Download Nucleotide Sequence of fruA, the Gene Specifying Enzyme IIfru of

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
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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
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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
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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
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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
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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
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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.
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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.
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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
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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.
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