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Journal of Bacteriology, February 2003, p. 1229-1235, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1229-1235.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

QscR, a LysR-Type Transcriptional Regulator and CbbR Homolog, Is Involved in Regulation of the Serine Cycle Genes in Methylobacterium extorquens AM1

Marina G. Kalyuzhnaya¹ and Mary E. Lidstrom^1,2*

Department of Chemical Engineering,¹ Department of Microbiology, University of Washington, Seattle, Washington 98195-1750²

Received 18 July 2002/ Accepted 24 November 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
A new gene, qscR, encoding a LysR-type transcriptional regulator that is a homolog of CbbR, has been characterized from the facultative methylotroph Methylobacterium extorquens AM1 and shown to be the major regulator of the serine cycle, the specific C₁ assimilation pathway. The qscR mutant was shown to be unable to grow on C₁ compounds, and it lacked the activity of serine-glyoxylate aminotransferase, a key enzyme of the serine cycle. Activities of other serine cycle enzymes were decreased during growth on C₁ compounds compared to the activities found in wild-type M. extorquens AM1. Promoter fusion assays, as well as reverse transcription-PCR assays, have indicated that the serine cycle genes belong to three separate transcriptional units, sga-hpr-mtdA-fch, mtkA-mtkB-ppc-mcl, and gly. Gel retardation assays involving the purified QscR have demonstrated the specific binding of QscR to the DNA regions upstream of sga, mtkA, gly, and qscR. We conclude that QscR acts as a positive transcriptional regulator of most of the serine cycle enzymes and also as an autorepressor.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The serine cycle is the pathway for C₁ assimilation found in many methylotrophic bacteria belonging to the alpha-subdivision of the Proteobacteria. Methylobacterium extorquens AM1 has served as a model system for studying the genetics of the serine cycle (1, 4-7, 13). Three regions coding for serine cycle enzymes have been identified so far. One of these regions (Fig. 1) contains the genes for serine glyoxylate aminotransferase (sga), hydroxypyruvate reductase (hpr), methylene tetrahydrofolate dehydrogenase (mtdA), malate thiokinase (mtkA and mtkB), phosphoenolpyruvate carboxylase (ppc), and malyl-coenzyme A (CoA) lyase (mcl). Two other enzymes of the serine cycle, serine hydroxymethyltransferase and glycerate kinase, are encoded by gly and gck, respectively, which are not linked to each other or to other serine cycle genes (6, 7). Prior to this study, little was known about the regulation of the serine cycle genes. Activities of the serine cycle enzymes specifically involved in methylotrophy were shown to be induced by about threefold during growth on C₁ compounds, compared to the activities present in cells grown on multicarbon compounds (1, 4), but the mechanisms for such regulation, or the regulators involved, remained unidentified.

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FIG. 1. Genetic maps of the chromosomal regions in M. extorquens AM1 analyzed in this work. Large arrows represent genes and direction of transcription. Small arrows above represent putative promoter regions analyzed in this study.

LysR-type transcriptional regulators are involved in the regulation of CO₂ fixation in the Calvin-Benson-Bassham (CBB) cycle in photo- and chemoautotrophic bacteria (15, 23, 27), and genes encoding such regulators have been designated as cbbR. CbbRs control transcription from the cbb operons. For Xanthobacter flavus, CbbR also controls the gap-pgk operon encoding glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase (18). For Synechocystis strain PCC6803, three CbbR orthologs are present, and they were shown to play an important role in the adaptation to inorganic carbon starvation and osmotic stress, via regulating the transcription of a Na⁺/H⁺ antiporter as well as NAD(P)H-dehydrogenase (9, 19). The expression from known cbb operons was demonstrated to be maximally induced in litho (H₂)- or organoautotrophically (methanol, formate)-grown cells (15, 23). The cbbR gene is typically located immediately upstream of the cbb operon and is divergently transcribed. All known cbbR genes encode polypeptides of similar size (32 to 36 kDa), which form either homodimers or homotetramers (14, 28). In all of them a helix-turn-helix DNA binding motif is present within the N-terminal region, and many require a small molecule acting as a coinducer. The exact nature of this metabolic signal is unknown, although some effectors have been suggested. NADPH was shown to enhance DNA binding of CbbR for X. flavus (28); however, no similar effect was observed for Ralstonia eutropha (10, 15). In Rhodobacter capsulatus, ribulose bisphosphate was suggested to be involved in CbbR-mediated control of the cbb genes (27).

Two genes potentially encoding CbbR-type regulators have been identified in the genome of M. extorquens AM1. One of them has been inactivated via allelic exchange, and the resulting mutant retained its ability to grow on C₁ compounds (L. Chistoserdova, unpublished data). Another cbbR homolog was inactivated by two different Tn5 insertions in the course of random transposon mutagenesis, and the resulting mutants have lost the ability to grow on C₁ compounds (17). In this study we investigated this latter cbbR homolog, which we designate qscR (for Quayle serine cycle regulation) in detail, by generating an allelic exchange mutant and analyzing transcription from the serine cycle genes in this mutant. The work presented here demonstrates that the product of qscR plays an essential role in expression of key serine cycle enzymes for M. extorquens AM1.

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Bacterial strains, vectors, and growth conditions. M. extorquens AM1 was grown in minimal medium described previously (11). Succinate (20 mM), methanol (100 mM), ethanol (50 mM), or ethylamine (20 mM) was used as the substrate. The following antibiotic concentrations were used for M. extorquens: tetracycline (TET), 12.5 µg ml^-1; kanamycin (KAN), 100 µg ml^-1; rifamycin, 50 µg ml^-1. For serine cycle enzyme induction in mutant strains unable to grow on C₁ compounds, succinate-grown cells were pelleted, washed, and exposed to methanol at 30°C with shaking for 16 to 18 h. M. extorquens CM82.1 (C. J. Marx, unpublished results) was grow on methanol or succinate in the previously described medium with addition KAN (100 µg ml^-1).

Escherichia coli strains JM109 (30), BL21(DE3) (Novagen, Madison, Wis.), S17-1 (25), and Top 10 (Invitrogen) were routinely cultivated at 37°C in Luria-Bertani (LB) medium (21). The following antibiotic concentrations were used: TET, 12.5 µg ml^-1; KAN, 100 µg ml^-1; and ampicillin, 100 µg ml^-1.

The following cloning vectors were used: pUC19 (Pharmacia) for cloning and subcloning, pAYC61 (2) as a suicide vector, pRK2013 (8) as a helper plasmid, pCR2.1 (Invitrogen) for cloning of PCR products, and pCM130 (16) for promoter fusion construction.

Triparental or biparental matings between E. coli and M. extorquens AM1 were performed overnight on nutrient agar at 30°C. Cells were then washed with sterile medium and plated on selective medium at appropriate dilutions. Rifamycin was used for E. coli counter-selection.

Construction of mutants. Data from the M. extorquens AM1 genome project (http://www.integratedgenomics.com/genomereleases.html#list6) were used for designing primers specific for the putative cbbR homolog, designated as qscR. The following primers were used: cbbrf (5'-CGGATCGTGGCGGCGGTGTC-3') and cbbRr (5'-TCGCGCACGAGGAAGGACTC-3'). The 890-bp fragment containing qscR was PCR amplified, cloned into pCR2.1, and then subcloned into pUC19 using appropriate restriction sites. After verification of the nucleotide sequence, a KAN resistance (Km^r) cassette from pUC4K was inserted into the unique XhoI site in qscR. The resulting construct, pUC19qscRKm^r, was ligated into the suicide vector pAYC61 using a unique KpnI site. The resulting plasmid was transformed into E. coli S17-1, and the resulting donor strain was mated with wild-type M. extorquens AM1, in a biparental mating. The Km^r recombinants were selected on succinate plates and checked for resistance to TET. TET-sensitive (Tc^s) recombinants were chosen as possible double-crossover recombinants. The identity of the double-crossover mutants was further verified by diagnostic PCR with primers specific to the insertion sites.

Construction of promoter fusions. Putative upstream promoter regions for sga (650 bp), hpr (477 and 930 bp), mtkA (740 bp), mcl (650 bp), gck (450 bp), ppc (700 bp), mdh (530 bp), eno (465 bp), fba (500 bp), and qscR (500 bp), qscR2, and qscR3 were amplified by PCR. The specific primer pairs for amplification were designed to contain additional restriction sites, PstI (the upstream primer) and HindIII (the downstream primer). The primers were as follows: orf1-sga intergenic region (sgaf, 5'-AAACTGCAGGAAGAGCGTCCGAACGCGGAT-3'; sgar, 5'-CCCAAGCTTGACGGGCGAGCGTGTTGGACA), sga-hpr intergenic region (hpr1f, 5'-AAACTGCAGAGAGATGTCGCTCATCGACAA-3'; hpr1r, 5'-CCCAAGCTTGCCCTGAAGGCGCTCGACGA-3'; hpr2f, 5'-AACCAATGCCTGCAGCACGCCTATCTGCGC-3'; hpr2r 5'-AGTCAAGCTTGGCGATGTCGTAGATCGGGTAAT-3'), mtkB-ppc intergenic region (ppcf, 5'-AAAACTGCAGGATCAAGGAGAACTTTTCCA-3'; ppcr, 5'-CCCAAGCTTAACCAGATGCCCTGCACCTGA-3'); ppc-mcl intergenic region (mclf, 5'-AAAACTGCAGCAGCGAGACCGACGAGGACA-3'; mclr, 5'-CCCAAGCTTGGATCATGGTCTTGTC-3'), orf3-gck intergenic region (gckf, 5'-AAAACTGCAGTCGTAGGCGATGTGACGGGA-3'; gckr, 5'-CCCAAGCTTCTCGCCACCGCGGCCATGCTG-3'), hpr-mtdA intergenic region (mtdAf, 5'-TGCTCCAGGCGCTCAAGGACGGCACCAT-3'; mtdAr, 5'-CATGTCGCCGCCGCCGACGAAGAT-3'), orf-mdh intergenic region (mdhf, 5'-AAACTGCAGACGTCTACCGCTACTTCCCCT-3'; mdhr, 5'-CCCAAGCTTGTCGACGGGAGCGGATTCGG-3'), orf-eno intergenic regions (enof, 5'-AAACTGCAGTACTATAGGCGAATCAGGTGTGTCG-3'; enor, 5'-CCCAAGCTTGATGTTCATCATCGGCACCGGG), fba-qscR intergenic region (qscRf, 5'-TGCACTGCAGAGCGCCCGCATGAAGCGCTT-3'; qscRr, 5'-CCCAAGCTTGGAGCTGGCGGATGCGTGCG-3'). The same primers (qscR-rev-f and qscR-rev-r) with modifications of the additional restriction sites (PstI to HindIII, and vice versa) were used for fba promoter fusions. The amplified PCR fragments were subsequently excised by PstI and HindIII and cloned into the promoter probe vector, pCM130, resulting in constructs containing the respective DNA fragments upstream of the promoterless reporter gene, xylE (16). Putative promoter regions for mtdA (520 bp) and gly (567 bp) were subcloned into pCM130 from pCM272 and pCM236, respectively (C. J. Marx, unpublished results). The resulting constructs were transferred into M. extorquens AM1 and the cbbR mutant via conjugation.

The following primers were used: fragment 1, qscR 1 (5'-AAAACTGCAGGTTCTTATCGCGCATGCGCG) and qscRr (see above); fragment 2, qscRf (see above) and cbbr2 (5'-CCCAAGCTTAGAAATCTTTCGACAGCC-3'); fragment 3, qscR3 (5'-CTCACGTCGATCGCGGCGGC-3') and cbbr2. The amplified PCR fragments 1 and 2 were subsequently excised by PstI and HindIII and cloned into the promoter probe vector pCM170 (C. J. Marx, unpublished results). The resulting plasmid was transformed into M. extorquens CM 82.1 by electroporation. The Tc^r recombinants were selected on succinate plates and checked for resistance to KAN. Km^s recombinants were chosen as recombinants with promoter-XylE inserts into the katA site of the chromosome.

Enzyme assays. For cell extract preparation, cells were suspended in 1 ml of Tris (25 mM Tris-HCl, 10 mM EDTA, 1 mM dithiothreitol [pH 7.8]) or phosphate (25 mM KH₂PO₄-Na₂HPO₄ [pH 7.5]) buffers and were disrupted by a French press at 1.2 x 10⁸ Pa. Cell extracts were centrifuged at 20,817 x g for 25 min at 4°C to remove cell debris. Serine cycle enzyme activities were assayed as described previously (1, 3, 4). Fructose-1,6-bisphosphatase (FBPase) was assayed according to the method described in reference 20. The phosphoribulokinase activity was assayed as described previously (26) with the following modifications. The reaction mixture contained 50 mM Tris-HCl (pH 8.0), 20 mM MgCl₂, 5 mM ribulose-5-phosphate, 0.05 mM NADH, 5 mM ATP, 0.05 U of ribulose-1,5-bisphosphate carboxylase (Sigma), and 10 mM NaH¹⁴CO₃. Every 2 min (total time, 10 min), 50 µl of mixture was transferred to a filter, and 100 µl of 0.5 M HCl was added. The filters were dried and counted for acid-stable radioactivity. Enolase was assayed as previously described (24). Phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities were determined as described previously (18). Since glyceraldehyde phosphate serves as a substrate for hydroxypyruvate reductase (HPR) in M. extorquens AM1 (unpublished results), which is present at high levels in both succinate and methanol-grown cells (3), the activity of GAPDH was measured in an HPR^- background (3, 4). Catechol 2,3-dioxygenase activity was determined as described previously (12). The protein concentration was measured by the Biuret reaction (29) using bovine serum albumin as a standard.

RNA isolation and reversed transcriptase-PCR assay. RNA was extracted from 25 ml of methanol-grown cultures of M. extorquens AM1 using the MasterPure RNA purification kit (Epicentre Technologies), followed by an additional purification step using the RNeasy kit (Qiagen). The reverse transcription-PCRs (RT-PCRs) were carried out with the ThermoScript RT-PCR System kit (Invitrogen) in a 50-µl mixture containing 0.2 µg of RNA template, a 1 mM concentration of a specific primer, and the reaction cocktail according to the manufacturer's instructions.

Expression and purification of QscR. The qscR gene was amplified by PCR, using the primers (5'-CATGCCATGGGCAATCTTTCGCTCAAGCAG-3' and 5'-AAGGAAAAAAGCGGCCGCATTCGCCCGC-3') containing additional NcoI and NotI sites, respectively. The PCR product was digested by NcoI/NotI and cloned into pET21d (Novagen). QscR was purified from E. coli BL21(DE3) carrying pET21d:cbbr. E. coli was grown in 100 ml of LB medium with 100 µg of ampicillin ml^-1 to an optical density at 600 nm of 0.5 to 0.8 and then was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 2 h at 37°C. Cells were harvested by centrifugation, resuspended in 1 ml of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 1 mM dithiothreitol, 10 mM imidazole [pH 8.0]), and disrupted by two passages through a French pressure cell at 1.2 x 10⁸ Pa. Soluble proteins were recovered by centrifugation at 15,000 x g for 30 min at 4°C. His-tagged QscR was purified using the Ni-NTA spin Kit (Qiagen). Proteins were separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and visualized by Coomassie brilliant blue staining.

Electrophoretic mobility shift assays. Target DNA fragments of about 400 to 500 bp were end labeled by phosphorylation using T4 DNA kinase (Promega). The purified QscR (1.5 to 2 µg) was incubated with the labeled DNA in gel shift binding buffer (Promega) [5 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 4% glycerol, 0.05-µg/ml poly(dI-dC)] for 30 min at room temperature. After incubation, the mixtures were loaded on a Novex 6% retardation gel (Invitrogen) or a 6% nondenaturing acrylamide gel in 0.5x Tris-borate-EDTA and electophoresed at 300 V. Gels were subsequently dried and exposed to X-ray film (Kodak).

Nucleotide sequence accession number. The nucleotide sequence of 900 bp containing qscR from M. extorquens AM1 has been deposited with GenBank under the accession number AF516903.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutation in qscR causes a C₁-negative phenotype. As part of a random transposon mutagenesis screen, a transposon insertion into a gene with identity to cbbR genes was identified that showed a C₁-negative phenotype (17). Transposon-derived mutants have slightly different phenotypes depending on the site of the Tn inserts. The mutant containing an insertion into the center of the cbbR homolog showed growth defects on all C₁ (no growth) and C₂ (reduced growth rate) substrates, but the mutant with an insertion close to the 3' end of the gene had a less severe phenotype (reduced growth rate on C₁ compounds and normal growth on C₂ compounds). In order to confirm that the Tn insertion into the cbbR gene is responsible for the growth defects on C₁ compounds, we constructed an insertion mutant in this gene via allelic exchange. Double-crossover recombinants (null mutants) were selected on succinate plates with a frequency of 30% relative to the total number of recombinants. One of these mutants was chosen for further analysis. The qscR knockout strain was not able to grow on the C₁ compound methanol or methylamine and grew slowly on the C₂ compounds, ethanol and ethylamine. This phenotype is typical for mutants with lesions in serine cycle genes (3-7). We have designated this gene qscR for Quayle serine cycle regulation. The qscR gene potentially encoding the LysR-type transcriptional regulator is located upstream of genes potentially encoding fructose-1,6-bisphosphatase and phosphoribulokinase and is divergently transcribed (Fig. 1). QscR shares the highest sequence identity with CbbR from Sinorhizobium meliloti (48%) and CbbRII from R. capsulatus (44%). As in other LysR-type transcriptional activators, a helix-turn-helix DNA binding motif is present within the N-terminal region of QscR (data not shown).

Mutation in qscR affects activities of key enzymes in the serine cycle. It is well documented that in chemoautotrophic bacteria, CbbRs are involved in the regulation of cbb genes (15, 18, 23, 27). Although the genome of M. extorquens AM1 does not contain a homolog for ribulose bisphosphate carboxylase/oxygenase, it does contain a single predicted homolog each of phosphoribulokinase (PRK), FBPase, GAPDH, and 3-phosphoglycerate kinase (PGK). We have tested whether QscR might regulate these enzymes in M. extorquens AM1. Low levels of PRK activity were detected for both QscR and wild-type strains (Table 1). Activity was detected only in the presence of NADH, as with facultatively anaerobic photosynthetic bacteria (26). Likewise, similar levels of FBPase, PGK, and GAPDH were observed for wild-type and mutant strains grown on succinate or induced with methanol (Table 1), suggesting that qscR is probably not important in regulation of these enzymes. The C₁-negative phenotype of the qscR mutant suggested that it might, instead, regulate enzymes of the serine cycle. Activities of the key enzymes of the serine cycle, serine-glyoxylate aminotransferase (SGAT), HPR, serine hydroxymethyltransferase (SHMT), malate thiokinase (MTK), enolase, and malate dehydrogenase (MDH) were measured in the wild type and the qscR mutant grown on succinate and induced with methanol. Activity of SGAT was not detectible in the qscR mutant under both conditions. HPR, SHMT, and MTK were present in succinate-grown cells of the mutant at wild type levels, but no increase was observed after methanol induction, in contrast to the wild-type M. extorquens AM1 (Table 1). The background level of HPR activity in succinate-grown cells has also been shown to be present in hpr mutants and is apparently due to a different gene product (3, 4). These data suggest that the mutation in qscR interferes with regulation of the methylotrophy-specific genes of the serine cycle. Activity of MDH, which is involved in both C₁ and multicarbon metabolism, was present in the mutant at levels similar to the wild-type levels.

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TABLE 1. Activities of serine cycle- and Calvin-Benson-Bassham cycle-associated enzymes for wild-type M. extorquens AM1 and the QscR mutant either grown on succinate or grown on succinate, washed, and induced in the presence of methanol

Mutation in qscR affects transcription of serine cycle genes. We further tested expression of the serine cycle genes in the qscR mutant, via transcriptional fusions to a promoterless reporter gene, xylE. First, the promoter regions were identified by testing regions 5' of serine cycle genes using promoter fusions, with the following exceptions. The genes encoding the two subunits of MTK, mtkA and mtkB, overlap; therefore, they were assumed to be cotranscribed, and no promoter fusion was constructed for mtkB. Only 97 bp separate mtdA and fch, and most probably no promoter exists in this region, so this region was also not tested. Two enzymes participating in the serine cycle, enolase (encoded by eno) and MDH (encoded by mdh) are also involved in multicarbon metabolism. As shown above, the activity of MDH was present in cells of the mutant at wild-type levels, suggesting that QscR was not important in transcription of mdh. To test that hypothesis, we included the promoter region for mdh in the analysis. The region upstream of qscR was also included in tests. The plasmids carrying these DNA fusions were introduced into wild-type M. extorquens AM1 and into the qscR mutant, and the resulting strains were assayed for XylE activity (Table 2).

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TABLE 2. Effect of the QscR mutant on transcription of serine cycle genes in cells either grown on succinate or grown on succinate, washed, and induced in the presence of methanol, determined via activity of the reporter enzyme, catechol dioxygenase

Promoter activity was detected for all tested regions except those upstream of ppc and mtdA. Relatively strong promoter activity in methanol-grown cells was detected upstream of sga, mtkA, and gly. Significantly higher activities were observed in the methanol-induced cells than in the succinate-grown cells for the promoter regions upstream of sga, mtkA, gly, eno, and qscR, while the opposite effect was observed for the promoter region upstream of mdh. For the mutant, transcription from only three promoter regions upstream of serine cycle genes was affected significantly. A very low level of transcription was observed from the sga promoter, which agreed with the SGAT activity data (Table 1). Transcription from two other promoter regions, for gly and mtkA, was at approximately the wild-type level in succinate-grown cultures, but no induction occurred in the cultures exposed to methanol, which also agreed with the enzyme activity data (Table 1). The rest of the promoter fusions exhibited similar levels of XylE for wild-type M. extorquens AM1 and the mutant.

The promoter fusion results suggested that some of these genes might be transcribed in groups. To test this hypothesis, RT-PCR assays were performed across the intergenic regions of several of these genes. We were able to demonstrate the presence of sga-hpr, hpr-mtdA, mtdA-fch, and mtkB-ppc intergenic regions in the cDNA synthesized by reverse transcriptase in the presence of total mRNA isolated from wild-type M. extorquens AM1 (Fig. 2). No RT-PCR products were obtained for the orf2-sga and fch-mtkA intergenic regions, in accordance with the presence of relatively strong promoters in these regions. However, we obtained a positive RT-PCR product for the ppc-mcl intergenic region, suggesting that mcl is transcribed both from its own promoter and from a longer transcript initiating upstream of mtkA.

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FIG. 2. RT-PCR analysis of the transcriptional organization of the sga, hpr, mtdA, fch, mtkA, ppc, and mcl genes in M. extorquens AM1. Lanes a, control PCR product, using DNA. Lanes b, RT-PCR product, using total RNAs prepared from a methanol-grown culture.

QscR binds to promoter regions of sga, mtkA, and gly and to its own. To examine whether QscR binds to the DNA regions upstream of sga, mtkA, gly, and qscR, gel retardation assays were performed with partially purified QscR. The qscR gene was cloned behind the T7lac promoter of the pET21d plasmid that allowed for overproduction of His-tagged QscR in E. coli. Overproduction of QscR in standard conditions resulted in formation of insoluble protein aggregates, which significantly lowered the yield of protein and caused a loss in its DNA-binding activity (data not shown). To obtain a soluble preparation of QscR, we increased the salt concentration in the lysis solution to 1 M NaCl, followed by purification via nickel affinity chromatography. This preparation containing partially purified soluble QscR was used for gel retardation experiments. As a control, a protein fraction of E. coli cells carrying pET21d without an insert was used. The following intergenic regions were employed in the analysis: orf2/sga, fch/mtkA, orf3-gly, sga/hpr, ppc/mcl, orf3/gck, mtk/ppc, orf/eno, and fba/qscR. These were amplified by PCR to produce specific products of about 0.5 kb in size. The DNA fragments were incubated with various concentrations of the purified QscR and loaded onto gels. No retardation was observed for the DNA regions upstream of hpr, mcl, ppc, eno, and gck. However, incubation of the DNA regions upstream of sga, mtkA, gly, and qscR with the purified QscR resulted in the electrophoresis mobility shift of these fragments (Fig. 3). In each case, the binding was decreased by the respective nonlabeled DNA but not by nonspecific DNA (data not shown), confirming specificity. These results directly demonstrate that QscR binds to four regulation target promoter regions, upstream of sga, mtkA, gly, and its own. These data are consistent with the enzyme activity measurements (Table 1) and promoter fusion assay results (Table 2).

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FIG. 3. Gel retardation assay for binding with partially purified QscR. (A) fba-qscR intergenic region; (B) orf3-gly intergenic region; (C) fch-mtkA intergenic region; (D) orf1-sga intergenic region. Lanes 1, DNA ladder (as in Fig. 3); lanes 2, no protein added; lanes 3, protein fraction from E. coli containing vector alone; lanes 4 to 7, increasing concentrations (1, 1.5, 2, and 2.5 µg of protein, respectively) of partially purified QscR.

QscR acts as autorepressor. Gel retardation assays showed that QscR binds to the regions upstream of its translational start site; however, no significant effect of the QscR mutation was observed on reporter gene activity in a qscR promoter-xylE plasmid fusion that covered the entire qscR-fba region. It is possible that an effect of QscR in the wild type might have been masked by the presence of extra copies of the QscR binding site on the plasmid. To examine this issue in more detail, we made chromosomal promoter fusions with different fragments in this region and tested the same fragments by gel retardation assays (Fig. 4). The results suggest that QscR binds only one site within the fba-qscR promoter region, located at the 5' end of qscR and/or immediately upstream of the translation start site. QscR does not bind the promoter region immediately upstream of fba, providing further supportive evidence that it is not involved in fba regulation. The transcriptional fusions with the fragment bound by QscR showed low levels of promoter activity, which increased significantly in the fragment missing the QscR binding site. These results suggest negative autoregulation of qscR transcription.

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FIG. 4. Gel retardation assay and transcriptional fusion analysis of the fba-qscR promoter region. Right, schematic representation of the fba-qscR region showing the various DNA fragments used. Labeled fragments of the fba-qscR promoter were incubated without (-) or with (+) 2 µg of protein. ND, not determined.

No coinduction of QscR is observed with serine cycle or energy metabolism intermediates. Most LysR-type transcriptional activators require a small molecule coinducer to promote transcription, and this function is commonly served by a metabolite or cofactor of the pathway regulated (22, 27, 28). We tested potential candidates for a coinducer function in the regulation of the serine cycle genes. The following intermediates of the serine cycle were tested: serine, hydroxypyruvate, phosphoenolpyruvate, acetyl-CoA, and glyoxylate. The following energy metabolism intermediates were also tested: ATP, ADP, AMP, NAD⁺, NADH, NADPH, and NADP⁺. These metabolites, in concentrations ranging from 10 to 200 µM, were included in the incubation reaction containing the purified QscR and the sga promoter fragments, followed by gel retardation analysis. None of the compounds listed above enhanced DNA-binding activity of QscR. NADP⁺, acetyl-CoA, and glyoxylate resulted in a decrease in DNA binding, whereas the rest of the metabolites tested did not affect QscR binding to DNA (Fig. 5).

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FIG. 5. Effect of intermediates of the serine cycle and energy metabolism (50 µM) on the DNA-binding characteristics of QscR. A gel retardation assay was carried out with a 560-bp sgaA upstream DNA fragment and QscR. (A) Lanes: M, DNA ladder; 1, no QscR added; 2, QscR; 3, QscR plus serine; 4, QscR plus hydroxypyruvate; 5, QscR plus glycerate; 6, QscR plus phosphoenolpyruvate; 7, QscR plus glyoxylate; 8, QscR plus acetyl-CoA. (B) Lanes: M, DNA ladder; 1, no QscR added; 2, QscR; 3, QscR plus ATP; 4, QscR plus ADP; 5, QscR plus AMP; 6, QscR plus NAD+; 7, QscR plus NADH; 8, QscR plus NADPH; 9, QscR plus NADP+.

Top Abstract Introduction Materials and Methods Results Discussion References

 
LysR-type transcriptional regulators represent a large family of prokaryotic regulatory proteins that control a wide range of cellular processes (22). Most of them positively regulate transcription from target genes and also act as autorepressors (22). CbbR regulators are a group of LysR-type regulators that generally serve as activators of the enzymes involved in CO₂ assimilation in chemoautotrophic and phototrophic bacteria (15, 23). Here we describe an ortholog of CbbR in the facultative methylotroph M. extorquens AM1 and demonstrate that it is involved in positive regulation of serine cycle gene expression. We have designated this gene qscR, for Quayle serine cycle regulation. Interestingly, qscR in M. extorquens AM1 is not linked to any of the known serine cycle gene clusters and is located upstream of fba and prk genes. Such gene organization is typical of the chemoautotrophic and phototrophic bacteria Nitrobacter vulgaris, Rhodobacter sphaeroides, and Rhodobacter capsulatus (23, 27). However, our data suggest that QscR is not involved in regulation of fba or prk, as is the case for the chemoautotrophic bacteria, but is specifically involved in regulation of serine cycle genes. The results presented here demonstrate that QscR is essential for transcription of the sga-hpr-mtdA-fch operon, since little detectable transcription occurs in the absence of QscR. QscR is also required for methanol-dependent induction of transcription from the mtdA-mtdB-ppc-mcl operon and from gly. Therefore, QscR is a major regulator of the serine cycle, involved in controlling expression of nine serine cycle genes. However, expression of the serine cycle genes mdh and eno encoding malate dehydrogenase and enolase, respectively, is not affected by QscR. This result is in agreement with the dual role of these genes in methylotrophy and in multicarbon metabolism. Our results also suggest that QscR acts as an autorepressor of its own synthesis.

Two genes encoding enzymes that function to interconvert formate and methylene H₄F (mtdA and fch) are cotranscribed with the two serine cycle enzymes sga and hpr and regulated by QscR. These results suggest that the serine cycle and this portion of H₄F-dependent interconversions are tightly interlinked.

We were not successful so far in determining a coinducer metabolite for QscR, but we show that NADP⁺, acetyl-CoA, and glyoxylate have an inhibitory effect on the binding of this regulator to the sga promoter in a gel retardation assay. These three compounds are all indicators of excess serine cycle activity and are logical candidates for negative signals. It is possible that an as-yet-untested metabolite serves as a positive signal for QscR function.

 
We thank L. Chistoserdova and N. Korotkova for help and discussion. We thank C. J. Marx for supplying the plasmids pCM130, pCM277, pCM170, and pCM272.

This work was supported by a grant from the NIH (GM58933).

 
* Corresponding author. Mailing address: Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1750. Phone: (206) 616-5282. Fax: (206) 616-5721. E-mail: lidstrom{at}u.washington.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, February 2003, p. 1229-1235, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1229-1235.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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