Summary
We have cloned the fru operon of Salmonella typhimurium, coding for the enzymes of the phosphoenolpyruvate: fructose phosphotransferase system (Fructose PTS. The fruFKA operon consists of three genes: fruF coding for FPr, fruK for fructose 1-phosphate kinase and fruA for Enzyme IIFru. Insertions of Tn5 in the different genes were isolated and the activities of the gene products were measured. Expression of the plasmid-encoded fru operon in the maxicell system resulted in the synthesis of three proteins with molecular weights of 47 kDa (fruA), 39 kDa (fruF) and 32 kDa (fruK). We have sequenced the fruF gene and the regulatory region of the fru operon. In contrast to previously published results, we have found that the fruF gene codes for a 39 kDa protein, FPr, that combines Enzyme IIIFru and pseudo-HPr activities. The N-terminal part of FPr is homologous to the cytoplasmic domain of the Escherichia coli Enzyme IIMtl, as well as several Enzymes IIIMtl from gram-positive bacteria. The C-terminal domain shows homology to HPr of E. coli and several gram-positive organisms. The fru operon is regulated by a repressor, FruR. We have constructed an operon fusion between fru and the galK gene and shown that regulation of the fru operon by FruR takes place at the level of transcription.
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References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bolshakova TN, Dobrynina OY, Gershanovitch VN (1979) Isolation and investigation of the Escherichia coli mutant with the deletion in the ptsH gene. FEBS Lett 107:169–172
Bramley HF, Kornberg HL (1987) Sequence homologies between proteins of bacterial phosphoenolpyruvate-dependent sugar phosphotransferase systems: Identification of possible phosphate-carrying histidine residues. Proc Natl Acad Sci USA 84:4777–4780
Büchel DE, Gronenborn B, Müller-Hill B (1980) Sequence of the lactose permease gene. Nature 283:541–545
Buschmeier B, Hengstenberg W, Deutscher J (1985) Purification and properties of 1-phosphofructokinase from Escherichia coli. FEMS Microbiol Lett 29:231–235
DeFranco AL, Koshland DE Jr (1980) Multiple methylation in processing of sensory signals during bacterial chemotaxis. Proc Natl Acad Sci USA 77:2429–2433
Dente L, Cesareni G, Cortese R (1983) pEMBL: a new family of single stranded plasmids. Nucleic Acids Res 11:1645–1657
De Reuse H, Roy A, Danchin A (1985) Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene. Gene 35:199–207
Ebner R, Lengeler JW (1988) DNA sequence of the gene scrA encoding the sucrose transport protein Enzyme IIScr of the phosphotransferase system from enteric bacteria: homology of the Enzyme IIScr and Enzyme IIBgl proteins. Mol Microbiol 2:9–17
El-Kabbani OAL, Waygood EB, Delbaere LTJ (1987) Tertiary structure of histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. J Biol Chem 262:12926–12929
Erni B, Zanolari B, Kocher HP (1987) The mannose permease of Escherichia coli consists of three different proteins. Amino acid sequence and function in sugar transport, sugar phosphorylation, and penetration of phage lambda DNA. J Biol Chem 262:5238–5247
Ferenci T, Kornberg HL (1971) Pathway of fructose utilization by Escherichia coli. FEBS Lett 13:127–130
Ferenci T, Kornberg HL (1974) The role of phosphotransferasemediated synthesis of fructose 1-phosphate and fructose 6-phosphate in the growth of Escherichia coli on fructose. Proc R Soc London Ser B 187:105–119
Geerse RH, Ruig CR, Schuitema ARJ, Postma PW (1986) Relationship between pseudo-HPr and the PEP: fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli. Mol Gen Genet 203:435–444
Harley CB, Reynolds RP (1987) Analysis of E. coli promoter sequences. Nucleic Acids Res 15:2343–2361
Klevit RE, Waygood EB (1986) Two-dimensional 1H-NMR studies of histidine-containing protein from Escherichia coli. 3. Secondary and tertiary structure as determined by NMR. Biochemistry 25:7774–7781
Kundig W, Roseman S (1971) Sugar transport. II. Characterization of constitutive membrane-bound enzymes II of the Escherichia coli phosphotransferase system. J Biol Chem 246:1407–1418
Lee CA, Saier MH Jr (1983) Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene. J Biol Chem 258:10761–10767
Lee LG, Britton P, Parra F, Boronat A, Kornberg H (1982) Expression of the ptsH + gene of Escherichia coli cloned on plasmid pBR322. A convenient means for obtaining the histidine-containing carrier protein HPr. FEBS Lett 149:288–292
Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 227:1435–1441
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Mandel M, Higa A (1979) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Mattoo RL, Waygood EB (1983) Determination of the levels of HPr and Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Can J Biochem Cell Biol 61:29–37
McKenney K, Shimatake H, Court D, Schmeissner U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In: Chirikian JG, Papas TS (eds) Gene amplification and analysis, Vol 2. Elsevier North Holland, Amsterdam, pp 383–415
Pas HH, Robillard GT (1988) S-phosphocysteine and phosphohistidine are intermediates in the phosphoenolpyruvate-dependent mannitol transport catalyzed by Escherichia coli Enzyme IIMtl. Biochemistry 27:5835–5839
Postma PW (1977) Galactose transport in Salmonella typhimurium. J Bacteriol 129:630–639
Postma PW, Lengeler JW (1985) Phosphoenolpyruvate: carbohydrate phosphotransferase system of bacteria. Microbiol Rev 49:232–269
Postma PW (1987) Phosphotransferase system for glucose and other sugars. In: Neidhardt FC, Ingraham JL, Magasanik B, Low KB, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium. Cellular and molecular biology. American Society for Microbiology, Washington, pp 127–141
Prior TI, Kornberg HL (1988) Nucleotide sequence of fruA, the gene specifiying Enzyme IIFru of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Escherichia coli. J Gen Microbiol 134:2757–2768
Reiche B, Frank R, Deutcher J, Meyer N, Hengstenberg W (1988) The Staphylococcal phosphoenolpyruvate-dependent phosphotransferase transferase system: purification and characterization of the mannitol-specific Enzyme IIIMtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the Enzyme IIMtl of Escherichia coli. Biochemistry 27:6512–6516
Reizer J, Saier MH Jr, Deutscher J, Grenier F, Thompson J, Hengstenberg W (1988) The phosphoenolpyruvate: sugar phosphotransferase system in Gram-positive bacteria: properties, mechanism and regulation. CRC Crit Rev Microbiol 15:297–338
Saffen DW, Presper KA, Doering TL, Roseman S (1987) Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes. J Biol Chem 262:16241–16253
Saier MH Jr, Simoni RD, Roseman S (1970) The physiological behavior of Enzyme I and heat-stable protein mutants of a bacterial phosphotransferase system. J Biol Chem 245:5870–5873
Saier MH Jr, Simoni RD, Roseman S (1976) Sugar transport. Properties of mutant bacteria defective in proteins of the phosphoenolpyruvate: sugar phosphotransferase system. J Biol Chem 251:6584–6597
Saier MH Jr, Grenier FC, Lee CA, Waygood EB (1985) Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. J Cell Biochem 27:43–56
Saier MH Jr, Yamada M, Erni B, Suda K, Lengeler J, Ebner R, Argos P, Rak B, Schnetz K, Lee CA, Stewart GC, Breidt F Jr, Waygood EB, Peri KG, Doolittle RF (1988) Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons. FASEB J 2:199–208
Sancar A, Wharton RP, Selzer S, Kacinski BM, Clark MD, Rupp WD (1981) Identification of the uvrA gene product. J Mol Biol 148:45–62
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Scholte BJ, Postma PW (1980) Mutation in the crp gene of Salmonella typhimurium which interferes with inducer exclusion. J Bacteriol 141:751–757
Scholte BJ, Schuitema ARJ, Postma PW (1981) Isolation of IIIGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system of Salmonella typhimurium. J Bacteriol 148:257–264
Sprenger GA, Lengeler JW (1984) L-sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose. J Bacteriol 157:39–45
Stock JB, Waygood EB, Meadow ND, Postma PW, Roseman S (1982) Sugar transport by the bacterial phosphotransferase system. The glucose receptors of the Salmonella typhimurium phosphotransferase system. J Biol Chem 257:14543–14552
Sutrina SL, Chin AM, Esch F, Saier MH Jr (1988) Purification and characterization of the fructose-inducible HPr-like protein FPr, and the fructose-specific Enzyme III of the phosphoenolpyruvate: sugar phosphotransferase system of Salmonella typhimurium. J Biol Chem 263:5061–5069
Waygood EB (1980) Resolution of the phosphoenolpyruvate: fructose phosphotransferase system of Escherichia coli into two components; Enzyme II fructose and fructose-induced HPr-like protein (FPr). Can J Biochem 58:1144–1146
Waygood EB, Mattoo RL, Peri KG (1984) Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIIMannose, IIIFructose, IIIGlucitol, and the phosphorylation of Enzyme IIIMannitol and Enzyme IIN-Acetylglucosamine. J Cell Biochem 25:139–159
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Yazyu H, Shiota-Niiya S, Shimamoto T, Kanazawa H, Futai M, Tsuchiya T (1984) Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli. J Biol Chem 259:4320–4326
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Communicated by J.W. Lengeler
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Geerse, R.H., Izzo, F. & Postma, P.W. The PEP: fructose phosphotransferase system in Salmonella typhimurium: FPr combines Enzyme IIIFru and pseudo-HPr activities. Mol Gen Genet 216, 517–525 (1989). https://doi.org/10.1007/BF00334399
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DOI: https://doi.org/10.1007/BF00334399