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• W1978516228 abstract "The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, theB. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His∼P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing asacB′-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity. The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, theB. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His∼P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing asacB′-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity. The Bacillus subtilis SacY protein is a transcriptional antiterminator involved in sucrose-promoted induction of thesacB gene, encoding levansucrase (1Shimotsu H. Henner D.J. J. Bacteriol. 1986; 168: 380-388Crossref PubMed Google Scholar, 2Steinmetz M. Aymerich S. Ann. Microbiol. ( Paris ). 1986; 137A: 3-14PubMed Google Scholar, 3Aymerich S. Steinmetz M. Mol. & Gen. Genet. 1987; 208: 114-120Crossref PubMed Scopus (21) Google Scholar, 4Arnaud M. Débarbouillé M. Rapoport G. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1996; 271: 18966-18972Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Transcription initiates constitutively from the sacB promoter, but in the absence of sucrose, most transcripts terminate at a rho-independent transcriptional terminator located in the leader region preceeding thesacB structural gene. In the presence of sucrose, SacY is activated and stabilizes a stem-loop structure of a ribonucleic acid antiterminator, thereby preventing alternative formation of the overlapping transcriptional terminator (5Aymerich S. Steinmetz M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10410-10414Crossref PubMed Scopus (114) Google Scholar). SacY activity is negatively controlled by enzyme I (EI) 1The abbreviations used are: EI, enzyme I; PTS, phosphoenolpyruvate:sugar phosphotransferase system; PEP, phosphoenolpyruvate; GST, glutathione S-transferase; IPTG, isopropyl-β-d-thiogalactopyranoside. and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a protein homologous to sucrose-specific PTS permeases (6Zukowski M.M. Miller L. Cogswell P. Chen K. Aymerich S. Steinmetz M. Gene ( Amst .). 1990; 90: 153-155Crossref PubMed Scopus (38) Google Scholar, 7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar). The PTS catalyzes both the transport and the phosphorylation of numerous carbohydrates in bacteria. Energy for these coupled reactions is provided by phosphoenolpyruvate (PEP). The phosphoryl group of PEP is sequentially transferred to the incoming sugar via EI, HPr, and two domains in each of the sugar-specific permeases. The modular carbohydrate-specific permeases are generally comprised of three proteins or domains, EIIA, EIIB, and EIIC (8Saier Jr., M.H. Reizer J. J. Bacteriol. 1992; 174: 1433-1438Crossref PubMed Google Scholar). The EIIC protein or domain is the integral membrane permease, whereas EIIA and EIIB are hydrophilic, peripherally membrane-associated proteins or domains that are phosphorylated during the phosphoryl transfer cascade. EIIB∼P transfers its phosphate to the incoming sugar during its translocation across the membrane (for reviews, see Refs. 9Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar and 10Saier Jr., M.H. Reizer J. Mol. Microbiol. 1994; 13: 755-764Crossref PubMed Scopus (171) Google Scholar). SacX is thought to be an EIIBC protein; the corresponding EIIA protein has not been characterized to date. Our long term interest in the multifaceted controls exerted by the PTS led us to investigate the molecular mechanism involved in PTS-mediated regulation of transcriptional antitermination by SacY. Previous genetic data (7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar) and comparison with results reported for the homologous BglG antiterminator of Escherichia coli (11Amster-Choder O. Houman F. Wright A. Cell. 1989; 58: 847-855Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 12Amster-Choder O. Wright A. Science. 1990; 249: 540-542Crossref PubMed Scopus (55) Google Scholar, 13Schnetz K. Rak B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5074-5078Crossref PubMed Scopus (75) Google Scholar) suggested that this control involves a cascade of phosphorylations. The E. coli bgl operon, required for transport and hydrolysis of β-glucosides, is inducible by β-glucosides through a BglG-dependent antitermination mechanism (14Mahadevan S. Wright A. Cell. 1987; 50: 485-494Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 15Schnetz K. Rak B. EMBO J. 1988; 7: 3271-3277Crossref PubMed Scopus (106) Google Scholar). Activity of BglG is negatively controlled by BglF, a β-glucoside permease with EIIBCA domain structure, that has been reported to phosphorylate BglG at an unknown residue in response to the external level of inducer (11Amster-Choder O. Houman F. Wright A. Cell. 1989; 58: 847-855Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 12Amster-Choder O. Wright A. Science. 1990; 249: 540-542Crossref PubMed Scopus (55) Google Scholar, 13Schnetz K. Rak B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5074-5078Crossref PubMed Scopus (75) Google Scholar, 14Mahadevan S. Wright A. Cell. 1987; 50: 485-494Abstract Full Text PDF PubMed Scopus (95) Google Scholar). Furthermore, phosphorylation of BglG has been shown to prevent its dimerization. Consequently the PTS was proposed to control the antitermination activity of BglG by modulating the level of its active, unphosphorylated, dimeric form (16Amster-Choder O. Wright A. Science. 1992; 257: 1395-1398Crossref PubMed Scopus (110) Google Scholar). Two other antiterminators in B. subtilis, in addition to SacY, belong to the BglG family: SacT mediates induction of thesacPA operon by sucrose (17Debarbouille M. Arnaud M. Fouet A. Klier A. Rapoport G. J. Bacteriol. 1990; 172: 3966-3973Crossref PubMed Google Scholar), and LicT mediates induction of the licTS and bglPH operons by β-glucosides (18Le Coq D. Lindner C. Krüger S. Steinmetz M. Stülke J. J. Bacteriol. 1995; 177: 1527-1535Crossref PubMed Google Scholar, 19Schnetz K. Stülke J. Gertz S. Krüger S. Krieg M. Hecker M. Rak B. J. Bacteriol. 1996; 178: 1971-1979Crossref PubMed Google Scholar). Interestingly, SacT and LicT are not active in mutants lacking EI (ptsI) or HPr (ptsH) (20Arnaud M. Vary P. Zagorec M. Klier A. Débarbouillé M. Postma P. Rapoport G. J. Bacteriol. 1992; 174: 3161-3170Crossref PubMed Google Scholar, 21Krüger S. Hecker M. J. Bacteriol. 1995; 177: 5590-5597Crossref PubMed Google Scholar, 22Krüger S. Gertz S. Hecker M. J. Bacteriol. 1996; 178: 2637-2644Crossref PubMed Google Scholar). Thus, PTS-mediated phosphorylation appears to exert positive control over SacT and LicT antitermination activities. Positive control by the PTS has been also demonstrated for LevR, a B. subtilisDNA-binding transcriptional regulator involved in fructose-promoted induction of the levanase operon (23Débarbouillé M. Martin-Verstraete I. Klier A. Rapoport G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2212-2216Crossref PubMed Scopus (78) Google Scholar, 24Stülke J. Martin-Verstraete I. Charrier V. Klier A. Deutscher J. Rapoport G. J. Bacteriol. 1995; 177: 6928-6936Crossref PubMed Google Scholar). Recent experiments have shown that SacT and LevR are phosphorylated by PEP in a PTS-catalyzed reaction that depends only on EI and HPr. These observations suggested that phosphorylation is involved in PTS-mediated positive control of these transcriptional regulators (4Arnaud M. Débarbouillé M. Rapoport G. Saier Jr., M.H. Reizer J. J. Biol. Chem. 1996; 271: 18966-18972Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 24Stülke J. Martin-Verstraete I. Charrier V. Klier A. Deutscher J. Rapoport G. J. Bacteriol. 1995; 177: 6928-6936Crossref PubMed Google Scholar). We note however that despite the sequence conservation between the antiterminators of the BglG family, SacY is not subject to PTS-mediated positive control sincesacB is constitutively expressed in a ptsHImutant (7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar). This fact renders SacY a good model protein for analysis of PTS-mediated negative control of antitermination. In this manuscript, we show that SacY is readily phosphorylated by PEP in a PTS-catalyzed reaction that depends only on EI and HPr. Additionally, computer-aided analyses led us to identify histidyl residues that are highly conserved in the modular SacY and in 13 distinct RNA- and DNA-binding proteins that bear duplicated SacY-like domains. Utilizing site-directed mutants of these conserved histidyl residues, we provide evidence demonstrating that His-99, His-207 and His-269 of SacY are phosphorylated by HPr(His∼P). Finally, we present data suggesting that PTS-dependent inactivation of SacY activity is mainly due to phosphorylation of His-99. Altogether, the data presented define the role of PTS-catalyzed phosphorylation of SacY in the modulation of its transcriptional antitermination activity. Since SacY-like domains as well as phosphorylatable histidyl residues are highly conserved in 14 distinct mRNA-binding and DNA-binding proteins, the reported data provide guidelines for studies of PTS-dependent transcriptional regulation by a unique family of functionally distinct regulatory proteins. A fragment carrying the entire sacY sequence, flanked with BamHI andEcoRI restriction sites, was generated by PCR with the DYB (5′-CGCGGATCCATGAAAATTAA-AAGAATCTTAAATC-3′) and FYE (5′-CCGGAATTCAGCGTGCGACTGACCGTTGG-3′) primers on pSL90 (7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar). Following digestion with BamHI and EcoRI, the 847-base pair fragment was cloned into pBluescript SK− (Stratagene, La Jolla, CA) generating pIC437. The CAT (His) codon at position 99, 158, 207, or 269 was substituted with a TAT (Tyr) codon by site-directed mutagenesis using the Muta-gene M13 in vitro mutagenesis kit (Bio-Rad, Hercules, CA). sacY alleles carrying two or three of these His to Tyr mutations were constructed by successive site-directed mutageneses or, when possible, by exchange of a relevant restriction fragment. The wild-type and mutated sacY genes were then cloned into the B. subtilis pSL90 integrative plasmid (7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar), generating a collection of plasmids that were used to integrate the different alleles at the sacY locus. For this, the BstBI/AccI fragment of pSL90, carrying the 3′ part of sacY, was replaced with theBstBI/EcoRV fragment of pIC437 or one of its derivatives carrying a sacY mutant allele. The BamHI/EcoRI fragment from pIC437 or its derivatives was inserted into pGEX-2T (Pharmacia, Uppsala, Sweden) to give a plasmid overproducing the wild-type or a mutant glutathioneS-transferase (GST)-SacY fusion protein. pGEX-2T derivatives overproducing the wild-type or the mutated P1 and P2 domains of SacY were constructed as follows. DNA fragments carrying the wild-type or the mutated sequences encoding the P1 and P2 domains flanked withBamHI and EcoRI restriction sites, were generated by PCR using pIC437 or its derivatives with the primers DYP1B (5′-CGCGGATCCACATTGCCTG-AAGACCAC-3′) and FYP1E (5′-CCGGAATTCAGGCATTGTTTCTTGCTGTG-3′) (for the P1 domain encoding sequence) or DYP2B (5′-CGCGGATCCGGCGATATGACACAAAC-3′) and FYE (for the P2 domain encoding sequence). These fragments were then digested withBamHI and EcoRI, and cloned into pGEX-2T. The entire sacY gene as well as the P1 and P2 encoding sequences in the different plasmids were verified by sequencing after site-directed mutagenesis or PCR. Plasmid pIC424, which carries the B. subtilis pts operon with a ptsI::spe disruption, was derived from pTS20 (25Gonzy-Tréboul G. Steinmetz M. J. Bacteriol. 1987; 169: 2287-2290Crossref PubMed Google Scholar) by insertion of a spectinomycin resistance gene,i.e. a SmaI fragment from pIC156 (26Steinmetz M. Richter R. Gene. 1994; 142: 79-83Crossref PubMed Scopus (180) Google Scholar), into theSstI site of ptsI. pSL151 is a B. subtilis replicative plasmid, carrying the pUB110 replication origin, the chloramphenicol resistance cat gene, and thesacX gene under the control of the spac promoter (27Yansura D.G. Henner D.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 439-443Crossref PubMed Scopus (229) Google Scholar). E. coli strains used for plasmid construction, mutagenesis and protein overproduction were DH5α, CJ236, and BL21(DE3,) respectively (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). B. subtilis GM152 (trpC2 sacB′-lacZ sacXYΔ3) was described previously (7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar). The B. subtilis strain GM1288 (sacXYΔ3 sacBΔ23 sacTΔ4 amyE::bglP′-lacZ Δ(licS-bglP)::aphA3) is deleted of all known antiterminator genes and derived from GM1271 (18Le Coq D. Lindner C. Krüger S. Steinmetz M. Stülke J. J. Bacteriol. 1995; 177: 1527-1535Crossref PubMed Google Scholar).B. subtilis GM1320 and GM1386 were derived from GM1288 by an in-frame deletion of the DraI/HpaI fragment ofptsH (deletion of codons 6–34) and by introduction of theptsI::spe disruption present in plasmid pIC424, respectively. The B. subtilis strains GM1288X, GM1320X, and GM1386X were obtained by transformation of GM1288, GM1320, and GM1386, respectively, with pSL151. LB medium (29Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) supplemented with appropriate antibiotics (chloramphenicol (4 μg/ml) or spectinomycin (100 μg/ml) forB. subtilis, and ampicillin (50 μg/ml) for E. coli) was routinely used for selection of transformants. BL21(DE3) transformants were grown at 30 °C in 2 × YT medium (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 50 μg/ml ampicillin. B. subtilis extracts for phosphorylation assays were prepared as follows. Cells were grown in liquid LB medium supplemented with sucrose (30 mm) and isopropyl-β-d-thiogalactopyranoside (IPTG) (0.5 mm) to allow sacX expression from thespac promoter carried by pSL151. Cells were harvested by centrifugation when the culture reached an A 600of 1.5 and resuspended in a B. subtilis lysis buffer (50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 1 mmdithiothreitol). Cells were ruptured by sonication, and extracts were then centrifuged at 10,000 × g for 5 min. Aliquots of the supernatants were stored at −80 °C until used. Enzyme I and HPr were prepared as described previously (30Reizer J. Sutrina S.L. Saier Jr., M.H. Stewart G.C. Peterkofsky A. Reddy P. EMBO J. 1989; 8: 2111-2120Crossref PubMed Scopus (94) Google Scholar, 31Reizer J. Sutrina S.L. Wu L.-F. Deutscher J. Reddy P. Saier Jr., M.H. J. Biol. Chem. 1992; 267: 9158-9169Abstract Full Text PDF PubMed Google Scholar). The wild type and mutant SacY proteins as well as the P1 and P2 domains were produced and purified as GST-fusions. For this purpose, BL21(DE3) was transformed with the pGEX-2T derivatives, and transformants were grown with shaking at 30 °C in 200 ml of 2 × YT liquid medium. Protein overproduction was induced by addition of IPTG (0.5 mm) at an A 600 of 0.5. After an additional 4-h incubation at 30 °C, cells were harvested by centrifugation and resuspended in an E. coli lysis buffer (50 mmTris-HCl, pH 7.5, 120 mm NaCl, 2 mm EDTA, 1 mm dithiothreitol, 5 mm benzamidine). Cells were lysed by sonication, and membranes were removed by a 30-min centrifugation at 100,000 × g. The supernatant was then applied to a prepacked glutathione-Sepharose 4B column (Pharmacia), and fusion proteins were purified as recommended by the manufacturer. Purity of the proteins was confirmed by electrophoresis on 12.5% SDS-polyacrylamide gels (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207472) Google Scholar). Protein concentrations were determined by the Bradford method (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217370) Google Scholar). Chemical synthesis of [32P]PEP from [32P]orthophosphate (ICN, Costa Mesa, CA) was performed as described previously (34Alpert C.A. Frank R. Stüber K. Deutscher J. Hengstenberg W. Biochemistry. 1985; 24: 959-964Crossref PubMed Scopus (46) Google Scholar). The reaction mixture was loaded onto an AG1 × 8 column (Bio-Rad), preequilibrated with 0.1 mammonium bicarbonate, and [32P]PEP was separated from [32P]orthophosphate using 5-ml aliquots of 0.2, 0.4, and 0.7 m ammonium bicarbonate. Fractions of 1 ml were collected, and eluted material was identified by thin layer chromatography on silica plates and tert-amyl alcohol/formate/H2O (3:2:1) as a solvent. Under these conditions, [32P]orthophosphate eluted in fractions 7 and 8 while [32P]PEP eluted in fractions 11–13. Purified SacY (2.8 μg, used as the GST-fusion or separated from GST after cleavage with thrombin) was phosphorylated by incubation at 37 °C in a reaction mixture (15 μl final volume) containing 50 mmpotassium phosphate buffer (pH 7.4), 2 mm dithiothreitol, 0.5 mm MgCl2, 2.5 μm (0.25 μCi) [32P]PEP, and either B. subtilis crude extract (17 μg) or the purified PTS proteins EI (3 μg) and HPr (1 or 0.1 μg). The reaction was terminated after 20 min by addition of SDS-electrophoresis sample buffer. This incubation time allowed for maximal protein phosphorylation, as determined by kinetic experiments (up to 60 min). Proteins were separated on 12.5% SDS-polyacrylamide gels. Autoradiography of dried gels were performed with Kodak x-ray film (X-OMAT) with exposure times ranging between 2 and 24 h at −80 °C. B. subtilis liquid cultures were grown in CgCH medium (7Crutz A.-M. Steinmetz M. Aymerich S. Richter R. Le Coq D. J. Bacteriol. 1990; 172: 1043-1050Crossref PubMed Google Scholar) supplemented with chloramphenicol (4 mg/l), tryptophan (50 mg/l), with or without 60 mm sucrose. Preparation of B. subtilis extracts and β-galactosidase assays were performed as described previously (29Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar, 35Piggot P.J. Curtis C.A.M. J. Bacteriol. 1987; 169: 1260-1266Crossref PubMed Google Scholar). Assays were repeated with at least three independent transformants for each construct. The mean value is given for each assay. Standard deviations were not more than 15% for activities equal to or higher than 3.5 Miller units and not more than 40% for lower activities. PTS-catalyzed phosphorylation of SacY was examined using in vitrophosphorylation assays. Purified SacY was incubated in the presence of [32P]PEP and a crude extract prepared from B. subtilis GM1288X. Phosphorylation of the 55-amino acid N-terminal domain of SacY fused to GST was examined under the same conditions. As shown, SacY was readily phosphorylated by PEP (Fig. 1,lane 3), whereas no phosphorylation of the 55-amino acid N-terminal peptide was detected (Fig. 1, lane 2). The involvement of the general PTS enzymes in this phosphorylation was examined by incubating SacY in the presence of a crude extract from GM1320X (ptsH) or GM1386X (ptsI). No phosphorylation could be detected when SacY was incubated in the presence of a GM1386X crude extract (Fig. 1, lane 4). SacY was also not phosphorylated when incubated with a crude extract prepared from GM1320X (Fig. 1, lane 5). Nevertheless, SacY phosphorylation was observed following addition of 0.1 or 1 μg of purified HPr to extracts of the ptsH mutant (Fig. 1,lanes 6 and 7). These results demonstrate that SacY is phosphorylated in a PTS-dependent reaction involving the general energy coupling proteins, EI and HPr.Figure 1SacY is phosphorylated by PEP in a PTS-dependent reaction. Autoradiogram of an SDS-polyacrylamide gel showing the radiolabeled proteins obtained by incubation of 14 μg of a crude extract from GM1288X (lanes 1–3) with [32P]PEP and 4 μg of the 55-amino acid N-terminal domain of SacY fused to the GST (lane 2) or 2.8 μg of the entire SacY protein (lane 3). 2.8 μg of the entire SacY protein was also incubated in the presence of [32P]PEP and 14 μg of crude extract from GM1386X (lane 4) or GM1320X (lanes 5–7) supplemented with 0.1 or 1 μg of purified B. subtilis HPr (lanes 6 and 7, respectively). The arrows indicate the positions of SacY and HPr.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the following phosphorylation experiments, the B. subtilis crude extract containing all the PTS components was replaced by the purified B. subtilis general energy coupling proteins EI and HPr. In these assays, EI, HPr, and SacY were incubated in all possible combinations in the presence of [32P]PEP. As shown in Fig. 2, SacY was readily phosphorylated in the presence of both EI and HPr, whereas no SacY phosphorylation was detected when at least one of the two general PTS proteins was absent. Interestingly, SacX appeared dispensable although the extent of SacY phosphorylation was somewhat higher in the presence of the crude extract (Fig. 1, lanes 6 and 7) than in the presence of the purified EI and HPr (see Fig. 2). These results show that SacY can serve as phosphoryl acceptor with the general energy-coupling protein, HPr(His∼P) serving as the phosphoryl donor, and that phosphorylation of SacY occurs outside of the first 55 N-terminal amino acids. The phosphate groups of phosphorylated SacY, like that of EI and HPr, were unstable under acidic conditions or during heat treatment (data not shown), suggesting that, as are EI and HPr, SacY is phosphorylated on histidyl residues. Studies described below were carried out to identify the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues were performed to derive guidelines for a biochemically based search of the phosphoryl acceptor residues in these proteins. Recent studies have identified the mRNA-binding domain of SacY as the N-terminal 55 amino acids of the antiterminator (36Manival, X., Yang, Y., Strub, M.-P., Kochoyan, M., Steinmetz, M., and Aymerich, S. (1997) EMBO J., in press.Google Scholar). Homologous N-terminal mRNA-binding domains are also present in six fully sequenced SacY homologues. Sequence analysis of these seven proteins revealed that their C-terminal regions consist of two homologous domains (∼100 residues each), herein designated P1 (the N-terminal domain) and P2 (the C-terminal domain; see Fig. 3). P domains are also present in four partially sequenced putative transcriptional regulatory proteins, one in GlcR ofStaphylococcus carnosus, one in LicT of Bacillus amyloliquefaciens, one in CasR of Klebsiella oxytoca, and two in CelR of Bacillus stearothermophilus. Two P domains are also present in each of three distinct transcriptional initiation regulatory proteins, the helix-turn-helix motif possessing DNA-binding LicR (previously named CelR; see Ref. 37Tobisch S. Glaser P. Krüger S. Hecker M. J. Bacteriol. 1997; 179: 496-506Crossref PubMed Google Scholar), LevR of B. subtilis, and MtlR of B. stearothermophilus (see Fig.3). Altogether, 25 homologous P domains have been identified in 14 modular transcriptional regulators. Significantly, homologous P domains could not be identified in eukaryotes or in bacteria lacking the PTS. Binary comparison scores calculated for all P1 and P2 domains revealed that (i) all P domains are homologous, (ii) the P1 domains are more closely related to themselves than to the P2 domains, and (iii) the antiterminator P2 domains, but not the DNA binding protein P2 domains, are more closely related to themselves than to the P1 domains (data not shown). These findings lead us to propose that intragenic duplication of the gene encoding the ancestral P domain gave rise to the present day mRNA-binding antiterminators bearing two homologous P domains and that the P2 domains of the antiterminators evolved to serve a function distinct from that of the P1 domains. Surprisingly, all six P domains of the DNA-binding proteins (LevR, LicR, and MtlR) exhibit higher similarity with the P1 domains than with the P2 domains of the mRNA-binding antiterminators (data not shown; see below for interpretation). The phylogenetic tree of all 25 sequenced P domains (Fig.4) confirmed the conclusions derived from the binary statistical analysis. Although the branch lengths vary substantially, the tree is largely symmetrical with all P1 domains depicted on the left and most P2 domains (except those of LevR, LicR, and MtlR) depicted on the right. The tree reveals (i) that the antiterminator P1 domains comprise a tighter cluster than the corresponding cluster of the P2 domains, (ii) that the P1 and P2 clusters of the antiterminators exhibit a near mirror image appearance, and (iii) that the P2 domains of the DNA-binding regulators also exhibit a near mirror image appearance, but they are more closely aligned with the antiterminator P1 domains. We propose that the antiterminator P2 domains evolved to assume a function dissimilar from that of the P1 domains while the P2 domains of the DNA-binding proteins did not. When the 25 sequenced P1 and P2 domains were multiply aligned, only three residues in the alignment proved to be conserved in at least 23 positions (Fig. 5). These amino acids, highlighted inblack, are (i) the histidyl residue at alignment position 8 that corresponds, respectively, to His-99 and His-207 in the P1 and P2 domains of SacY (conserved in all 25 P domains), (ii) the arginyl residue at alignment position 15 (conserved in 23 P domains), and (iii) the glutamyl residue at alignment position 63 (conserved in 24 P domains). An additional histidyl residue (alignment position 74 in Fig.5) that corresponds to His-158 and His-269 in the P1 and P2 domains of SacY, respectively, is conserved in all P1 domains and in all but three of the P2 domains (those of BglR, BglG, and LevR). It is noteworthy, however, that a histidyl residue is present in two of these P2 domains close to alignment position 74, i.e. in position 82 of P2BglG and in position 81 of P2LevR (see Fig. 5). Additionally, we suspect that a frameshift error was inadvertently introduced in the C-terminal region of BglR. The “correct” sequence at this region contains histidyl, glutamyl, and arginyl residues that can be aligned with the corresponding conserved C-terminal residues of the P2 domains (data not shown). Altogether, the data provide evidence for a modular organization of the mRNA-binding antiterminators and the DNA-binding transcrip" @default.
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• W1978516228 date "1997-07-01" @default.
• W1978516228 modified "2023-09-30" @default.
• W1978516228 title "Multiple Phosphorylation of SacY, a Bacillus subtilisTranscriptional Antiterminator Negatively Controlled by the Phosphotransferase System" @default.
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• W1978516228 doi "https://doi.org/10.1074/jbc.272.27.17230" @default.
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