* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Nucleotide Sequence of fruA, the Gene Specifying Enzyme IIfru of
Ancestral sequence reconstruction wikipedia , lookup
Magnesium transporter wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Gene expression wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genetic code wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Molecular cloning wikipedia , lookup
Genomic library wikipedia , lookup
Community fingerprinting wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Homology modeling wikipedia , lookup
Restriction enzyme wikipedia , lookup
Proteolysis wikipedia , lookup
Metalloprotein wikipedia , lookup
Western blot wikipedia , lookup
Expression vector wikipedia , lookup
Phosphorylation wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Point mutation wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Biochemistry wikipedia , lookup
Biosynthesis wikipedia , lookup
Journal of General Microbiology (1988), 134, 2757-2768. Printed in Great Britain 2757 Nucleotide Sequence of fruA, the Gene Specifying Enzyme IIfruof the Phosphoenolpyruvate-dependentSugar Phosphotransferase System in Escherichia coli K12 By T R E V O R I. P R I O R A N D H A N S L. K O R N B E R G * Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 l Q W, UK (Received 9 March 1988; revised 27 June 1988) The Enzyme IIfruof the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system (PTS), which catalyses the uptake of fructose and its concomitant phosphorylation to fructose 1phosphate by Escherichia coli, is specified by a gene designatedfmA. The nucleotide sequence of a 2.5 kb PvuII restriction fragment spanningfruA+,cloned on a plasmid, was determined. This fragment contained three open reading frames (ORFs) but only one complete ORF, 1689 base pairs long, which was preceded by a well-defined Shine-Dalgarno sequence and ended with a rho-independent transcription terminator. The amino acid sequence deduced from this DNA correspondsto that of a protein of 563 amino acids (57.5 kDa), which has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.40) and which is characterized by a number of well-marked hydrophobic loops that may correspond to membrane-spanning regions. There is relatively little overall homology between this protein and those of other Enzymes I1 of the PTS but there is considerable correspondence between the region surrounding one of the six histidine residues of Enzyme IIfruand those surrounding the particular histidines of other Enzymes 11, and of HPr, known to be involved in phosphorylation. A plasmid carrying the completefmA+ nucleotide sequence, but not that of any other functional protein, fully restored the ability offruA mutants to grow on fructose and of extracts of fruA mutants to phosphorylate fructose, which confirms that the nucleotide sequence determined specifies Enzyme IIfru. INTRODUCTION The phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the uptake and concomitant phosphorylation of a number of carbohydrates, such as glucose, fructose, hexitols, N-acetylglucosamine and P-glucosides, by Escherichia coli and by other facultative and obligate anaerobes (for a recent review, see Postma & Lengeler, 1985). The PTS comprises the sugar-nonspecific,cytoplasmic phosphoproteins Enzyme I and, in the general case, HPr, and one of a range of sugar-specific membrane-associated Enzymes I1 or Enzyme II/Enzyme I11 pairs. In the case of fructose uptake, HPr is replacid by a fructose-specific HPr-like protein (Walter & Anderson, 1973; Saier et al., 1976)termed FPr (Waygood, 1980). These components effect the stepwise transfer of a phosphate group from phosphoenolpyruvate (PEP) to the incoming sugar. The genes encoding the fructose-specific components of the PTS are located at approximately minute 46 on the linkage map of E. coli K12 (Bachmann, 1983). They specify the activities of FPr, of Enzyme IIIfru,of fructose-1-phosphate kinase, and of the membrane-spanning Enzyme IIfru.These genes have been designated in a variety of ways. We adopt the suggestion of Saier (1985) that they be termed, respectively,fruH, fruB, fmK and fruA. An additional gene, fruR, Abbreviations: ORF, open reading frame ; PEP, phosphoenolpyruvate; PTS,phosphotransferase system. 0001-4759 0 1988 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 2758 T. I . P R I O R AND H . L . K O R N B E R G specifying a repressor of the operon (Geerse et al., 1986; Kornberg & Elvin, 1987) maps at minute 2 on the E. coli linkage map. Mutation offruR+ tofmR results in constitutive expression of the fructose operon (Reiner, 1977). It is the main aim of this paper to report the molecular cloning and DNA sequence offmA and the deduced amino-acid sequence of Enzyme IIfru.This sequence is compared with those of other Enzymes I1 of the PTS. METHODS Bacterial strains andplasmids. The bacterial strains used are derivatives of E. coli K 12 and are listed in Table 1. P1-mediated transduction was performed as described by Miller (1972). Tetracycline-sensitive derivatives of tetracycline-resistant (TetR) strains were selected as organisms able to grow in in the presence of 7chlorotetracycline under the conditions described by Bochner et al. (1980). Such derivatives are designated ChlTet+ in Table 1. DNA transformations were performed using either the CaC1, method (Maniatis et al., 1982) or the procedure described by Hanahan (1983). Bacteria were grown at 37 "C in one of the following media: nutrient broth (Oxoid no. l), Luria Broth, or basal medium (Ashworth & Kornberg, 1966) supplemented with 10 mM carbon source except where stated otherwise, and required amino acids at 40 pg ml-I . When appropriate, tetracycline, ampicillin and chloramphenicol were inoculated at 10, 100 and 10 pg per ml of medium respectively. Table 1. E. coli strains and plasmids Strain or plasmid E. coli ED8654 Genotype HK994 HK1108 HK1112 HK1145 HK 1287 LA5523 PB11 TP5 lac YI or (lacI-Z)6 galK2 galT22 metBl hsdR514 trpR55 1- supE44 supF58 F32burF dsdA)/tonA2 or tonl4, lacy1 or lacZ4, tsx-23 or -25, supE44 A- purFl aroC4 dsdAl rpsL8,9 or 14 malT2(AR)xyl-7 mtl-2 argHI thi-1 ptsH2 ptsM zeg : : TnlO argHBCE thr leu fruHI fruBl fruR ptsH2 his trp thr argHBCE f m R ptsH2 his trp thr argHBCE cir : :TnlO f m R ptsH2 arg thr cir : :TnlO fruAl fruR lacA (his,gnd)" araD zeg : :TnlO ptsH2 ptsM cysA argHBCE f m A 3 fruR metB pyrE uhpC cir : :TnlO f m K 1 uvrA gutA : :TnlO recA lacA (his g d A araD fruA4 f m K 2 TP7 lac" (his gnd)A araDfruA.5fmB2fruH2fruK3 TP9 la& (his gnd)" araD f m A 4 cir : :TnlO f m K 2 TPI 1 TP26 lac" (his gnd)A araD fruA5 cir : :TnlO fruB2 fmH2fmK3 as ED8654 cir : :TnlO f m A 4 f m K 2 TP28 as HK994 cir : :TnlO fruA5 fruB2 f m H 2 f m K 3 TP32 as HK994 cir : :TnlO f m A 4 f m K 2 TP33 TP41 as ED8654 f m A 4 f m K 2 as ED8654 f m A 4 f m K 2 gutA : :TnlO recA TP 120 as HK 1287f m A 3 gutA : :TnlO recA Plasmids pBR322 pTP3 pTP6 pTP 10 ApR TetR ApR TetRfruA+ ApR TetRf m A + ApR TetRf m A + EM2001 HK881 Source Borck et al. (1976), received as CGSC 6512 from B. Bachmann McFall(l967), received as CGSC 4210 from B. Bachmann Kornberg (1986) Laboratory stock Laboratory stock Laboratory stock Laboratory stock Laboratory stock Middenhorf et al. (1984) P. Britton ChlTet+ Fru- derivative of HK1145 ChlTet+ Fru- derivative of HK1145 TetR transductant Pl(HKllO8) x TP5 TetR transductant Pl(HK1108) x TP7 TetR, Fru- transductant Pl(TP9) x ED8654 TetR Fru- transductant Pl(TPl1) x HK994 TetR Fru- transductant P1(TP9) x HK994 ChlTet+ derivative of TP26 TetR recA transductant Pl(PB11) x TP33 TetR recA transductant Pl(PB11) x HK1287 Bolivar et al. (1977) This study This study This study Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 Sequence of EnzIIfmof E. coli 2759 Fructose-negative strains, used for screening plasmids for the presence of fruA+, were constructed as tetracycline-sensitive derivatives of strain HKll45, which carries the tetracycline-resistance transposon (zeg : :TnZO), and testing these for concomitant loss of the ability to grow on 2 mM-fructose.This low concentration of fructose was chosen as, at > 5 mM, fructose can enter the cells also via the mannose-specific transport system (Kornberg & Jones-Mortimer, 1975; Curtis & Epstein, 1975). Enzymes. Restriction endonucleases, T4 DNA ligase, RNAase (DNAase-free), calf-intestinal alkaline phosphatase, and DNA polymerase large fragment were purchased from New England Biolabs, Amersham, Boehringer Mannheim, Sigma and Pharmacia. Incubations were carried out according to manufacturers' instructions. Sequenase was obtained from Cambridge Bioscience. Enzyme I was the generous gift of Professor E. B. Waygood (University of Saskatchewan, Saskatoon, Canada). Construction of episomal gene library. F32-episomal DNA (McFall, 1967)was purified by the method described by Dardel et al. (1984) except that DNA was obtained from a 1 litre culture of strain EM2001 grown overnight on basal medium supplemented with fructose and arginine. The purified episomal DNA was partially digested using Tag1 and fragments larger than 9.4 kb were separated by electrophoresis through a 0.5% (w/v) agarose gel and isolated using DE-81 (Whatman) paper (Dretzen et al., 1981). The purified fragments were ligated into dephosphorylated ClaI-cut pBR322. Strain TP41 was transformed with the library and the resulting ampicillin-resistant colonies screened for their ability to grow on fructosesupplemented medium. DNA preparation and sequencing. The 2.5 kb PvuII fragment obtained from the plasmid pTPlO was purified by electrophoresis through a 1% (w/v) agarose gel as described earlier. The purified fragment was self-ligated, sonicated to give random fragments and ligated into the SmaI site of M13mplO (Bankier & Barrell, 1983). The nucleotide sequence was determined by the dideoxy chain-termination method using modified T7 DNA polymerase (Tabor & Richardson, 1987). Electrophoresis was performed either using gradient gels of 0.2-0.6 mm thickness run in an LKB 2010 Macrophor electrophoresis unit at 55 "C or using buffer-gradient gels according to Bankier & Barrell (1983). Data were compiled using the Staden package of computer programs (reviewed in Staden, 1987). Biochemical assays. Fructose-l-phosphate kinase was assayed as described by Ferenci & Kornberg (1971), and PEP-dependent sugar phosphorylation as described by Waygood et al. (1984) as modified by Kornberg (1986); protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as standard. R E S U L T S AND DISCUSSION Preparation and characterization of fructose mutants Strain HK 1 145 contains a transposon (zeg : :TnlO) integrated in the genome at a site which maps very close to the region specifyingthe enzymes of fructose uptake and phosphorylation (min 46). When tetracycline-sensitive derivatives of such tetracycline-resistant organisms were selected, some of them had also lost the ability to utilize fructose as sole carbon source, presumably through inversion or deletion of the relevant region of the chromosome. Two such mutant strains, TP5 and TP7, were studied further. The mutations in these strains were transferred by phage-mediated transduction into strain HK994, which expresses the fructose operon constitutively VruR),and yielded the derivatives TP28 and TP32. Sonic extracts of these derivatives were separated into membrane and cytoplasmic fractions; these could be tested for the presence of active FPr/Enzyme 111"" (cytoplasmic) and Enzyme IIfru(membrane), by measuring their ability to complement extracts of mutants impaired in specific known components of the fructose-PTS (Table 2). The results indicate that neither strain TP28 nor strain TP32 contains Enzyme IIfruactivity (Table 2) and both strains lack fructose-l-phosphate kinase activity (Table 3). However, the extract from strain TP32, but not that from TP28, restored fructose phosphorylation to an extract of the mutant HK881 known to be greatly impaired in FPr and devoid of Enzyme IIIfruactivities (Kornberg, 1986; Sutrina et al., 1988). These results thus support the gene order fruB-fruH/fruK-fruA (Geerse et al., 1986) and show that fruK and fruA are adjacent. Isolation and characterization of plasmids A gene bank was prepared from the F32 episome that covers thepurF-dsdA region (McFall, 1967) by a TaqI partial digest inserted into the unique CIaI site of plasmid pBR322. The library was used to transform competent cells of strain TP41 VruAfruKrecA). Selection was for Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 2760 T. I. PRIOR A N D H. L. K O R N B E R G Table 2. PEP-dependent phosphorylation of fructose by extracts of E. coli Cytosol and membrane fractions, from cells of fruR strains of E. coli grown in nutrient broth, were prepared as described by Kornberg (1986). The cytosol fraction contained approximately 12 mg protein ml-l and the membrane fraction contained approximately 8 mg protein ml-l. The ability of such fractions to catalyse the phosphorylation of [ 14C]fructosewas assayed by the method of Waygood et al. (1984) as modified by Kornberg (1986). For each assay, 0.3 mg cytosolicprotein was mixed with 0.2 mg membrane protein, extra Enzyme I was added as recommended, and the amounts of [14C]fructose1phosphate formed in 15 min at 30 "C were measured. Components of assay mixture 7--- Cytosol fraction P Strain TP28 TP28 TP28 TP28 HK881 HK994 HK1112 TP32 TP32 TP32 TP32 HK881 HK994 HK1112 Presence of FPr/Enzyme IIP - + + + + + ++ + Membrane fraction 3 I Strain TP28 HK881 HK994 HKlll2 TP28 TP28 TP28 TP32 HK881 HK994 HK1112 TP32 TP32 TP32 Presence of Enzyme IIfru [ 14C]Fructose 1-phosphate + +- 12 9 39 7 2 6 4 3 89 70 8 1 2 2 formed (nmol) Table 3, Fructose I-phosphate kinase activity in extracts of E. coli The fructose-1-phosphate kinase activity of cell-free extracts of the strains listed below was assayed as described by Ferenci & Kornberg (1971). Strain Fructose 1,dbisphosphate formed [nmol min-l (mg protein)-'] HK994 TP28 TP32 188 2 3 ampicillin resistance in the first instance followed by test of the ability of the transformed colony to utilize fructose as sole carbon source. From this procedure, a plasmid, pTP3, was identified. The restriction map of the plasmid was established (Fig. 1) and the 10.1 kb insert was subcloned, first by deletion of the 5.9 kb CZaI fragment to yield plasmid pTP6. The 2.5 kb PuuII fragment in pTP6 was subsequently isolated (pTP10) and identified as being able to complement a strain with a mutation in f m A (TP120). Experiments with cell-free extracts (Table 4) confirm that pTPlO codes for functional Enzyme IIfru. The DNA sequence The 2.5 kb PuuII fragment excised from pTPlO was sequenced with 100%coverage on both strands and with each base covered a minimum of two times. Analysis of the DNA sequence revealed the presence of three open reading frames (ORFs). All ORFs are on the same strand. The DNA sequenceof ORF2, of the intercistronic region between ORF2 and ORF3, and of the end of ORFl and the beginning of ORF3, are shown in Fig. 2. ThefruA sequence. ORF2 is the only complete open reading frame and codes for a protein of 563 amino acids. This is the DNA encodingfmA+, specifying Enzyme IIfru, as this is the only Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 276 1 Sequence of EnzIIfruof E. coli Al CI c3 PI C2 PI A2 PSI I Sml Si A3 P2 Ps I I Ps 1 C3 Sml SI A3 P2 HI A4 Ps2 A3 SI A4 BI S2 AS Ps2 CI pTP3 B1 H2 H2 C1 B1 S2 A5 P3 - S2 A5 Ps2 CI pTP6 I kb PI pTPlO 1 kb Fig. 1. Restriction maps of plasmids pTP3, pTP6 and pTP10. Plasmid pTP3 was isolated from the F32 episomal DNA library as described in the text. The plasmid was subcloned first by deletion of the 5.9 kb Cl-C3 fragment to yield plasmid pTP6, and secondly, by cloning the 2.5 kb Pl-P2 fragment into the PvuII site of plasmid pBR322. This plasmid, pTP10, contains the fruA+ gene. The 2.5 kb Pl-P2 fragment was re-isolated from pTPlO and sequenced as described in the text. The D N A derived from E. coli is shown as a solid line and that from pBR322 as a solid box. A, AvaI; B, BamHI; C, ClaI; H, HindIII; P, PvuII; Ps, PstI; S, SalI; Sm, SmaI. Table 4. Efect of the fruA+ plasmid pTP10 on the PEP-dependent phosphorylation of fructose by extracts of E . coli devoid of Enzyme IIf" activity The fruR-strains TP120 and TP120(pTP10) were grown on nutrient broth (supplemented with ampicillin, where appropriate, to stabilize the plasmid). Cytosol- and membrane-fractions were prepared from these cultures and assayed, as described in the legend to Table 2. Components of assay mixture I A Cytosol from TP 120 TP 120 TP 120(pTP10) TP120(pTP10) \ Membrane from TP120 TP120(pTP10) TP 120 TP120(pTP10) [ 14C]Fructose 1-phosphate formed (nmol) 4 30 9 42 complete ORF in the 2.5 kb PvuII fragment the expression of which restores the growth of fruA mutants on 2.5 mM-fructose and the ability of sonic extracts of the fruR fruA strain TP120 (Table 4) to form fructose 1-phosphate from PEP and fructose. In addition, the polypeptide sequence deduced from the nucleotide sequence of ORF2 has the properties expected of an integral membrane protein, and shows at least one important area of homology with a number of Enzymes I1 of the PTS. There is a potential Shine-Dalgarno ribosomal recognition site (Stormo et al., 1982) with the sequence AGGAGAGG, located five bases upstream of ORF2. It is also significant that although there are two other initiation codons (18 and 43) located near the proposed amino-terminus of the translation product, neither is preceded by a possible Shine-Dalgarno sequence. A statistical study of the positional base preferences (Stormo, 1987) in the three phases on this strand indicate that ORF2 is the most likely to be expressed in this section of the DNA (results not shown). The other phases of this strand, and the three phases on the opposite strand, are interrupted frequently by stop codons. The intercistronic region. ORF2 is separated from ORF3 by 322 base pairs. Immediately followingfruA there is a possible stem loop sequence (Fig. 3) which may be a rho-independent Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 2762 T. I . PRIOR A N D H. L . KORNBERG Ser Cys C y s Ser Pro G l y G l y L y s Ser L y s G l n Cys G l y Tyr Tyr A r g Ser Ser A l a V a l G l y AGC TGT TGC AGC CCT GGC GGT AAG TCA AAG CAA TGT GGG TAT TAC CGA TCG TCC GCA GIT GGC ... 1 Met L y s T h r L e u A r g L e u Thr Thr P h e CGA CTT ACA ACC TTT TAA c~(;AcAGc$EE~ZEZZATA ATG AAA ACG CTG 20 L e u G l y G l n Ala A r g Ala Tyr Met Ala L y s T h r Leu Leu G l y Ala CTC GGT CAG GCA CGC GCC TAT ATG GCG AAG ACC CTG CTG GGC GCG 40 L e u G l u I l e I l e A s p A s n Pro A s n A s p A l a G l u Met A l a I l e V a l CTG GAA ATC ATC GAC AAT CCG AAC GAC GCT GAA ATG GCG ATT GIT 60 Asn Asp Ser Ala L e u Asn G l y L y s Asn V a l T r p Leu G l y Asp Ile AAC GAC AGC GCG CTG AAC GGT AAA AAT GTC TGG CTG GG€ GAT A T T 80 P r o G l u L e u P h e L e u Ser G l u A l a L y s G l y H i s A l a L y s Pro Tyr CCT GAG CTG TTC CTG AGT GAA GCC AAA GGC CAT GCG AAA CCT TAC 100 T h r Ala Pro V a l Ala Ala Ser G l y Pro L y s A r g V a l V a l Ala V a l ACA GCA CCA GTT GCC GCC AGC GGT CCG AAA CGC GTA GTT GCG G'IG 110 120 130 T h r A l a C y s Pro T h r G l y V a l A l a H i s T h r P h e Met A l a A l a G l u A l a I l e G l u Thr G l u A l a ACT GCT TGC CCG ACT GGC GTA GCA CAC ACC TTT ATG GCG GCT GAA GCC ATT GAA ACC GAA GCG A r g Asn Asp G l y A l a A r g CGC AAT GAT GGC GCG CGT 10 Leu I l e Ile Asp A l a A s n C'IG ATT ATT GAC GCT AAT 30 Ala Ala Arg Lys Ala Lys GCG GCG CGA AAA GCA AAA 50 Leu G l y Asp Ser Ile P r o CTC GGT GAT TCC ATC CCG 70 Ser A r g A h V a l A l a H i s TCC CGG GCA GI" GCG CAC 90 Thr Ala Pro V a l Ala Ala ACT GCG CCG GTC GCT GCG 150 140 L y s L y s A r g G l y T r p T r p V a l L y s V a l G l u T h r A r g G l y Ser V a l G l y Ala G l y A s n Ala I l e AAA AAA CGT GGC TGG TGG GTG AAA GTT GAA ACC CGT GGT TCT GTT GGC GCG GGT AAT GCA ATC Thr Pro G l u G l u V a l A l a A l a A l a ACT CCC GAA GAA GTC GCA GCA GCG 180 Ala L y s P h e A l a G l y L y s Pro M e t GCG AAA TTT GCT GGT AAA CCG ATG 200 G l n G l u Leu Asp Lys A l a V a l A l a CAG GAA CTG GAT AAA GCG G" GCT 220 Ala T h r T h r G l u Ser L y s L y s G l u GCG ACC ACT GAA AGT AAG AAA GAG 240 Tyr Met L e u Pro Met V a l V a l A l a TAT ATG CTG CCG A T , GTC GI" GCA 260 G l u Ala P h e L y s G l u Pro G l y T h r GAA GCG 'ITT AAA GAG CCG GGT ACG 280 P h e A l a L e u Met V a l Pro V a l L e u TIT GCG CTG ATG GTG CCG GTA CTG 300 Leu T h r Pro G l y Leu Ile G l y G l y CTC ACT CCG GGT C'X ATT GGC GGT 320 Ile Ile A l a G l y Phe L e u A l a G l y ATT ATT GCG GGC TTC CTG GCT GGT G l n Ser Met G l u Ala Leu L y s Pro CAG AGT ATG GAG GCG CIG AAA CCG Leu Ala Met Ile Tyr L e u Ile G l y CTG GCG ATG ATC TAC CTG ATC GGT Leu G l n Thr M e t G l y C'IG CAG ACC ATG GGG Thr A s p Met G l y G l y ACT GAC ATG GGC GGT G l n T h r Tyr G l y Pro CAA ACC TAT GGC CCG 4 50 Leu Ala T h r Met V a l CIG GCA ACA ATG GTG 470 Leu V a l L e u G l y L e u C'JX GTA TlX GGA c% 390 T h r Ala Asn ACT GCG AAT 4 10 Pro V a l A s n CCG GTA AAC 430 Met A l a Ala ATG GCG GCG A l a Arg Arg GCG CGT CGC Cys P h e I l e TGC TI'C ATT 160 170 A s p Leu V a l Ile V a l A l a A l a A s p Ile G l u V a l GAT CTG GTG ATT GTG GCG GCA GAT ATC GAA GTG 190 Tyr A r g T h r Ser Thr G l y Leu Ala Leu L y s L y s TAT CGT ACC TCT ACC GGT CTG GCG C E AAG AAA 210 G l u A l a T h r Pro Tyr G l u Pro A l a G l y L y s A l a GAA GCA ACG CCG TAT GAA CCG GCG GGC AAA GCT 230 Ser Ala G l y Ala 'ryr A r g H i s Leu Leu Thr G l y AGT GCA GGC GCA TAC CGT CAC YE C'X ACG GGC 250 G l y G l y L e u Cys I l e A l a L e u Ser Phe A l a Phe GGT GGT CTG TGT ATC GCG CTT TCT TIT GCT TIT 270 L e u Ala A l a Ala Leu M e t G l n Ile G l y G l y G l y Tn; GCT GCG GCG C'X ATG CAG ATT GGT GGT GGT 290 A l a G l y Tyr I l e A l a Phe Ser I l e A l a A s p A r g GCA GGT TAT ATT GCC TIT TCC ATT GCC GAT CGT 310 M e t Leu Ala V a l Ser Thr G l y Ser G l y Phe Ile ATG CIG GCG GTC AGC ACC GGT TCT GG€ TTC A?T 330 Tyr I l e A l a L y s L e u I l e Ser Thr G l n L e u L y s TAC ATT GCG AAG TTA ATC AGT ACG CAA TIY; AAA 350 I l e Leu I l e I l e Pro Leu Ile Ser Ser Leu V a l A X C'IG ATC ATT CCG CTA ATT TCC AGT c*II; GTG 370 380 L y s Pro V a l A l a G l y I l e L e u G l u G l y Leu Thr AAA CCA GIT GCT GGC ATT CTC GAA GGG CTG ACT 400 A h V a l Leu Leu G l y Ala Ile Leu G l y G l y Met GCG GTT CTG C E GGG GCG ATC CTC GGT GGC ATG 420 L y s A l a A l a Tyr A l a Phe G l y V a l G l y L e u L e u AAA GCA GCG TAC GCA TTC GGT G E GGT CTG Crc; 440 Ile Met Ala Ala G l y M e t V a l Pro P r o Leu Ala ATT ATG GCG GCA GGT ATG GTG CCA CCG CK GCA 460 L y s P h e Asp L y s Ala G l n G l n G l u G l y G l y L y s AAA TTC GAC AAA GCG CAG CAG GAA GGT GGC AAA 480 Ser G l u G l y A l a I l e Pro Phe A l a A l a A r g Asp TCG GAA GGT GCA ATT CCG TTT GCT GCT CGT GAT Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 Asp Leu GAT CTG T h r Ala ACC GCG G l n Thr CAA ACG V a l Ser GTT TCT G l y Ile GGT ATC Ser Ala TCA GCC Pro G l y CCG GGC Gly Gly GGT GGT 340 L e u Pro (3% CCA 360 Val Gly GTC GGT His Trp CAC 'IGG M e t Cys ATG TGT Ser Thr AGT ACT Met G l y ATG GGT Ala A l a GCC GCT P r o Met CCG ATG 2763 Sequence of EnzIIfm of E. coli 490 500 A r g V a l Leu Pro Cys Cys I l e V a l G l y G l y Ala Leu T h r G l y Ala Ile Ser Met Ala Ile G l y cI;T G'IG CIG CCG TGC TGT A'TC GTG GGT GGG GCG CTG ACT GGC GCA ATC TCA A X GCG A W GGT 510 520 Ala L y s L e u Met A l a Pro H i s G l y G l y L e u Phe V a l L e u L e u I l e Pro G l y A l a Ile Thr Pro GCG AAA CTG ATG GCA CCG CAC GGT GGT CTG TIT GI"l' CTG CTG ATC CCT GGC GCT ATT ACG CCG 530 540 550 V a l Leu G l y Tyr Leu V a l Ala Ile Ile A l a GTA 'MG GGT TAC C'E GTA GCA ATT ATT GCC 560 Leu L y s A r g Pro G l u V a l A s p A l a V a l Ala CTG AAA CGT CCG GAA G ! E GAC GCA GTA GCG Lys A l a Ala AAA GCA GCG TAA TAAAAGG'TGTGITACAGGGCAGAAATT T A C ~ T C ~ A A ~ G l y T h r Leu V a l Ala G l y L e u Ala Tyr Ma Phe GGT ACG CIG GTG GCG GGT "TG GCC TAT GCC ?ry: ... ~ ~ G C G C A A A G A G Met A r g G l u L y s A s p A A T G CITTGCAGTATCTATATTC~TGTGGATTG ATG CGC GAA AAG GAT 10 20 Tyr V a l V a l Ile Ile G l y Ser Ala Asn Ile Asp V a l Ala G l y Tyr Ser H i s G l u Ser Leu Asn TAT GTC GTA ATT ATA GGT TCG GCG AAT ATT GAT GTC GCC GGA TAT TCA CAT GAA TCA TTA AAT 30 40 Tyr A l a Asp Ser Asn Pro G l y L y s Ile L y s Phe T h r Pro G l y G l y V a l G l y A r g Asn I l e A l a TAT GCG GAT 50 G l n Asn Leu CAA AAC Cn; 70 Gly G l n Ser GGT CAA TCG TCA AAT CCA GGT AAA ATA AAA TIT ACG CCT GGT GGA GTA GGG CGC AAT ATT GCA 60 Ala Leu Leu G l y Asn L y s Ala T r p Leu Leu Ser Ala V a l G l y Ser Asp Phe ?yr GCG !PIC CIG GGl' AAC AAA GCC TGG CTA C'E AGC GCC GTA GGC AGT GAT TTT TAT 80 L e u L e u Thr G l n Thr A s n G l n Ser G l y V a l l'yr V a l Asp L y s Cys L e u I l e V a l Cn; CTA ACG CAA ACC AAT CAA TCT GGC GI"I' TAT GTC GAT AAA Tc;c C E ATT GTG 90 100 110 Pro G l y G l u Asn Thr Ser Ser Tyr Leu Ser Leu Leu Asp Asn Thr G l y G l u M e t Leu V a l Ala CCG GGA GAA AAT ACG TCG AGT TAT TTA TCA TTA CTC GAT AAT ACC GGl' GAA ATG CIG GTT GCT 120 Ile Asn Asp Met A s n Ile Ser A s n A l a Il e Thr A l a ATA AAT GAC ATG AAT ATT AGC AAC GCT ATT ACA GCT G Fig. 2. DNA sequenceof the 2-5 kb PuuII fragment spanningfnrA and showing the deduced amino acid sequence of the gene product. Potential Shine-Dalgarno sequences are boxed. Stop codons are symbolized by three dots. The potential rho-independent transcriptional terminator is underlined once. The 12 base pair repeat prior to ORF3 is underlined twice. A A A 5' U U C U G*C A*U COG G U G-C G-C A*U COG A*U U*A U*A G-C G U G U o A UU U 3 ' Fig. 3. The structure of the rho-independent terminator following fmA. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 2764 T. I. PRIOR A N D H . L . KORNBERG transcription terminator (Rosenberg & Court, 1979). Ninety-six base pairs prior to the initiation codon for ORF3 there is the 12 base pair sequence CACATGTTTAAA (Fig. 2) which is separated by six bases from the identical sequence. Of these six bases, five (TATGA) are found also to precede the first of these 12 base pair sequences, and only in the sixth base does one purine (A) differ from another (G). The significance of this repeat is not known. The uther ORFs. ORF3 codes for an incomplete protein of 122 amino acid residues; its function and identity are as yet unknown. It is unlikely to form part of the fructose operon as fruA+ is believed to be the gene most distal to the promoter (Geerse et al., 1986). Moreover, it is separated from ORF2 by the potential RNA-transcription terminator discussed earlier. ORFl may code for the carboxy-terminal 36 amino acids of an incomplete protein; it may thus represent the end of fruK. Amino acid sequence of Enzyme IIfw The amino acid sequence deduced for Enzyme IIfruis shown in Fig. 2. Direct analysis of the protein specified by ORF2, purified from a strain of E. coli overexpressing it, confirms that the N-terminal sequence is MKTLLII.. . (T. I. Prior & H. L. Kornberg, unpublished). It has been pointed out by Saier et al. (1985) that the sugar-specific proteins of the PTS consist of either an Enzyme I1 of approximately 65 kDa, or an Enzyme II/III pair with a combined molecular mass of 65kDa. The calculated molecular mass of Enzyme IIfruis 57.5 kDa, a value similar to the 54.0 kDa calculated for Enzyme IPUt(Yamada & Saier, 1987),which has been shown to function in conjunction with Enzyme IIIgut (13.3 kDa). The presence of Enzyme IIIfru-likeactivity in Salmonella typhimurium has been reported (Geerse et al., 1986); we have also obtained evidence to this effect in E. coli (Kornberg, 1986; Kornberg & Elvin, 1987). It might thus be expected that the Enzyme IIIfruwould also be small. However, Waygood et al. (1984) described the induction by fructose of two soluble proteins with approximate molecular masses of 40 kDa (identified as Enzyme IIIfru)and 8 kDa (possibly FPr). The identity of the former has been confirmed by Sutrina et al. (1988), who have purified Enzyme IIIfrufrom S. typhimurium and have shown it to be a 40 kDa protein. The combined Enzyme II/IIIfrumass of 97.5 kDa thus greatly exceeds the previously established trend. The hydropathic profile (Kyte & Doolittle, 1982) of Enzyme IIfruis shown in Fig. 4. The average hydropathy is 0.40, which is between that for EnzymeIImtl(0.33) (Lee & Saier, 1983)and that for EnzymeIIbgl(0.62) (Bramley & Kornberg, 1987a) and is well within the range reported for integral membrane proteins. There are at least eight well-defined alternating hydrophilic and hydrophobic regions towards the carboxy-terminal region, as expected from a membrane protein. The hydrophobic regions are shown in Fig. 5 as membrane-spanning helices. Since they are rich in both glycine and proline, this representation may not be justified. However, it is also possible that the consequential perturbations of the helices may help to provide a channel for fructose to traverse the membrane for phosphorylation in the cytoplasm. The histidine assumed to be primarily involved in this phosphorylation (His38l ) appears to be advantageously located for this to occur. Comparison with other amino acid sequences The DIAGON algorithm was used to compare the amino acid sequence of Enzyme 11"" with those of a number of membrane transport proteins of the PTS including the Enzymes I1 for mannitol (Lee & Saier, 1983), glucose (Emi & Zanolari, 1986), mannose (Erni et al., 1987), glucitol (Yamada & Saier, 1987), P-glucosides (Bramley & Kornberg, 1987a) and N-acetylglucosamine (Rogers et al., 1988), as well as the Enzymes I11 for glucose (Nelson et al., 1984) and mannose (Erni et al., 1987). Significant homology was observed over only a few brief sections between Enzyme IIfru and the Enzymes I1 for mannitol, glucose, @-glucosidesand N-acetylglucosamine (Fig. 6) but there was no significant homology between Enzyme IIfruand either the Enzymes I1 for mannose and glucitol, or the Enzymes 111. It has been suggested that the PEP-dependent PTS evolved from a primordial fructose PTS (Saier et al., 1985;Saier, 1985). Our data offer little or no support for this hypothesis. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 200 300 Amino acid residue number 400 500 I I1 1v V VI VII VIII Fig. 5 . Proposed structure of the membrane-spanning domain of Enzyme IIfru.The histidine residue (381) postulated to be involved in phosphate transfer is boxed. The numbered membrane-spanning helices correspond to those shown in Fig. 4. Periplasm Fig. 4. Hydropathy plot of the deduced Enzyme IIfruamino acid sequence. The plot was generated using a span of 11 residues plotted every 3 residues. The horizontal line is at -0.4 (the average hydropathy of a soluble protein). The histidine residue (381) postulated to be involved in phosphate transfer from Enzyme HIfruto the sugar is indicated by the arrow. 100 2766 T . I . PRIOR A N D H . L . K O R N B E R G 252 125 30 30 28 282 155 60 60 53 31 2 185 90 90 83 31 8 215 120 120 100 --- - 328 225 128 130 110 - Fig. 6. Alignment showing homologies within a 130 amino acid stretch from the amino-termini of the Enzymes I1 for mannitol, glucose and N-acetylglucosamine; from a region beginning at amino acid residue 96 of Enzyme I P ; and from the mid-portion of Enzyme IIfru.Apart from the data shown, no other significant degrees of homology between Enzyme IIfruand other Enzymes I1 were found. Identical residues are boxed; conservativesubstitutions are not marked. Standard one-letter amino acid symbols are used; dashes indicate gaps inserted for maximal alignment. HPr Enzl lnag Enzl P I c Enzl lbg' Enzl lfru Enzl lscr Enzl l m t ' Enzl Put 21 195 21 8 31 2 386 31 5 20 1 196 Fig. 7. Alignment of the conserved histidine regions of HPr and the Enzymes I1 for N-acetylglucosamine,glucose, B-glucosides, fructose, sucrose, mannitol and glucitol. However, there is a high degree of homology in the regions of many Enzymes I1 that probably contain a histidine residue involved in phosphorylation (Fig. 7). The regions surrounding such histidine residues in the Enzymes I1 for mannitol (His19 5 ) , glucose (His2 I ) and P-glucosides (His306)were shown to correlate with the region adjacent to His1 of HPr (Bramley & Kornberg, 1987b), which has been shown to be phosphorylated (Weigel et d.,1982). The region flanking His381of Enzyme IIfru also shows a high degree of homology with these conserved regions. Marked homology is observed also with the regions adjacent to His309 in the Enzyme I1 for Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 Sequence of EnzIIfw of E. coli 2767 sucrose (Ebner & Lengeler, 1988) to in the Enzyme I1 for glucitol, and to His189in the Enzyme I1 for N-acetylglucosamine, although the homology between Enzymes I1 for mannitol and glucitol and the other proteins is less pronounced. These observations support the view that the histidine residues thus identified are involved in phosphate transfer. We thank Dr N. J. Gay for much practical advice, Dr P. J. F. Henderson for helpful discussions, Professor W. Boos (Konstanz University) for the gift of organisms containing TnlO, and Professor E. B. Waygood (University of Saskatchewan) for generous gifts of Enzyme I purified from S. typhimurium. This work was done during the tenure by T.I.P. of an Overseas Scholarship of the Royal Commission for the Exhibition of 1851, and was supported by a personal Grant-in-Aid to H. L. K. from Unilever Ltd. REFERENCES ASHWORTH, J. M. & KORNBERG, H. L. (1966). The anaplerotic fixation of carbon dioxide by Escherichia coli. Proceedings of the Royal Society of London B165, 179- 188. BACHMANN, B. J. (1983). Linkage map of Escherichia coli K-12, edition 7. Microbiological Reviews 47,180230. BANKIER,A. T. & BARRELL,B. G. (1983). Shotgun DNA sequencing. Nucleic acid biochemistry B508,134. In Technique in the Life Sciences BS. Edited by R. A. Flavell. Elsevier Scientific Publishers Ireland Ltd. BOCHNER, B. R., HUANG,H.-C., SCHIEVEN, G. L. & AMES,B. N. (1980). Positive selection for loss of tetracycline resistance. Journal of Bacteriology 143, 926-933. BOLIVAR,F., RODRIGUEZ,R. L., GREENE,P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER,H. W., S. (1977). Construction and CROSA, J. H. & FALKOW, characterization of new cloning vectors. 11. A multipurpose cloning system. Gene 2, 95-1 13. BORCK,K., BEGGS,J. D., BRAMMAR, W. J., HOPKINS, N. E. (1976). The construction in A. S. & MURRAY, vitro of transducing derivatives of phage lambda. Molecular and General Genetics 146, 199-207. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. BRAMLEY,H. F. & KORNBERG,H. L. (1987a). Nucleotide sequence of bglC, the gene specifying EnzymeIP' of the PEP :sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product. Journal of General Microbiology 133,563-573. BRAMLEY, H. F. & KORNBERG, H. L. (19876). Sequence homologies between the proteins of bacterial phosphoenolpyruvate-dependent sugar phosphotransferase systems: identification of possible phosphatecarrying histidine residues. Proceedings of the National Academy of Sciences of the United States of America 84, 4777-4780. CURTIS, S. J. & EPSTEIN, W. (1975). Phosphorylation of Dglucose in Escherichia coli mutants defective in glucose phosphotransferase, mannose phosphotransferase, and glucokinase. Journal of Bacteriology 122, 1189-1 199. DARDEL,F., FAYAT,G. & BLANQUET, S. (1984). Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthetase gene. Journal of Bacteriology 160, 1115-1 122. DRETZEN, G., BELLARD, M., SARSONNE-CORSI, J. K. & OVERATH,P. (1981). A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Analytical Biochemistry 112, 295298. EBNER,R. & LENGELER, J. W. (1988). DNA sequence of the gene scrA encoding the sucrose transport protein EnzymeIIScrof the phosphotransferase system from enteric bacteria: homology of the EnzymeIISCr and EnzymeIIB9' proteins. Molecular Microbiology 2, 9- 17. B. (1986). Glucose permease of ERNI,B. & ZANOLARI, the bacterial phosphotransferase system : gene cloning, overproduction and the amino acid sequence of enzymeIIGlC.Journal of Biological Chemistry 261, 16398-16403. ERNI,B., ZANOLARI, B. & KOCHER,H. P. (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 L DNA. Journal of Biological Chemistry 262, 5238-5247. H. L. (1971). Pathway of FERENCI, T. & KORNBERG, fructose utilization by Escherichia coli. FEBS Letters 13, 127-130. GEERSE,R. H., RUIG,C. R., SCHUITEMA, A. R. J. & POSTMA, P. W. (1986). Relationship between pseudoHPr and the PEP :fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli. Molecular and General Genetics 203, 435-444. HANAHAN, D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166, 557-580. KORNBERG, H. L. (1986). The roles of HPr and FPr in the utilization of fructose by Escherichia coli. FEBS Letters 194, 12-15. H.L. & ELVIN,C. M. (1987). Location and KORNBERG, function offnrC, a gene involved in the regulation of fructose utilization by Escherichia coli. Journal of General Microbiology 133,341-346. KORNBERG, H. L. & JONES-MORTIMER, M. C. (1975). PtsX: a gene involved in the uptake of glucose and fructose by Escherichia coli. FEBS Letters 51, 1-4. KYTE,J. & DOOLITTLE, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-132. LEE, C. A. & SAIER,M. H., JR (1983). Mannitolspecific enzyme11 of the bacterial phosphotransferase system. 111. The nucleotide sequence of the permease gene. Journal of Biological Chemistry 258, 10761-10767. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04 2768 T . I . PRIOR A N D H . L . K O R N B E R G MANIATIS, T., FRITSCH,E. F. & SAMBROOK, J. (1982). Molecular Cloning : a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. MCFALL,E. (1967). Dominance studies with stable merodiploids in the D-serine deaminase system of Escherichia coli K12. Journal of Bacteriology 94, 1982-1988. MIDDENHORF, A., SCHWEIZER, H., VREEMAN, J. & Boos, W. (1984). Mapping of markers in the gyrAhis region of Escherichia coli. Molecular and General Genetics 197, 175-1 81. MILLER, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. NELSON,S . O., SCHUITEMA, A. R. J., BENNE,R., VAN DER PLOEG,L. H. T., PLIJTER,J. J., AAN, F. & POSTMA, P. W. (1984). Molecular cloning, sequencing and expression of the err gene; the structural gene for IIIGICof the bacterial PEP :glucose phosphotransferase system. EMBO Journal3, 1587-1 593. POSTMA, P. W. & LENGELER, J. W. (1985). Phosphoenolpyruvate :carbohydrate phosphotransferase system of bacteria. MicrobiologicalReviews 49,232-269. REINER,A. M. (1977). Xylitol and D-arabitol toxicities due to derepressed fructose, galactitol and sorbitol phosphotransferase of Escherichia coli. Journal of Bacteriology 132, 166-1 73. J. & SOLL,D. ROGERS,M. J., OHGI,T., PLUMBRIDGE, (1988). Nucleotide sequences of the Escherichia coli nagE and nagB genes : the structural genes for the Nacetylglucosamine transport protein of the bacterial phosphoenolpyruvate :sugar phosphotransferase system and for glucosamine 6-phosphate deaminase. Gene 62, 197-207. ROSENBERG, N. & COURT,D. (1979). Regulatory sequences involved in the promotion and termination of RNA transcription. Annual Review of Genetics 13, 319-353. SAIER,M. H., JR (1985). Mechanisms and Regulation of Carbohydrate Transport in Bacteria. New York : Academic Press. SAIER,M. H., JR, SIMONI, R. D. & ROSEMAN, S. (1976). Sugar transport. Properties of mutant bacteria defective in proteins of the phosphoenolpyruvate : sugar phosphotransferase system. Journal of Biological Chemistry 251, 6584-6597. SAIER,M. H., JR, GRENIER,F. C., LEE, C. A. & WAYGOOD, E. B. (1985). Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate :sugar phosphotransferase system. Journal of Cellular Biochemistry 27, 43-56. STADEN,R. (1987). Computer handling of DNA sequencing projects. In Nucleic Acid and Protein Analysis: a Practical Approach, pp. 173-217. Edited by M. J. Bishop & C. J. Rawlings. Oxford: IRL Press. STORMO, G. D. (1987). Identifying coding sequences. In Nucleic Acid and Protein Analysis : a Practical Approach, pp. 231-258. Edited by M. J. Bishop & C. J. Rawlings. Oxford: IRL Press. T. D. & GOLD, L. M. STORMO,G. D., SCHNEIDER, (1982). Characterization of translational initiation sites in Escherichia coli. Nucleic Acids Research 10, 297 1-2996. SUTRINA, S. L., CHIN,A. M., ESCH, F. & SAIER,M. H., JR (1988). Purification and characterization of the fructose-inducible HPr-like protein, FPr, and the fructose-specificEnzyme I11 of the phosphoenolpyruvate :sugar phosphotransferase system of Salmonella typhimurium. Journal of Biological Chemistry 263, 5061-5069. TABOR,S. & RICHARDSON, C. C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America 84, 47674771. WALTER,R. W., JR & ANDERSON, R. L. (1973). Evidence that the inducible phosphoenolpyruvate :Dfructose 1-phosphate phosphotransferase system of Aerobacter aerogenes does not require ‘HPr’. Biochemical and Biophysical Research Communications 52, 93-97. WAYGOOD, E. B. (1980). Resolution of the phosphoenolpyruvate :fructose phosphotransferase system of Escherichia coli into two components; Enzyme I1 fructose and fructose-induced HPr-like protein (FPr). Canadian Journal of Biochemistry 58, 40-48. WAYGOOD, E. B., MATTOO,R. L. &PERI,K. G. (1984). Phosphoproteins and the phosphoenolpyruvate :sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImann”, IIIfructose,IIIglucitol,and the phosphorylation of EnzymeIImannitoland EnZymeIIN-acetylglucosamine. Journal of Cellular Biochemistry 25, 139-1 59. WEIGEL,N., POWERS,D. A. & ROSEMAN, S. (1982). Sugar transport by the bacterial phosphotransferase system. Primary structure and active site of a general phosphocarrier protein (HPr) from Salmonella typhimurium. Journal of Biological Chemistry 257, 1449914509. YAMADA, M. & SAIER,M. J., JR (1987). Glucitolspecific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon. Journal of Biological Chemistry 262, 54555463. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 10 May 2017 19:35:04