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

Abstract

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 using GI698 and the were grown in M9 salts-based medium casamino 1% and M9 at 30 °C. was to the culture medium when the culture an and were were used at the 30 strains and plasmids and or or E. M. J. J.D. J. Science. 1997; D. S. A. S. 1993; Peterkofsky A. S. Seok Y.-J. A. 1998; from S. R. S. A. 1990; of P. Peterkofsky A. K. Nucleic Res. in for P. N. E. Peterkofsky A. in for with in for in in a new the from to of the E. coli frsA gene was by using to an at the and a from the The at and was by to that this not in any amino acid The product, with and was the P. Peterkofsky A. K. Nucleic Res. in the for FrsA in which FrsA expression is the of its the its and was by using to an of the frsA and a from the The product, with and was the of interaction of IIAGlc and its binding was by surface plasmon resonance using a 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, D. Peterkofsky A. S. Seok Y.-J. J. 2001; Scholar). IIAGlc and were immobilized the surface of a IIAGlc and in were to a at to the to the by a of was by of that resonance to a surface of the IIAGlc and were immobilized to a surface of and The was and and all were at a rate of The surface was between by of to the in interaction between IIAGlc and FrsA, GI698 FrsA or were grown in of M9 salts-based rich medium to an of and with 30 of containing The were in of the in the of The was in a and at for 30 at °C, and the was used as the The was with of the was a the was with the with containing The to the were with the containing and by by with and using serum in of were and to with to the of J. J. J.W. in Press, Scholar). were to on a previously described H. 1998; Scholar) high liquid coupled to a mass with an a at and with a of 30 was acid and was acid was at The mass was in the in which the was to a between and and of the in the were using the were the the J. and J. of Washington, of Molecular were to a of mass to for of and enzyme was to were using the of of were by was by the acid protein E. coli GI698 was used for of FrsA. culture and of protein was as described previously Y.-J. Zhu Peterkofsky A. S. A. Scholar). The from of culture containing FrsA was in containing and three through a at The was of by at for The soluble was through a using a of The containing of FrsA were and in a The was purified on a using a of containing FrsA were and The was on a with A. a was used with of to FrsA The of FrsA from of culture was EI, HPr, IIAGlc, and were purified as described previously D. Peterkofsky A. S. Seok Y.-J. J. 2001; Scholar, Y.-J. Zhu Peterkofsky A. S. A. Scholar, P. N. E. Peterkofsky A. Scholar). of was in a system containing either of IIAGlc or of FrsA or both in containing were for on and through a with the was at at a rate of of were of FrsA to its interaction with IIAGlc were in a was used as the binding of FrsA, and IIAGlc was to at for 30 This was with of glycerol) and in the for at the was with of Glucose were using a glucose the with some as described previously B.-M. Seok Y.-J. J. Microbiol. 2001; 39: 24-30Google Scholar). were and at for were used to glucose in the culture glucose in the of culture was in and of was with of and at 37 for 30 was at the of enzyme containing of glucose of and was in of and with in Disruption of frsA gene was using by and E. coli as described previously D. S. A. Scholar). The frsA gene from the was by a gene was from with the and and GI698 were by of the of Disruption of the frsA gene was by of by and of were grown in of M9 medium containing glucose or other sugars and casamino acids with and of culture were at the and were at for at °C. were used to the extracellular products. The culture at were with and to the The were at a resonance of by of a The of in culture was as described previously 1983; Scholar) with The in a of in and and or to a The were and to for at 37 °C. the at of kinase was to a of The were and at 37 for the at The in was and to by from a IIAGlc with a in E. coli of ligand-fishing by (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 by IIAGlc. a in from the interaction of a soluble protein with protein to a IIAGlc, immobilized on a was used as the between the and IIAGlc, a cytoplasmic from a culture of an E. coli strain a of the S. A. 1990; Scholar) was through a using a of was to the immobilized IIAGlc for we found that a in IIAGlc with glycerol that the interaction might be to of the glycerol when purified glycerol kinase was to the immobilized IIAGlc in the of D.W. Meadow N.D. Roseman S. Remington S.J. 1998; Scholar) for interaction was not the ptsHIcrr is in strain S. A. 1990; the of interaction of HPr and IIAGlc was also This us to this new IIAGlc-binding of the with to of the that with IIAGlc was on from were for binding to immobilized IIAGlc using the E. coli strain was grown to in of at 37 °C. The was and in containing this was three through a at The was at for to the soluble cytoplasmic This was through a 30 using a of interaction with immobilized these were and in a The was purified on a using a of interaction with immobilized IIAGlc, and these were and as described The was on a with A. the were using SPR, and affinity immobilized IIAGlc were and as described for previous a a was used with a in A. The interaction with IIAGlc, were and the protein was by to a of was to in The IIAGlc-binding the yafA were in and to of the with a protein with an mass of in addition to a to The in the of the 47-kDa protein were of the and to were by to in the was as on the of the and in the was as a of on the of the and and on the of the and the of the we that the was a from the protein and that the was to to the IIAGlc-binding The of the mass by of the IIAGlc-binding protein with the mass of was with this and of the frsA the that the IIAGlc-binding protein to the product of the yafA gene refer to yafA as we report that its gene product as a fermentation/respiration the gene was an expression that of the protein be For of FrsA, we the In this are the of the binding P. Peterkofsky A. K. Nucleic Res. Scholar, Y.-J. Zhu Peterkofsky A. S. A. Scholar). E. coli GI698, with the was used for of the gene This strain was grown as described and the protein with mass to of the protein sequential as described protein was as by not The purified protein to immobilized IIAGlc with high that the product of the frsA gene is the IIAGlc-binding The for the interaction between IIAGlc and its binding protein, using was to be interaction of the forms of both IIAGlc and its binding protein not the of of the IIAGlc-binding protein immobilized IIAGlc in with that of the purified FrsA, the cellular of the IIAGlc-binding protein was to be in FrsA to IIAGlc to a the regulatory functions of the PTS on the phosphorylation of the protein (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 effect of phosphorylation of IIAGlc on the affinity for its binding protein was by and immobilized IIAGlc, PEP, EI, and HPr were and for and this was to the immobilized IIAGlc IIAGlc was binding of the IIAGlc-binding to the immobilized IIAGlc surface was while there was binding to the unphosphorylated form of IIAGlc The of the binding was by the that FrsA not to immobilized D. Peterkofsky A. S. Seok Y.-J. J. 2001; Scholar). EI, HPr, or the soluble was to a surface immobilized with FrsA, was not The interaction by a high affinity interaction of IIAGlc and its binding was that such a complex might Consequently, experiments using were The to FrsA the in IIAGlc was FrsA the of IIAGlc in the in not to a of EI, HPr, or with the FrsA was with IIAGlc the in the of EI, HPr, and the to the complex between IIAGlc and FrsA was and in These the previous that FrsA specifically with IIAGlc and that this interaction on the phosphorylation of IIAGlc. The interaction between IIAGlc and FrsA was also by The from a of the complex was with of FrsA as a at to the form while IIAGlc at IIAGlc and FrsA were in a with and the a at 1983; Scholar, S. A. 1990; Scholar, D.W. Meadow N.D. Roseman S. Remington S.J. 1998; Scholar) to this were on a two at the for IIAGlc and its binding protein to IIAGlc. the and to the complex were the as a at the that the complex was its from the two not on the of the complex through the and the on of the that IIAGlc with FrsA in a ratio to form a for the in between IIAGlc and in experiments described a phosphorylation state-dependent high affinity interaction of IIAGlc with FrsA. of the complex in FrsA with histidine was in the frsA The from these was to a using shown in IIAGlc, but not its was with to the of IIAGlc in its phosphorylated form as from while IIAGlc was from the when the was from FrsA. These results that the phosphorylation state-dependent interaction between FrsA and IIAGlc also in of frsA on the of frsA on phenotype was using phenotype This involves the of cellular using respiration as the The a to the respiration of of this to respiration growth results in of a and in the well the the are as a of the respiration P. E. Res. 2001; Scholar, J. Bacteriol. Scholar). the the of in growth and the of respiration a system for were on with the strain in a of containing or this of respiration to cellular in a be at The results that using such as glucose, and its strain while there was in other these was that, of an in respiration rate on several sugars is the only phenotype to the of frsA frsA expression the an expression in which frsA expression is the of its was and the from of the frsA to of the was by and The expression level of FrsA from this was to be but that of IIAGlc by not the phenotype we phenotype of and using containing activities of the three strains the rate of was from strain to using the glucose, lactose, and as from the described is that of the strain resulted in the phenotype as the that the increased respiration rate of on several sugars was with of the frsA gene from not FrsA of of growth and glucose we found that the frsA strain consumed glucose the or while a growth rate the other strains in M9 medium containing glucose as the culture of several either or expression of FrsA not the expression level of IIAGlc or IICBGlc not these results suggest that FrsA might flux through the also be that in not with growth respiration is not coupled to growth P. E. Res. 2001; Scholar). the for the between growth and glucose utilization in these we spectroscopy of the growth medium and and were in the glucose medium for for in which the frsA gene is of these fermentation the In the growth medium containing as the of and increased in strain of the of in growth medium also these The of from was or in medium containing all the except for These results suggest that FrsA mixed-acid fermentation in E. coli by as a fermentation/respiration switch that respiration and fermentation protein of the E. coli PTS in glucose uptake been shown to the of a protein by a These regulatory functions of the PTS on the phosphorylation of the the phosphorylation of PTS been shown to in the and in the of a PTS sugar The ratio of phosphorylated to in as for the of these physiological EI with and regulates the of to PTS R. K. K. Lengeler J.W. S. A. Scholar). IIAGlc inhibits glycerol kinase and several sugar by a mechanism termed inducer (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, J.H. Faber H.R. Worthylake D. Meadow N.D. Roseman S. Pettigrew D.W. Remington S.J. Science. 1993; 259: 673-677Google Scholar). IIAGlc (6Peterkofsky A. Reizer A. Reizer J. Gollop N. Zhu P.P. Amin N. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 31-65Google Scholar). HPr with and the of glycogen phosphorylase in (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, 10Seok Y.-J. Koo B.-M. Sondej M. Peterkofsky A. J. Mol. Microbiol. Biotechnol. 2001; 3: 385-393Google Scholar) and in B.-M. Seok Y.-J. J. Microbiol. 2001; 39: 24-30Google Scholar). was also discovered that the membrane-bound glucose enzyme IICBGlc with to the cellular and of the protein in a phosphorylation state-dependent D. Peterkofsky A. S. Seok Y.-J. J. 2001; Scholar, S.J. J. J. Scholar, K. Aiba H. J. Scholar). have been in for other physiological activities by the PTS in E. coli. Consequently, we ligand-fishing experiments aimed at detecting soluble protein(s) exhibiting binding to IIAGlc with a affinity to glycerol kinase M.J. Saier Jr., M.H. J. Bacteriol. Scholar). The employing SPR, as used to the high affinity interaction of HPr with glycogen phosphorylase (8Seok Y.-J. Sondej M. Badawi P. Lewis M.S. Briggs M.C. Jaffe H. Peterkofsky A. J. Biol. Chem. 1997; 272: 26511-26521Google was that the interaction between glycerol kinase and IIAGlc was by only in the of not This is by the previous report that the between glycerol kinase and IIAGlc D.W. Meadow N.D. Roseman S. Remington S.J. 1998; Scholar). is that interaction was when purified EI was to immobilized HPr in the in our previous study (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) or when purified HPr or were to the IIAGlc surface in this study not the were to be between EI and HPr Seok Y.-J. Peterkofsky A. 1997; Scholar) and between HPr and IIAGlc Sondej J. Seok Y.-J. Peterkofsky A. J. Scholar) and between IIAGlc and M. Jr., Peterkofsky A. J. Biol. Chem. Scholar). that was that an interaction with a in this be by our The search for protein(s) employing ligand-fishing by in the of a protein with the to experiments the novel protein as the product by the yafA gene, which we now refer to as frsA This gene was previously only as a protein of of the protein with other glycerol and interacting with IIAGlc the frsA gene the of the to the expression Consequently, FrsA be to of the of the protein, using a of several resulted in protein not The interaction between FrsA and IIAGlc was on the phosphorylation of IIAGlc, and only the of IIAGlc an interaction with FrsA, both in and in search that of FrsA exist only in some such as E. coli, and acid at is of that all of these species are facultative anaerobes belonging to the γ-proteobacterial and of are FrsA been to as a protein and to this protein have not previously been Thus, of the of FrsA might us the of facultative and in these search that FrsA might be an enzyme of the which and purified FrsA, we not any of these activities the for these enzymes from the FrsA amino acid is that the cellular of FrsA is while that of IIAGlc is Meadow N.D. Roseman S. Postma P.W. J. Biol. Chem. Scholar). there is a interaction of the two proteins, is that all of the cellular FrsA protein in a complex with IIAGlc when PTS sugars are transported condition which the PTS are mainly in their we that with the of phosphorylation of the PTS. For facultative anaerobes E. coli, between and fermentation pathways be important for utilization of sugars available in the the physiological of the interaction between FrsA and IIAGlc, we E. coli strains with the of FrsA not been and its are found only in facultative we This be used to new functions of by for a of using respiration as the system J. Bacteriol. Scholar). on phenotype experiments was found that of frsA resulted in an of cellular respiration in medium containing glucose or some other sugars as the with of the strain resulted in the phenotype as the that increased respiration rate of on several sugars was with of the frsA gene from not and that fermentation are in the growth medium of the strain the and strains with all PTS and several non-PTS sugars also the phosphorylated forms of the PTS (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 the interaction between IIAGlc and FrsA FrsA to utilization of sugars through or pathways in in this study suggest that FrsA fermentation and that IIAGlc with to the previous study that the in the of glucose is to the of to in that the the of through to pathways the mechanisms not be T. Mol. Microbiol. 1994; Scholar). The the mechanism for this are of glucose or other IIAGlc exist in its and the FrsA protein be of IIAGlc and thus be FrsA as a of respiration or of fermentation the of of and in this of amino acid of IIAGlc and FrsA in facultative anaerobes that regulation of the switch from respiration to pathways glucose by IIAGlc might be a general mechanism in facultative experiments be to the by FrsA, to regulation by IIAGlc. of for the of E. coli