spd-7 allele revealed the presence of a potential operon (rfdRTS) predicted to encode a two-component regulatory system (RfdRS) and an additional protein (RfdT) with no predicted function (Fig. 2a); these correspond to open reading frames named RSP2880 to RSP2882 in the R. sphaeroides genome sequence (http://genome.ornl.gov/microbial/rsph/).

View larger version (25K): [in this window] [in a new window]   FIG. 2. Physical map of the region around rfdRTS and afdRTS in R. sphaeroides. Hypothetical genes are indicated by their gene numbers in the annotated R. sphaeroides genome (http://genome.ornl.gov/microbial/rsph/). The sizes of the genes (in base pairs) are indicated above the map, and the direction of transcription is indicated by the arrows below. (A) Open reading frames corresponding to rfdRTS are indicated in the boxes (RSP2882 to RSP2880). The positions of mutations in rfdR, rfdT, and rfdS used in this study are indicated by the triangles. (B) The top shows the region surrounding afdRTS, including RSP2575 to RSP2596 on R. sphaeroides chromosome I. The bottom shows the region surrounding afdRTS. The places where Tpr genes were inserted to generate the afd-1 and afd-2 alleles are indicated by the triangles.

RfdRTS negatively regulates adhI expression. The reduced expression of adhI-cycI when RfdRTS are present in cells containing the spd-7 allele predicts that these proteins are negative regulators of GSH-FDH expression. If RfdRTS negatively regulated adhI transcription, then the loss of RfdRS should increase adhI expression. To test this hypothesis, we constructed strains that lack RfdRTS (RfdR1, rfd-1 allele) or RfdS (RfdS1, rfd-2 allele). Expression of adhI in mutant and wild-type strains was monitored by using a previously described transcriptional fusion of adhI promoter sequences (–296 to +14 relative to the transcriptional start site) to a promoterless lacZ gene on a low-copy plasmid (pTY296) (6).

Aerobically grown wild-type cells display levels of ß-galactosidase activity that are well above background (Table 2), as reported previously (6). Aerobic cells lacking RfdRTS have three- to fourfold-higher levels of ß-galactosidase activity than wild-type cells grown in a succinate-based minimal medium. This result supports the hypothesis that RfdRTS negatively regulate adhI transcription. Cells lacking only RfdS also have 3-fold-higher levels of activity from the adhI reporter gene (Table 2), suggesting that the presumed kinase activity of RfdS is necessary for negative regulation of adhI transcription. The amount of adhI transcription in each of these mutants is restored to wild-type levels by introducing a plasmid (pRfd4) containing wild-type rfdRTS, confirming that the loss of RfdRTS is responsible for the increase in cells lacking either RfdRTS or RfdS (Table 2). The presence of rfdRTS on this 6- to 10-copy plasmid does not decrease adhI expression compared to the uninduced levels seen in the wild type (Table 2). This result is likely due to the fact that adhI expression is repressed in uninduced wild-type cells, so the presence of 6 to 10 copies of the genes encoding this potential negative regulator does not cause an additional decrease in adhI expression.

adhI expression in cells lacking RfdRTS or RfdS is increased by metabolic sources of formaldehyde. We know that metabolic sources of formaldehyde, like methanol, increase adhI transcription (6). To determine if rfdRTS was responsible for this increase, we measured ß-galactosidase activity from the adhI reporter in aerobically grown wild-type cells and those lacking either RfdRTS or RfdS in the presence or absence of this metabolic source of formaldehyde. We found that adhI expression is induced 15-fold by adding methanol to aerobic cells grown in a succinate-based minimal medium (Table 2), similar to the increase reported previously (6). In addition, we found that adhI expression in aerobic cells lacking either RfdRTS or RfdS is increased 10-fold by the addition of methanol (Table 2). This finding indicates that RfdRTS are not essential for increased adhI transcription in the presence of metabolic sources of formaldehyde. The similar level of adhI transcription in either wild-type or RfdRTS mutant cells in the presence of metabolic sources of formaldehyde could reflect the maximal activity of the adhI promoter in vivo. In summary, these results show that RdfRS are negative regulators of adhI expression but that these proteins are not required for increased adhI transcription in response to metabolic sources of formaldehyde.

Genes encoding possible signal transduction proteins are within 8 kb of adhI. The ability of formaldehyde to increase adhI transcription in cells lacking RfdRS suggests that cells contain an additional pathway that increases activity of this promoter in the presence of formaldehyde. To search for such an additional regulator of adhI expression, we queried the R. sphaeroides genome sequence (http://genome.ornl.gov/microbial/rsph/) for gene products related to proteins that are predicted to control similar pathways in other proteobacteria.

This analysis identified additional potential regulators of adhI expression near a putative formaldehyde metabolism gene cluster (Fig. 2b). In the region surrounding adhI-cycI are genes that could encode enzymes to oxidize methanol to formaldehyde (XoxF), periplasmic c-type cytochromes (CycI and CycB), a protein to facilitate the formation of S-hydroxymethylglutathione from formaldehyde and GSH (GfaA), and open reading frames which encode homologs of proteins of unknown function that are derived from genes typically linked to those encoding GSH-FDH family members in other eubacteria (14, 15, 17). This region also contains genes (RSP2591 to RSP2593) predicted to encode a sensor histidine kinase, a response regulator, and a predicted integral membrane protein of unknown function (Fig. 2b). We called these latter genes afdRTS, for activator of glutathione-dependent formaldehyde dehydrogenase, based on the results of the following analyses.

The predicted sensor histidine kinase, AfdS, has significant amino acid sequence similarity to RfdS (39% identity, 52% similarity) and to a P. denitrificans protein, FlhS (51% identity, 64% similarity), that is required to increase GSH-FDH expression (see Fig. 6) (17). The potential response regulator, AfdR, is predicted to contain a C-terminal DNA-binding domain in the NarL response regulator family with significant amino acid sequence similarity to RfdR (44% identity, 59% similarity) and P. denitrificans FlhR (61% identity, 76% similarity). The predicted integral membrane protein has significant amino acid sequence similarity to RfdT and the uncharacterized gene product encoded by P. denitrificans orf2.

View larger version (64K): [in this window] [in a new window]   FIG. 6. The putative sensor domain of RfdS is different from those of AfdS and FlhS. Amino acid sequence alignment of the N-terminal regions of R. sphaeroides RfdS, P. denitrificans FlhS, and R. sphaeroides AfdS is shown. Identical residues are shaded in black, and functionally similar residues are shaded in grey. A consensus sequence is shown below the alignment.

The results of previous experiments led us to propose that formaldehyde, or possibly the glutathione adduct S-hydroxymethylglutathione, is an inducer of adhI transcription (6). Thus, the failure of methanol to stimulate adhI expression in cells lacking AfdRTS could reflect an inability of these mutants to generate formaldehyde from methanol or a defect in transcriptional control by formaldehyde. To distinguish between these possibilities, we monitored the ability of sublethal concentrations of formaldehyde to increase adhI expression. If the defect in cells lacking AfdRTS reflected an inability in generating this inducer from methanol, adhI expression should be increased by formaldehyde in these mutants. However, if formaldehyde were a potential signal molecule for AfdRTS, then cells lacking these proteins would be defective in stimulating adhI expression when formaldehyde was added.

In wild-type cells, adhI transcription is stimulated 10-fold within 1.5 h after the addition of 50 µM formaldehyde (Table 2). However, there was no significant increase in adhI expression when 50 µM formaldehyde was added to cells lacking either AfdRTS or AfdS (Table 2). Placing a single copy of afdRTS in either mutant restored induction of adhI expression by formaldehyde (Table 2), suggesting that AfdRS responds to this compound.

To test if AfdRS was also required for the methanol-dependent increases in adhI expression seen in cells lacking RfdRTS, we compared the ability of this compound to increase adhI expression in cells lacking both AfdRS and RfdRS (JWH1) to that of wild-type cells or stains lacking one of these pathways. In the absence of methanol, strains lacking both RfdRS and AfdRS have a level of adhI expression similar to that of wild-type cells but less than that observed in an RfdRTS mutant (Table 2). When either methanol or formaldehyde was added to induce adhI expression, there was no detectable increase in transcription in cells lacking both RfdRTS and AfdRTS (Table 2). Thus, we conclude that AfdRTS is necessary for the increased adhI expression in the presence of formaldehyde that is seen in both wild-type cells and strains lacking RfdRTS.

AfdR activates adhI transcription. To test if AfdR is a direct regulator of adhI expression, we asked if this protein stimulates activity of this promoter in vitro. To accomplish this, recombinant AfdR was added to in vitro transcription reactions containing R. sphaeroides RNA polymerase holoenzyme (20 nM) and an adhI-specific template. Many response regulators activate transcription only when phosphorylated, so reactions were performed with both AfdR and protein that was incubated with acetyl phosphate prior to the in vitro transcription assays.

No adhI-specific transcript was detected in assays lacking AfdR or when up to 5 µM AfdR that was not incubated with acetyl phosphate was used (Fig. 3). However, the addition of 2 to 5 µM concentrations of AfdR that was incubated with acetyl phosphate (AfdR-Pi) resulted in the production of an adhI-specific transcript of the expected size (184 nucleotides) based on the position of the known transcription initiation site relative to a transcription terminator on the plasmid template (Fig. 3). Of equal significance, the addition of acetyl phosphate-treated AfdR caused no detectable increase in the abundance of control RNA1 transcript that is contained on the plasmid template, suggesting that this increase in adhI expression is not due to a general increase in transcription.

View larger version (55K): [in this window] [in a new window]   FIG. 3. AfdR treated with acetyl phosphate activates adhI transcription in vitro. Positions of the adhI-specific and RNA1-specific transcripts are indicated by arrows. The adhI transcript appears as two discrete products due to RNA polymerase terminating transcription at adjacent bases downstream of the terminator on the plasmid template (2). The amount of AfdR in reactions and treatment with (+) acetyl phosphate (Acetyl-P) is listed at top. Transcript levels were quantified by using ImageQuant software (Molecular Dynamics). Indicated increases in adhI transcript levels were calculated relative to background in each lane.

RfdR binds the adhI promoter. The increased adhI promoter activity in cells lacking RfdR suggested that this protein was a repressor of GSH-FDH expression (see above). To provide evidence supporting this hypothesis, we tested the ability of RfdR to interact with the adhI promoter by a gel mobility shift assay. To accomplish this, we used a truncated form of RfdR that contains only its putative C-terminal DNA binding domain (RfdR-CTD). Control experiments indicate that that half-life of ³²P-labeled full-length RfdR is less than 1 min (data not shown), suggesting that it would be difficult to maintain phosphorylation of full-length RfdR during the time needed to observe any interactions with adhI promoter sequences by a gel mobility shift assay. Thus, the use of RfdR-CTD meant that this protein should not need to be treated with acetyl phosphate in order to observe an interaction with the adhI promoter.

A DNA fragment containing adhI promoter sequences that extend from –296 to +180 relative to the known adhI transcription start site was incubated with purified recombinant RfdR-CTD prior to separating any DNA-protein complexes from promoter DNA on a 6% polyacrylamide gel. A shift in the mobility of the adhI promoter fragment can be seen when 0.5 µM RfdR-CTD is added to the reaction (Fig. 4). The amount of this slower-migrating species increases when 1.0 µM RfdR-CTD is present. This finding indicates that RfdR-CTD is capable of interacting with the adhI promoter at micromolar concentrations. This interaction between RfdR-CTD and the adhI promoter appears to be specific due to the fact that a 300-fold excess of a nonspecific competitor was present in these reactions.

View larger version (37K): [in this window] [in a new window]   FIG. 4. RfdR-CTD binds to adhI promoter sequences. The amounts of RfdR-CTD added to the binding reaction are indicated above the lanes. The arrows indicate the migration position of the adhI promoter fragment and migration position of the presumed complex between DNA and RfdR-CTD. In this assay, 2 ng of end-labeled DNA (extending from –296 to +180 relative to the adhI transcription initiation site) was present in each reaction (6).

To test if RfdR was a direct inhibitor of adhI transcription, we monitored the abundance of this transcript when increasing amounts of RfdR-CTD (0.5 to 4 µM) were added to multiple-round transcription reactions containing R. sphaeroides RNA polymerase holoenzyme (95 nM), an adhI-specific template, and AfdR (2 µM) that was treated with acetyl phosphate (Fig. 5). As expected, pretreatment of AfdR with acetyl phosphate prior to the assay resulted in the accumulation of an adhI transcript. There was a concentration-dependent decrease in the amount of the adhI transcript when increasing amounts of RfdR-CTD were present in the reactions. This negative effect of RfdR-CTD did not appear to reflect a general decrease in transcription since the addition of this protein did not reduce the amount of the control RNA1 transcript in a concentration-dependent manner (Fig. 5). If one measures the amount of adhI transcript present at different concentrations of RfdR-CTD relative to the level of RNA1 transcript that is present in each of these assays, 2 µM RfdR-CTD is sufficient to reduce the amount of this product by 50% (Fig. 5). By comparing the ability of RfdR-CTD to interact with adhI promoter DNA (Fig. 4) and its ability to inhibit transcript production from this promoter (Fig. 5), it appears that the apparent affinity of this truncated protein is in the micromolar range. Thus, we conclude that RfdR is a direct inhibitor of adhI transcription because it binds to this promoter and can act as a repressor of GSH-FDH expression.

View larger version (56K): [in this window] [in a new window]   FIG. 5. RfdR is a direct inhibitor of adhI transcription in vitro. The top panel indicates positions of the adhI-specific and RNA1-specific transcripts with arrows. The presence of AfdR (2 µM) and the concentration of RfdR-CTD (see the text) present (+) or absent (–) in reactions and pretreatment with acetyl phosphate (Acetyl-P) are indicated above the figure. Transcript levels were quantified by using ImageQuant software (Molecular Dynamics). The graph at the bottom shows the relative decrease in adhI transcript levels as the concentration of RfdR-CTD is increased, after correction for background and the relative amount of RNA1 transcript that is produced in each assay.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Our results predict that one regulator of adhI expression, RfdRS, is a repressor of adhI expression, while the other, AfdRS, activates adhI transcription. Both systems contain a predicted sensor histidine kinase (RfdS and AfdS) and a response regulator (RfdR and AfdR). The R. sphaeroides genome sequence predicts that AfdRS is encoded within a putative five-gene operon that includes afdRTS, RSP2594, and RSP2595. The function of AfdT and the latter two genes are unknown, but homologs exist in P. denitrificans (17). If these five genes are cotranscribed from one promoter, RSP2594 and RSP2595 are not necessary for AfdRS function under the conditions tested since afdRTS complements cells containing a polar insertion in afdR.

Features of RfdRTS and AfdRTS. The deduced amino acid sequence of AfdS and RfdS predict that they are soluble sensor histidine kinases. Of the histidine kinases that have been described, RfdS and AfdS display the greatest similarity to the presumed sensor in a pathway that activates P. denitrificans GSH-FDH expression, FlhS (17). The C termini of RfdS, AfdS, and FlhS are each predicted to also contain an additional receiver domain that is found on the N terminus of response regulators (RfdS residues 618 to 720 and AfdS residues 350 to 470). The predicted presence of a receiver domain in these presumed sensors suggests that these are hybrid histidine kinases that use multiple phosphotransfer reactions as part of their signaling pathway. AfdS and FlhS each have a relatively small N-terminal sensor domain (residues 1 to 83) compared to that of RfdS (residues 1 to 373) (Fig. 6). The possible significance of this larger sensor domain in RfdS is presented below.

The amino acid sequence of RfdR and AfdR predicts that they each contain typical N-terminal receiver domains and C-terminal helix-turn-helix motifs related to the DNA binding domain of NarL. AfdR appears to require phosphorylation to activate adhI transcription. This is consistent with the behavior of the related E. coli NarL protein and many other response regulators (3). It is also likely that the related RfdR protein also requires phosphorylation to interact with DNA since it is also a response regulator in the NarL family. It is unknown where either AfdR or RfdR binds within the adhI promoter and whether they contain one or multiple binding sites as is the case for NarL at some promoters (12). There is also considerable amino acid sequence identity in the C-terminal domains of AfdR and RfdR, so it may be possible that they have similar or overlapping target sites for DNA binding.

RfdT and AfdT are not related to any characterized proteins, but hydropathy analysis (using the Kyte-Doolittle scale [http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html]) indicates that they contain at least one transmembrane domain. An in-frame deletion in R. sphaeroides rfdT had no detectable effect on adhI expression in the absence or presence of known inducers. Therefore, a role for AfdT/RfdT in R. sphaeroides and the P. denitrificans homolog (Orf2) remains to be determined.

Multiple pathways control adhI expression. Transcription of R. sphaeroides adhI is induced by formaldehyde or several metabolic sources of this compound (6), suggesting that a metabolic intermediate acts as an inducer of gene expression. It appeared that an early pathway intermediate was responsible for this increase since the addition of formate or bicarbonate does not induce adhI transcription (4). Additionally, mutants lacking GSH-FDH have elevated adhI expression even in the absence of exogenous formaldehyde, indicating that a pathway intermediate upstream of GSH-FDH is stimulating this promoter. Because of this, it was proposed that formaldehyde, or its glutathione adduct S-hydroxymethylglutathione, is a potential metabolic signal that induces adhI expression. Our results suggest that the AfdRS pathway is responsible for this induction since these proteins were needed to increase adhI expression in response to metabolic or exogenous sources of formaldehyde in vivo (Fig. 7). We further propose that phosphorylated AfdR is responsible for this increase since this protein was a direct activator of adhI transcription in vitro.

View larger version (13K): [in this window] [in a new window]   FIG. 7. Multiple two-component systems regulate adhI expression. AfdRS activates adhI transcription in response to a signal derived from formaldehyde metabolism, possibly formaldehyde (HCHO) or S-hydroxymethylglutathione (HMGSH). RfdRS represses adhI transcription. The signal that relieves repression by RfdRS is unknown.

While we identified R. sphaeroides RfdRS for its ability to reduce adhI-cycI expression and prevent photosynthetic growth of cells containing the spd-7 allele, inactivating rfdRTS does not restore photosynthetic growth to a cytochrome c₂ mutant (data not shown). Thus, it appears that the unknown signal which relieves the negative effect of RfdRS on adhI-cycI expression would be unable to allow sufficient production of isocytochrome c₂ to support photosynthetic growth in the absence of cytochrome c₂.

Other homologs of AfdRS and RfdRS. There are important similarities and differences in the regulation of formaldehyde metabolism between R. sphaeroides and other proteobacteria. In R. sphaeroides, GSH-FDH expression is directly regulated by multiple two-component regulatory circuits (RfdRS and AfdRS) that appear to respond to different signals. To date, only a single positive regulator of GSH-FDH has been described in P. denitrificans (FlhRS), and it has not been demonstrated that FlhR directly activates transcription of the GSH-FDH gene in this organism (17).

If FlhRS is a direct activator of GSH-FDH expression in P. denitrificans, there appears to be differences in the control of formaldehyde metabolism. For example, the loss of FlhRS in P. denitrificans abolishes expression of formyl-GSH hydrolase, cytochrome c_553i, and methanol dehydrogenase (17). As a result, P. denitrificans requires FlhRS for growth on formaldehyde-generating carbon sources like methanol, methylamine, and choline (17). It does not appear that the loss of AfdRS blocks expression of a putative methanol dehydrogenase and the formyl-GSH hydrolase in R. sphaeroides since afdRS mutants of this bacterium can use methanol as a sole photosynthetic carbon source but at a reduced growth rate compared to that of the wild-type (data not shown). Possibly, the basal levels of GSH-FDH expression in afdRS mutants are sufficient to support growth under these conditions. Also supporting this notion is the fact that both adhI transcription in vivo and activity of GSH-FDH (data not shown) are still detectable in the AfdRS mutant strain.

There are several predicted homologs of AfdRS and RfdRS in the microbial genome database. In virtually every microbial genome that contains an AfdRS homolog, the genes are adjacent to regions predicted to encode either GSH-FDH enzymes or putative PQQ-dependent alcohol dehydrogenases that could produce formaldehyde from methanol. Among the bacteria for which genomes have been sequenced, R. sphaeroides is the only one predicted to contain homologs of both RfdRS and AfdRS. Thus, it will be interesting to see why this facultative -proteobacterium contains pathways to negatively and positively regulate GSH-FDH expression compared to other organisms which appear to contain only an AfdRS or RfdRS system.

In conclusion, we have identified multiple two-component systems that directly regulate expression of the R. sphaeroides GSH-FDH structural gene adhI. One of these systems, AfdRS, is a direct activator of adhI transcription that appears to respond to an intermediate in formaldehyde oxidation, while RfdRS is a direct negative regulator of GSH-FDH expression in response to an unknown signal. These observations indicate that regulation of GSH-dependent formaldehyde oxidation in R. sphaeroides is an intricate process with multiple two-component systems that could each respond to different physiological signals. The AfdRS system shares similarities with other two-component regulators, but this is the first report of a transcription factor acting directly to regulate transcription of a formaldehyde-inducible gene. We are now in a position to determine how AfdRS senses signals from formaldehyde metabolism and regulates genes needed for metabolism of this compound. Additional work to identify the signal that controls RfdRS function will characterize the role of this two-component system and could help elucidate additional roles for GSH-FDH in cells.

	ACKNOWLEDGMENTS

 
This work was supported by grant DE-FG02-97ER20262 from the U.S. Department of Energy to T.J.D. During these studies, J.W.H. was supported by the William H. Peterson memorial fellowship awarded by the Department of Bacteriology, University of Wisconsin—Madison, and M.D. was supported by predoctoral training grant 5 T32 GM07133 from NIGMS.

We thank Federico Rey for critical reading of the manuscript.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, Room 390, 420 Henry Mall, Madison, WI 53706. Phone: (608) 262-4663. Fax: (608) 262-9865. E-mail: tdonohue{at}bact.wisc.edu.

Present address: Department of Microbiology, University of Iowa, Iowa City, IA 52242.

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