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A Novel Fermentation/Respiration Switch Protein Regulated by Enzyme IIAGlc in Escherichia coli

2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês

10.1074/jbc.m405048200

1083-351X

Byoung‐Mo Koo, Mi‐Jeong Yoon, Chang‐Ro Lee, Tae‐Wook Nam, Young‐Jun Choe, Howard Jaffe, Alan Peterkofsky, Yeong‐Jae Seok,

Amino Acid Enzymes and Metabolism

The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIAGlc (IIAGlc)) mediates some of these regulatory phenomena. In this report, we characterize a novel IIAGlc-binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as “function unknown.” FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIAGlc, with the highest affinity of any protein thus far shown to interact with IIAGlc. Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the γ-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIAGlc regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA. The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIAGlc (IIAGlc)) mediates some of these regulatory phenomena. In this report, we characterize a novel IIAGlc-binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as “function unknown.” FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIAGlc, with the highest affinity of any protein thus far shown to interact with IIAGlc. Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the γ-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIAGlc regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA. The bacterial phosphoenolpyruvate(PEP) 1The abbreviations used are: PEP, phosphoenolpyruvate; PTS, phosphoenolpyruvate:sugar phosphotransferase system; EI, enzyme I of the PTS; IIAGlc, the glucose-specific enzyme IIA of the PTS; HPr, histidine phosphocarrier protein; FrsA, a novel fermentation/respiration switch protein interacting with IIAGlc described in this work; SPR, surface plasmon resonance; LC, liquid chromatography; MS, mass spectrometry; BSA, bovine serum albumin; PM, phenotype microarray. 1The abbreviations used are: PEP, phosphoenolpyruvate; PTS, phosphoenolpyruvate:sugar phosphotransferase system; EI, enzyme I of the PTS; IIAGlc, the glucose-specific enzyme IIA of the PTS; HPr, histidine phosphocarrier protein; FrsA, a novel fermentation/respiration switch protein interacting with IIAGlc described in this work; SPR, surface plasmon resonance; LC, liquid chromatography; MS, mass spectrometry; BSA, bovine serum albumin; PM, phenotype microarray.:sugar phosphotransferase system (PTS) plays an important role in the transport of a variety of sugar substrates. This system catalyzes phosphorylation coupled to translocation of numerous simple sugars across the cytoplasmic membrane. The PTS is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein, HPr, which are used for all sugars, and, in addition, some sugar-specific components collectively known as enzymes II (1Postma P.W. Lengeler J.W. Jacobson G.R. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 1149-1174Google Scholar). The glucose-specific enzyme II of Escherichia coli consists of two components: soluble enzyme IIAGlc (IIAGlc) and membrane-bound enzyme IICBGlc (IICBGlc). Thus, glucose transport in E. coli involves three soluble PTS components (EI, HPr, and IIAGlc, encoded by the ptsHIcrr operon) and one membrane-bound protein, enzyme IICBGlc (encoded by the ptsG gene). Glucose uptake entails sequential phosphoryl transfer via the PTS, as follows: phosphoenolpyruvate (PEP) → EI → HPr → IIAGlc → IICBGlc → glucose. Components of the PTS also participate in several regulatory mechanisms (1Postma P.W. Lengeler J.W. Jacobson G.R. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 1149-1174Google Scholar). Catabolite repression allows for the preferential utilization of sugars transported by the PTS. Consequently, when E. coli are cultured in a medium containing both glucose and a non-PTS sugar, the glucose is consumed first. The currently accepted mechanism for this effect is that, when PTS sugars are transported, the steady-state condition of IIAGlc is mainly in the dephospho-form. The unphosphorylated form of IIAGlc inhibits transport of non-PTS sugars such as lactose, maltose, melibiose, and raffinose by interacting with transporters for these sugars (a process termed inducer exclusion) (1Postma P.W. Lengeler J.W. Jacobson G.R. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 1149-1174Google Scholar, 2Saier Jr., M.H. Novotny M.J. Comeau-Fuhrman D. Osumi T. Desai J.D. J. Bacteriol. 1983; 155: 1351-1357Google Scholar, 3Dean D.A. Reizer J. Nikaido H. Saier Jr., M.H. J. Biol. Chem. 1990; 265: 21005-21010Google Scholar, 4Titgemeyer F. Mason R.E. Saier Jr., M.H. J. Bacteriol. 1994; 176: 543-546Google Scholar). Other allosteric regulatory functions of IIAGlc include inhibition of the phosphorylation of glycerol by binding to glycerol kinase (5Hurley J.H. Faber H.R. Worthylake D. Meadow N.D. Roseman S. Pettigrew D.W. Remington S.J. Science. 1993; 259: 673-677Google Scholar) and either inhibition or activation of adenylyl cyclase (6Peterkofsky A. Reizer A. Reizer J. Gollop N. Zhu P.P. Amin N. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 31-65Google Scholar). E. coli IIAGlc is a protein with a Mr, predicted from its amino acid sequence, of 18,250 and which is phosphorylated by P-HPr at the N-3 position of His-90. In E. coli, the level of phosphorylated IIAGlc reflects not only the availability of extracellular glucose but also the intracellular ratio of [PEP] to [pyruvate] (7Hogema B.M. Arents J.C. Bader R. Eijkemans K. Yoshida H. Takahashi H. Aiba H. Postma P.W. Mol. Microbiol. 1998; 30: 487-498Google Scholar). In addition to the regulatory roles of IIAGlc listed above, we thought that there might be regulation of other physiological activities by IIAGlc in E. coli. This perspective stimulated us to embark on ligand-fishing experiments aimed at detecting other soluble protein(s) exhibiting high affinity binding to IIAGlc. In our previous study, employing ligand-fishing in E. coli extracts with surface plasmon resonance spectroscopy and immobilized HPr as the bait, we discovered a high affinity binding of glycogen phosphorylase to HPr; thus, HPr mediates cross-talk between sugar uptake through the PTS and glycogen breakdown (8Seok Y.-J. Sondej M. Badawi P. Lewis M.S. Briggs M.C. Jaffe H. Peterkofsky A. J. Biol. Chem. 1997; 272: 26511-26521Google Scholar, 9Koo B.-M. Seok Y.-J. J. Microbiol. 2001; 39: 24-30Google Scholar, 10Seok Y.-J. Koo B.-M. Sondej M. Peterkofsky A. J. Mol. Microbiol. Biotechnol. 2001; 3: 385-393Google Scholar). In this study, ligand-fishing, using surface plasmon resonance spectroscopy, was also employed to search for a high affinity binding of IIAGlc to a protein in E. coli extracts. Consequently, we discovered a new IIAGlc-binding protein in E. coli extracts. The protein was purified and established to be the previously uncharacterized yafA gene product, which we now refer to as a fermentation/respiration switch protein (FrsA). This report also describes experiments aimed at deducing the locus of action of FrsA. Strains, Plasmids, and Growth—The bacterial strains and plasmids used in this study are listed in Table I. Luria-Bertani (LB) medium was used for routine bacterial growth except with strain GI698 and its derivatives. M9 salts-based rich medium (supplemented with 2% casamino acids and 1% glycerol) was used for routine culture of GI698 and its derivatives. DY330, GI698, and their derivatives were grown at 30 °C, while other strains listed in Table I were grown at 37 °C. For the overproduction of proteins using GI698 and the pRE1-based vector system, cells were grown in M9 salts-based induction medium (0.2% casamino acids, 1% glycerol, and M9 salts) at 30 °C. Tryptophan (final concentration: 100 μg/ml) was added to the culture medium when the culture reached an A600 = 0.4, and cells were harvested 18–20 h after induction. Antibiotics were used at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 20 μg/ml; chloramphenicol, 30 μg/ml.Table IBacterial strains and plasmids usedStrains and plasmidsRelevant genotype or phenotypeSource or Ref.StrainsMG1655Wild type E. coli32Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Google ScholarDY330W3110 ΔlacU169 gal490 λCI857 Δ(cro-bioA)17Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Google ScholarGI698F- λ- lacIq lacPL8 ampC::Ptrp cI33LaVallie E.R. DiBlasio E.A. Kovacic S. Grant K.L. Schendel P.F. McCoy J.M. Bio/Technology. 1993; 11: 187-193Google ScholarGI698ΔptsGI698 Δ(ptsH, ptsI, crr), Kmr34Nosworthy N.J. Peterkofsky A. König S. Seok Y.-J. Szczepanowski R.H. Ginsburg A. Biochemistry. 1998; 37: 6718-6726Google ScholarSR702ΔptsGaraD139 ΔargF-lacU169rpsL150 thiA1 relA1 flbB5301 deoC1 ptsF25 rbsR suhX1 ΔptsG::cat CmrGift from S. R. RyuTP2811F-, xyl, argH1, ΔlacX74, aroB, ilvA, Δ(ptsH, ptsI, crr), Kmr19Levy S. Zeng G.Q. Danchin A. Gene (Amst.). 1990; 86: 27-33Google ScholarBM100DY330 ΔfrsA::cat, CmrThis workBM101MG1655 ΔfrsA::cat, CmrThis workBM201GI698 ΔfrsA::cat, CmrThis workPlasmidspRE1Expression vector under control of λPL promoter, Ampr11Reddy P. Peterkofsky A. McKenney K. Nucleic Acids Res. 1989; 17: 10473-10488Google ScholarpR3crr (NdeI/BamHI) in pRE1, Ampr, overexpression vector for IIAGlc16Reddy P. Fredd-Kuldell N. Liberman E. Peterkofsky A. Protein Expression Purif. 1991; 2: 179-187Google ScholarpKY103frsA (NdeI/BamHI) in pRE1, Ampr, overexpression vector for FrsAThis workpKY103HfrsA (NdeI/BamHI) with N-terminal 6 hisditines in pRE1, Ampr, overexpression vector for His-FrsAThis workpBR322pKY513frsA in pBR322, AmprThis work Open table in a new tab To construct pKY103, the DNA sequence from nucleotide 3227 to 4541 of the E. coli frsA gene (GI:2367098) was amplified by PCR, using mutagenic primers to create an NdeI site (underlined) at the ATG start codon (5′-TCTGGAGGCTGCACATATGCACAGGCAAA-3′) and a BamHI site (underlined) 39 nucleotides downstream from the TAA stop codon (5′-TATCTCCTGTTGGGATCCAACTGTTTTACC-3′). The internal NdeI site located at codons 186 and 187 was removed by mutagenesis (CATATG to CACATG) so that this did not result in any amino acid changes. The PCR product, digested with NdeI and BamHI, was cloned into the vector pRE1 (11Reddy P. Peterkofsky A. McKenney K. Nucleic Acids Res. 1989; 17: 10473-10488Google Scholar), resulting in the recombinant plasmid pKY103 for FrsA overproduction. To construct pKY513, in which FrsA expression is under the control of its own promoter, the sequence covering its own promoter and coding regions was amplified by PCR using mutagenic primers to create an EagI site (underlined) 432 nucleotides upstream of the frsA start codon (5′-TGCCGTAAGCCGTGGCGGCCGGGTACCGGG-3′) and a BamHI site (underlined) 39 nucleotides downstream from the TAA stop codon (5′-TATCTCCTGTTGGGATCCAACTGTTTTACC-3′). The PCR product, digested with EagI and BamHI, was cloned into the vector pBR322. Measurement of Protein-Protein Interaction—The interaction of IIAGlc and its binding factor was monitored by surface plasmon resonance (SPR) detection using a BIAcore 3000 (BIAcore AB) as described previously (8Seok Y.-J. Sondej M. Badawi P. Lewis M.S. Briggs M.C. Jaffe H. Peterkofsky A. J. Biol. Chem. 1997; 272: 26511-26521Google Scholar, 12Nam T.-W. Cho S.-H. Shin D. Kim J.-H. Jeong J.-Y. Lee J.-H. Roe J.-H. Peterkofsky A. Kang S.-O. Ryu S. Seok Y.-J. EMBO J. 2001; 20: 491-498Google Scholar). IIAGlc and IIBGlc were separately immobilized onto the carboxymethylated dextran surface of a CM5 sensor chip. IIAGlc and IIBGlc (80 μl, 100 μg/ml) in coupling buffer (10 mm sodium acetate, pH 4.0) were allowed to flow over a sensor chip at 10 μl/min to couple the proteins to the matrix by a N-hydroxysuccinimide/N-ethyl-N′(3-diethylaminopropyl)-carbodiimide reaction (80 μl of mix). Unreacted N-hydroxysuccinimide was inactivated by injecting 80 μl of 1 m ethanolamine HCl, pH 8.0. Assuming that 1000 resonance units correspond to a surface concentration of 1 ng/mm2, the proteins IIAGlc and IIBGlc were immobilized to a surface concentration of 1.5 and 1.8 ng/mm2, respectively. The standard running buffer was 10 mm HEPES, pH 7.4, 150 mm NaCl, 10 mm KCl, 1 mm MgCl2, and 1 mm dithiothreitol, and all reagents were introduced at a flow rate of 10 μl/min. The sensor surface was regenerated between assays by injecting 10 μl of 2 m NaCl to remove bound analyte. To examine the in vivo interaction between IIAGlc and FrsA, GI698 cells harboring pKY103 expressing FrsA or pKY103H expressing His-FrsA were grown in 200 ml of M9 salts-based rich medium to an A600 of 1.5, harvested, and washed with 30 ml of 20 mm HEPES buffer, pH 8.0, containing 100 mm NaCl. The cell pellets were resuspended in 3 ml of the buffer in the presence of 500 μm phenylmethylsulfonyl fluoride. The cell suspension was disrupted in a French pressure cell and centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was used as the crude extract. The crude extract was incubated with 200 μl of BD TALON™ resin. After the mixture was loaded onto a Poly-Prep chromatography column (8 × 40 mm) (Bio-Rad), the column was washed twice with the wash buffer (20 mm HEPES, pH 7.0, with 300 mm NaCl) containing 10 mm imidazole. The proteins bound to the column were eluted with the wash buffer containing 200 mm imidazole and analyzed by SDS-PAGE followed by staining with Coomassie Blue and Western blotting using anti-IIAGlc serum raised in mice. Identification of Coomassie Blue-stained Bands—Coomassie Blue-stained gel bands were excised and subjected to in-gel proteolytic digestion with trypsin according to the method of Moritz et al. (13Moritz R.L. Eddes J. Hong J. Reid G.E. Simpson R.J. Crabb J.W. Techniques in Protein Chemistry VI. Academic Press, San Diego, CA1995: 311-319Google Scholar). Digests were subjected to LC-MS analysis on a previously described (14Jaffe H. Veeranna Shetty K.T. Pant H.C. Biochemistry. 1998; 37: 3931-3940Google Scholar) Michrom Bioresources Magic 2002 model microbore high performance liquid chromatography coupled to a model LCQ ion trap mass spectrometer (Finnigan) equipped with an electrospray interface utilizing a 0.3 × 150 mm Magic MS C18 column (Michrom Bioresources) eluted at 8 μl/min and 40 °C with a linear gradient of 2–65% Solvent B over 30 min. Solvent A was 10/10/980/1/0.005 CH3CN/1-PrOH/H2O/acetic acid/hexafluorobutyric acid (v/v/v/v/v) and Solvent B was 700/200/100/0.9/0.005 CH3CN/1-PrOH/H2O/acetic acid/hexafluorobutyric acid (v/v/v/v/v). Column effluent was monitored at 215 nm. The mass spectrometer was operated in the “Top Five” mode in which the instrument was set up to automatically acquire (a) a full scan between m/z 300 and m/z 1300 and (b) tandem MS/MS spectra (relative collision energy = 35%) of the five most intense ions in the full scan. MS/MS spectra were analyzed using the Bioworks software package (Finnigan). Individual uninterpreted MS/MS spectra were searched against the non-redundant data base utilizing the SEQUEST program (by J. Eng and J. Yates, University of Washington, Department of Molecular Biotechnology, Seattle, WA). Search parameters were set to a static modification of +71 atomic mass units to allow for acrylamide alkylation of cysteine and enzyme specificity was set to “no enzyme.” Fragment ions were labeled using the Bioworks software. Purification of Overexpressed Proteins—The steps of purification were followed by SDS-PAGE. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce). E. coli GI698 harboring pKY103 was used for overexpression of FrsA. Cell culture and induction of protein overexpression was done as described previously (15Seok Y.-J. Lee B.-R. Zhu P.-P. Peterkofsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 347-351Google Scholar). The cell pellet obtained from 500 ml of culture containing overexpressed FrsA was resuspended in buffer A (10 mm Tris·HCl, pH 7.5, containing 50 mm NaCl) and then passed three times through a French pressure cell at 10,000 p.s.i. The lysate was cleared of cell debris by centrifugation at 100,000 × g for 90 min. The soluble fraction was chromatographed through a DEAE-Sepharose (Sigma) column (2.5 × 10 cm) using a gradient of 50–500 mm NaCl (220 ml). The fractions containing substantial amounts of FrsA were pooled and concentrated in a 3 K Macrosep centrifugal concentrator (Pall Gelman Laboratory, Ann Arbor, MI). The concentrated pool was further purified on a hydroxyapatite (Bio-Rad) column (1.5 × 10 cm) using a gradient of 0–500 mm potassium phosphate buffer, pH 7.5 (80 ml). Fractions containing FrsA were pooled and concentrated. The concentrated fraction was chromatographed on a HiLoad 16/60 Superdex 75 prepgrade column (Amersham Biosciences) equilibrated with buffer A. Finally, a Mono Q 5/5 (Amersham Biosciences) column was used with 15 volumes of NaCl gradient (50–300 mm) to obtain homogeneous FrsA (>98% pure). The yield of pure FrsA from 500 ml of culture was ∼2 mg. EI, HPr, IIAGlc, and EIIBGlc were purified as described previously (12Nam T.-W. Cho S.-H. Shin D. Kim J.-H. Jeong J.-Y. Lee J.-H. Roe J.-H. Peterkofsky A. Kang S.-O. Ryu S. Seok Y.-J. EMBO J. 2001; 20: 491-498Google Scholar, 15Seok Y.-J. Lee B.-R. Zhu P.-P. Peterkofsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 347-351Google Scholar, 16Reddy P. Fredd-Kuldell N. Liberman E. Peterkofsky A. Protein Expression Purif. 1991; 2: 179-187Google Scholar). Gel Filtration Chromatography of IIAGlc-FrsA Complex—Gel filtration chromatography was performed in a AKTA-FPLC system (Amersham Biosciences). One-ml samples containing either 4 mg of IIAGlc or 4 mg of FrsA or both proteins in 20 mm Tris-HCl, pH 8.0, containing 50 mm NaCl were incubated for 10 min on ice and injected through a Superose 12 column (25 × 500 mm; Amersham Biosciences) equilibrated with the same buffer. Filtration was performed at room temperature at a flow rate of 1 ml/min. Fractions of 3 ml were collected. Native Polyacrylamide Gel Electrophoresis—Mobility shifts of FrsA due to its interaction with IIAGlc were demonstrated in a nondenaturing 4–20% gradient gel (Novex). Tris·glycine, pH 8.3, was used as the running buffer. A binding mixture (20 μl) of 100 mm Tris·HCl, pH 7.5, 2 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, 200 μg/ml FrsA, and 50 μg/ml IIAGlc was allowed to incubate at room temperature for 30 min. This mixture was combined with 5 μl of 5× loading buffer (0.01% bromphenol blue, 0.5 m Tris·HCl, pH 6.8, 50% glycerol) and electrophoresed in the gel for 2 h at 100 V. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. Measurement of Glucose Concentration—Glucose concentrations were measured using a glucose assay kit (Sigma) following the supplier's instruction with some modifications as described previously (9Koo B.-M. Seok Y.-J. J. Microbiol. 2001; 39: 24-30Google Scholar). Cells were withdrawn every 2 h and centrifuged at 10,000 × g for 5 min. Supernatant solutions were used to determine glucose remaining in the culture medium. To determine glucose in the medium, each aliquot of culture broth was diluted 100-fold in water, and 20 μl of diluted solution was mixed with 200 μl of combined enzyme-color reagent solution and incubated at 37 °C for 30 min. After color development, absorbance was measured at 450 nm. To prepare the combined enzyme-color reagent solution, 200 mg of enzyme mixture containing 100 units of Aspergillus niger glucose oxidase, 20 Purpurogalin units of horseradish peroxidase, and buffer salts was dissolved in 20 ml of distilled water, and mixed with 320 μl O-dianisidine dihydrochloride solution (2.5 mg/ml in distilled water). Disruption of frsA—The frsA gene was disrupted using electroporated linear DNA amplified by PCR and E. coli DY330 as described previously (17Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Google Scholar). The frsA gene (nucleotides 7–1164 from the start codon) was replaced by a chloramphenicol acetyltransferase gene (cat). cat was amplified from SR702 (ptsG::cat) with the following primers; forward primer, 5′-GCGGGTTACA ATAGTTTCCA GTAAGTATTC TGGAGGCTGC ATCCATGACA GAGAAAAAAA TCACTGGATA-3′ and reverse primer, 5′-GATTTCCTGA AGACCTTTGT CAAAATTCCG ATACACCGGG TTAAATGGGA CGCCCCGCCC TGCCACTCAT-3′. MG1655 ΔfrsA (BM101) and GI698 ΔfrsA (BM201) were constructed by P1 transduction of the Cmr region of BM100. Disruption of the frsA gene was confirmed by PCR. Analysis of Metabolic Products by NMR Spectroscopy and Measurement of Acetate Concentration—Cells were grown in 200 ml of M9 salts medium containing 0.5% glucose or other indicated sugars and 0.2% casamino acids supplemented with 1 mm MgCl2, 0.1 mm CaCl2 and 100 μg/ml ampicillin. Aliquots of culture were taken at the indicated time and were centrifuged at 3000 × g for 10 min at 4 °C. Supernatant solutions were used to analyze the extracellular metabolic products. The cell-free culture solutions (0.9 ml each) taken at 12 h after inoculation were mixed with D2O (0.1 ml) and subjected to the NMR experiments. The spectra were recorded at a 1H resonance frequency of 500 MHz by use of a Bruker DRX500 spectrometer. The concentration of acetate in culture supernatants was measured as described previously (18Clarke P.M. Payton M.A. Anal. Biochem. 1983; 130: 402-405Google Scholar) with slight modifications. The assay mixture contained in a final volume of 3 ml (final concentration in parentheses): potassium phosphate, pH 7.6 (150 mm), MgCl2 (20 mm), PEP (0.5 mm), NADH (0.5 mm), ATP (5 mm), pyruvate kinase/lactate dehydrogenase (4.4 and 6.0 units/ml, respectively), and samples or 10–150 μm sodium acetate to make a standard curve. The reagents were mixed and allowed to equilibrate for 10 min at 37 °C. After noting the absorbance at 365 nm (A1), 20 μl of acetate kinase was added to a final concentration of 1.5 units/ml. The reagents were mixed again and incubated at 37 °C for 60 min before noting the final absorbance at 365 nm (A2). The change in absorbance (ΔA = A1 – A2) was calculated and related to concentration by reading from a standard plot. Immobilized IIAGlc Complexes with a Factor in E. coli Extracts—The technique of ligand-fishing by SPR (8Seok Y.-J. Sondej M. Badawi P. Lewis M.S. Briggs M.C. Jaffe H. Peterkofsky A. J. Biol. Chem. 1997; 272: 26511-26521Google Scholar) was used to search for a protein that might interact with and be regulated by IIAGlc. SPR detects a change in refractive index resulting from the interaction of a soluble protein with another protein covalently linked to a surface. IIAGlc, immobilized on a CM-5 sensor chip (see “Experimental Procedures”), was used as the bait. To minimize nonspecific interactions between the crude extract and IIAGlc, a cytoplasmic crude extract from a stationary phase culture of an E. coli strain (TP2811) carrying a deletion of the pts operon (see “Experimental Procedures”) (19Levy S. Zeng G.Q. Danchin A. Gene (Amst.). 1990; 86: 27-33Google Scholar) was chromatographed through a Mono Q 10/10 column (Amersham Biosciences) using a linear gradient of 50–500 mm NaCl (80 ml total volume). Each fraction was allowed to flow over the immobilized IIAGlc for 10 min; we found that fractions eluting near 100 mm NaCl showed a detectable increase in SPR response (Fig. 1A). Since IIAGlc interacts with glycerol kinase, it seemed possible that the detected interaction might be due to formation of the glycerol kinase-IIAGlc complex. However, when purified glycerol kinase (120 μlof10 μg/ml protein) was allowed to flow over the immobilized IIAGlc in the absence of Zn2+ (20Pettigrew D.W. Meadow N.D. Roseman S. Remington S.J. Biochemistry. 1998; 37: 4875-4883Google Scholar) for 10 min, no interaction was detectable (data not shown). Furthermore, because the ptsHIcrr operon is deleted in strain TP2811 (19Levy S. Zeng G.Q. Danchin A. Gene (Amst.). 1990; 86: 27-33Google Scholar), the possibility of interaction of HPr and IIAGlc was also excluded. This result prompted us to purify this new IIAGlc-binding protein. Purification of the Factor Interacting with IIAGlc—The approach to purification of the factor that interacts with IIAGlc was based on SPR analysis; fractions from each purification step were examined for binding to immobilized IIAGlc using the BIAcore (Fig. 1). E. coli strain TP2811 was grown to stationary phase in 6 liters of Luria broth at 37 °C. The harvested cell pellet was washed and resuspended in buffer A (10 mm Tris·HCl, pH 7.5, containing 50 mm NaCl); this suspension was passed three times through a French pressure cell at 10,000 p.s.i. The lysate was centrifuged at 100,000 × g for 90 min to obtain the soluble cytoplasmic fraction. This fraction was chromatographed through a DEAE-Sepharose (Sigma) column (8 × 30 cm) using a linear gradient of 50–500 mm NaCl (2 liters total volume). Fractions eluting around 100 mm NaCl demonstrated interaction with immobilized IIAGlc; these fractions were pooled and concentrated in a 3 K Macrosep centrifugal concentrator (Pall Gelman Laboratory). The concentrated pool was further purified on a hydroxyapatite (Bio-Rad) column (1.5 × 10 cm) using a linear gradient of 0–500 mm potassium phosphate buffer, pH 7.5 (80 ml total volume). Fractions eluting around 100 mm potassium phosphate demonstrated interaction with immobilized IIAGlc, and these fractions were pooled and concentrated as described above. The concentrated pool was chromatographed on a Superdex 75 column (Amersham Biosciences) equilibrated with buffer A. Once again, the eluted fractions were analyzed using SPR, and fractions showing affinity toward immobilized IIAGlc were pooled and concentrated as described for previous steps. As a final purification step, a Mono Q 5/5 column (Amersham Biosciences) was used with a 15-volume NaCl gradient (50–300 mm) in buffer A. The fractions eluting near 100 mm NaCl, showing interaction with IIAGlc, were pooled, and the protein was precipitated by adding acetone to a final concentration of 80% after BSA (1 mg/ml) was added to assist in recovery. The IIAGlc-binding Protein Is the yafA Gene Product—The precipitated proteins were solubilized in SDS-loading buffer and subjected to SDS-PAGE. Staining of the gel with Coomassie Blue revealed a protein band migrating with an apparent molecular mass of ∼47 kDa in addition to a band corresponding to BSA. The bands in the vicinity of the 47-kDa protein were cut out of the gel and subjected to in-gel proteolytic digestion. Digests were analyzed by LC/MS/MS (see “Experimental Procedures”) to identify proteins. Protein in the slower migrating (presumptive BSA) band was identified as BSA on the basis of the following tryptic peptides: (K)SEIAHR, (K)DLGEEHFK, (R)FKDLGEEHFK, (K)HLVDEPQNLIK, (R)KVPQVSTPTLVEVSR, and (K)TSESGELHGLTTEDK. Protein in the faster migrating band was identified as a mixture of BSA on the basis of the following tryptic peptides: (K)HLVDEPQNLIK, (R)KVPQVSTPTLVEVSR, (R)RPCFSALTPDETYVPK, (K)LVNELTEFAK, (R)RHPEYAVAVLLR, (K)LGEYGFQNAILVR, and (K)TSESGELHGTLTTEDK and YAFA_ECOLI (P04335) on the basis of the following tryptic peptides: (R)LGMHDASDEALR, (K)GDDLAEQAQALSNR, (R)VAAFGFR, (R)TDDDLYDTVIGYR, and (M)TQANLSETLFKPR (see Fig. 1B). On the basis of the sequence analyses, we concluded that the BSA sequence was a carryover from the added protein (see above) and that the YAFA_ECOLI sequence was likely to correspond to the IIAGlc-binding factor. The correspondence of the apparent molecular mass by SDS-PAGE of the IIAGlc-binding protein with the calculated molecular mass of YAFA_ECOLI (47,008 Da) was consistent with this prediction. Overexpression and Purification of the frsA Gene Product—To further explore the possibility that the IIAGlc-binding protein corresponded to the product (YAFA_ECOLI) of the yafA gene (we refer to yafA as frsA, since we report here that its gene product (FrsA) acts as a fermentation/respiration switch), the gene was cloned into an expression vector so that sizeable amounts of the pure protein could be produced. For overexpression of FrsA, we constructed the pRE1-based recombinant plasmid, pKY103. In this plasmid, genes are under the control of the strong λPL promoter-cII ribosome binding site combination (11Reddy P. Peterkofsky A. McKenney K. Nucleic Acids Res. 1989; 17: