From the Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland
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ABSTRACT |
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The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IICMan-IIDMan complex and the cytoplasmic IIABMan subunit. IIABMan has two flexibly linked domains, IIAMan and IIBMan, each containing a phosphorylation site (His-10 and His-175). Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6' OH of the transported hexose. Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions. IIBMan contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis. The IIA domains have no conserved arginines. The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity. All five IIB mutants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6' OH of Glc) and hexose transport activity. R168Q has no hexose transport and strongly reduced phosphotransferase activity. R204K has no transport but almost normal phosphotransferase activity. R304Q has only slightly reduced transport activity. R190K behaves like wild-type IIABMan. Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1998) J. Mol. Biol. 276, 591-602) (Protein Data Bank accession code 1BLE). Arg-204 is at the putative interface between IIBMan and the IICMan-IIDMan complex.
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INTRODUCTION |
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Protein phosphorylation plays an important role in energy transduction, signal transduction and enzyme regulation. P-type ATPases transport cations across the cell membrane and are transiently phosphorylated on an aspartic acid during turnover (1). Protein kinases and protein phosphatases regulate the activity of enzymes and membrane bound receptors by phosphorylation and dephosphorylation (2, 3). In the phosphoenolpyruvate-dependent phosphotransferase system of bacteria (PTS)1 active transport and signaling are two functions of a protein phosphorylation cascade comprising four phosphoprotein units. The four components, enzyme I, HPr, IIA, and IIB sequentially transfer phosphoryl groups from phosphoenolpyruvate to carbohydrates that are accumulated across the cell membrane by a mechanism coupling translocation to phosphorylation. Whereas enzyme I and HPr are two energy coupling components, IIA and IIB together with the transmembrane IIC (and sometimes an additional IID) units form the sugar-specific transport complexes. IIA, IIB, and IIC occur either as protein subunits or as domains of a multidomain protein. Escherichia coli for example has over 30 genes for transporter complexes that differ in substrate specificity, amino acid sequence, and subunit/domain composition. The phosphorylation sites are histidines in enzyme I, HPr and the different IIA components, cysteines in the IIBs belonging to the glucose, mannitol, and lactose family, and histidines in the IIBs belonging to the mannose family of PTS transporters (for reviews see Refs. 4 and 5).
Phosphate-binding sites and phosphate-catalytic sites frequently
contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions (6).
Amino acid sequence comparisons with respect to invariant arginines in
PTS proteins from different bacteria reveal the following picture. The
N-terminal domain of enzyme I has Arg-126, Arg-186, and Arg-195, which
are completely invariant in 17 sequences. Arg-186 and Arg-195 are close
to the phosphorylation site His-189 (7). Arg-131 is conserved in 14 and
Arg-136 is conserved in 15 sequences. The C-terminal domain has 7 completely invariant arginines (Protein Domain Data Base:
protein.toulouse.inra.fr/prodom/prodom.html). The functional
role of these residues in enzyme I has not been explored. HPr is a
9-kDa open faced four-stranded antiparallel -sheet with three
-helices on one face. Arg-17, which is close to the active site
His-15, is invariant in all 22 known sequences. When Arg-17 is replaced
by Ser or Glu, phospho-donor and -acceptor activity of HPr are reduced
to between 6% and less than 0.1% of the control (8). The phosphoryl
group bound to His-15 of HPr is most likely complexed by the guanidino
group of Arg-17, as inferred from its restricted conformational freedom
observed upon phosphorylation of HPr, and from 31P chemical
shift changes (9). However, more recently this stabilization of the
phosphoryl group by the guanidino group has been questioned, based on a
molecular dynamics simulation in water and refined NMR data of
phospho-HPr (10, 11). No invariant arginines occur in the 21 IIA
components of the glucose family and in the five IIA domains of the
mannose family of PTS transporters. Two arginines, Arg-424 and Arg-426,
are invariant in the IIB components of the glucose family. They are
close to the active site Cys-421 (residues numbers refer to the
E. coli IICBGlc subunit), and both are essential
for phospho-donor activity toward glucose but not for phospho-acceptor
activity (12). Five arginine residues, Arg-168, Arg-172, Arg-190,
Arg-204, and Arg-304 (numbers refer to E. coli
IIABMan subunit), are invariant in the IIB domains of the
PTS transporters of E. coli, Klebsiella
pneumoniae (13), and Bacillus subtilis (14) belonging
to the mannose family. Arg-168 and Arg-172 are close in sequence to the
active site His-175 of IIABMan. In summary, it appears that
arginines close to the active site residues occur in the even numbered
phosphorylation sites (HPr and IIB) of the glucose and mannose specific
PTS and possibly in enyzme I but that arginines are absent from the IIA
domains.
The mannose transporter complex of E. coli consists of three
subunits. The IICMan and IIDMan subunits
comprise six and one putative membrane-spanning segments (15). They
contain the substrate binding site, and they facilitate the penetration
of bacteriophage DNA by an as yet unknown mechanism (16, 17). Two
IIABMan subunits form a stable homodimer, which reversibly
binds to the IICMan-IIDMan complex
(KD 5-10 nM). The IIABMan
subunit consists of two protein domains (IIA and IIB), which are
connected by a 20-residue-long hinge peptide rich in alanine and
proline (18). The two domains can be separated by limited trypsinolysis
at two lysine residues in the hinge. The IIAMan domain is
phosphorylated by phospho-HPr at His-10 and donates the phosphoryl
group to His-175 of the IIBMan domain. IIBMan
then donates the phosphate to the sugar. Although IIBMan
can accept a phosphate from IIAMan in the absence of the
IICMan-IIDMan complex, it can donate the
phosphate to the sugar only in the ternary complex with
IICMan and IIDMan. The IIAMan
domain has an
/
open pleated sheet structure. The central
-sheet consists of four parallel strands from one subunit and one
antiparallel strand from the second subunit in the dimer (19). This
strand exchange makes the dimer highly resistant to subunit
dissociation. While this work was in progress, the x-ray structure of
IIBLev of B. subtilis was solved (20). It has an
open pleated
/
fold consisting of a strongly twisted
seven-stranded
-sheet with helices on both faces (Fig. 4). The
IIBMan domain has 47% amino acid sequence identity with
IIBLev, and the two proteins complement each other (20).
Here we characterize the functions of the invariant arginines in the
IIBMan domain of the mannose transporter.
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EXPERIMENTAL PROCEDURES |
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Media, Strains, and Plasmids--
E. coli
K12-UT580LPM (
manXYZ::cat) and
ZSC112L
LPM(
manXYZ::cat ptsG glk) carry a
deletion of the chromosomal manXYZ allele. Gene replacement
was done in E. coli DPE271 (recD) as described (21) with the following modifications. (i) The
HindIII-KpnI fragment encoding residues 111-323
of IIABMan, the complete IICMan, and residues
1-241 of IIDMan was removed from plasmid pTSML1 (22) and
replaced by KpnI-HindIII fragment from plasmid
pMc5-8 (23) encoding cat (the KpnI site was
introduced into the original pMc5-8 by gapped duplex site-directed mutagenesis). (ii) The recombinant plasmid pTS
LPM::Cm was
linearized with NdeI and used to transform E. coli DPE271 (recD). (iii) Transformants defective in
mannose uptake were selected on McConkey indicator plates containing
mannose and chloramphenicol and tested for the loss of ampicillin
resistance. (iv) The cat gene was then P1 transduced into
UT580 and ZSC112L. ZSC112
LPM was transformed with plasmid pTSPM6
(encoding IICMan and IIDMan) (18) and a second
plasmid encoding the IIABMan mutants, and the strains were
used for in vivo experiments. WA2127
HIC (manXYZ,
ptsH ptsI crr) (24) was used as host for protein expression. Cells were grown at 37 °C in LB medium containing appropriate antibiotics. XL1-blue (Stratagene) was used for cloning and plasmid amplification.
Site-directed Mutagenesis-- IIABMan mutants R168Q,R172Q, R304Q were constructed using the gapped duplex procedure (23, 25). Mutant clones were identified by way of diagnostic restriction sites FspI, PvuII and SacI respectively. Mutants R190K and R204K were constructed by overlap extension mutagenesis (26). Mutant clones were identified by DNA sequencing. The HindIII-SnaBI segments carrying the mutations were cloned into the expression vector pJFL (encoding manX under the control of tacP, identical with pTACL293) (27).
Overproduction and Purification of Proteins--
E.
coli WA2127HIC was transformed with derivatives of pJFL
encoding wild-type and mutant IIABMan. IIABMan
was overexpressed and purified as described (25). Enzyme I and HPr were
purified as described (24). The IICMan-IIDMan
complex was purified by metal chelate affinity chromatography (28).2
Assay for Phosphotransferase Activity-- In vitro phosphorylation of [14C]Glc was assayed by ion-exchange chromatography as described (25). 100 µl of incubation mixture contained 0.5 µg of enzyme I, 2.8 µg of HPr, and 0.5 µl of crude membranes (~4 µg protein) containing the IICMan-IIDMan complex.
Transport Assay-- Uptake of [14C]dGlc by bacteria was assayed as described (29). The [14C]dGlc concentration was 120 µM, the specific activity was 6600 dpm/nmol. The cell density in the assay mixture was A600 = 15. 100-µl aliquots were removed at the indicated time points, diluted in ice-cold minimal salts medium, and applied to glass fiber filters under suction.
Assay for Protein Phosphorylation and Dephosphorylation-- The rate and the extent of protein phosphorylation and dephosphorylation was measured in a filter binding assay (21). The incubation mixture (50 mM NaPi, pH 7.4, 5 mM MgCl2, 2.5 mM NaF, 2.5 mM dithiothreitol) contained 15 µg of enzyme I, 2.5 µg of HPr, and 85 µg of IIABMan per 250 µl. The phosporylation reaction was started by adding to the incubation mixture at 24 °C [32P]PEP (~1200 dpm/nmol) to a final concentration of 80 µM. Aliquots of 40 µl were withdrawn at the indicated time points and diluted into 1 ml of 80% ammonium sulfate solution at 4 °C. The protein precipitates were collected on glass microfiber filters (GF/F, Whatman) under suction. The precipitate was washed with 2× 1 ml of ice-cold 80% ammonium sulfate, and the filters were counted in a liquid scintillation counter. To measure the dephosphorylation rate of PIIABMan (rate of P transfer to mannose) the reaction mixture was first incubated with 80 µM [32P]PEP for 5 min (pulse). At time 0 the following components were added together: a 50-fold molar excess of unlabeled PEP (chase); 1 µg of IICMan/IIDMan; an aqueous suspension of E. coli lipids (Sigma) to a final concentration of 0.5 mg/ml; Glc to a final concentration of 0.4 mM. 40-µl aliquots were withdrawn and processed as indicated above. Control reactions containing all components with the exception of IICMan/IIDMan were done in parallel. The background counts due to enzyme I and HPr (less than 10%) were subtracted from the counts of the complete system. In parallel, the phosphorylated proteins were analyzed on 17.5% polyacrylamide gels as described (21). 20-µl incubation mixtures contained 100 µM [32P]PEP (<1200 dpm/nmol), 1 µg of enzyme I, 1 µg of HPr, 6 µg of IIABMan, and where indicated 2 µg of IICMan/IIDMan. [32P]PEP was prepared as described (30).
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RESULTS |
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In Vivo Transport Activity--
Expression of the three subunits
of the mannose transporter was directed by two compatible plasmids
encoding the soluble IIABMan subunit and the transmembrane
IICMan-IIDMan complex. The expression of
IICMan/IIDMan was constitutive. Expression of
IIABMan was induced with 30 µM
isopropyl-1-thio--D-galactopyranoside, at which
concentration the cell growth rate on a mineral salts medium containing
mannose as the only carbon source was maximal.2 Cells
expressing wild-type IIABMan or mutants R190K and R304Q
produced red colonies on McConkey mannose indicator plates, whereas
mutants R168Q and R172Q produced yellow colonies. R204K produced yellow
colonies with a small red center. The initial rates of
[14C]dGlc uptake by wild-type and R190K expressing cells
were very similar, whereas the R304Q mutant had about 50% of wild-type
activity (Fig. 1A). The
arginine mutants R168Q, R172Q and R204K had less than 5% transport
activity. The results from McConkey plates and in vivo
transport studies are consistent and indicate that arginines 168, 172, and 204 are essential for transport activity of the mannose
transporter, whereas arginines 190 and 304 are dispensable when
replaced by lysine and glutamine, respectively. As expected, these
conservative replacements did not have a major impact on structural
stability and folding since all five mutant proteins could be
overexpressed and also retained the strong affinity for phosphocellulose characteristic of IIABMan (not shown).
They differ from the H175C active site mutant, which is no longer
phosphorylated and displays a reduced affinity for phosphocellulose
(25).
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In Vitro Phosphotransferase Activity-- In the intact cell the PTS transporters catalyze vectorial transport coupled to phosphorylation of the substrate. Observations made with the glucose transporter indicate that phosphorylation of intracellular substrates by the PTS (nonvectorial phosphorylation) also occurs in intact cells. The physiological significance of this activity is unknown (31, 32). Phosphorylation without transport can be assayed with purified proteins. Purified IIABMan proteins were incubated in the presence of PEP and [14C]Glc, enzyme I, HPr, and crude membrane vesicles containing the IICMan-IIDMan complex, and the formation of [14C]Glc6P was measured (Fig. 1B). The R190K and R304Q mutants had the same activity as wild-type IIABMan, R204K had 70% and R168Q had 10% activity. The R172Q mutant was completely inactive. These in vitro results are consistent with the observations made with intact cells with one exception; the R204K mutant, which had a very low in vivo transport activity, retained 70% nonvectorial phosphorylation activity.
Protein Phosphorylation--
The IIABMan subunit
consists of two independent domains (IIA and IIB) each containing one
phosphorylation site. IIB accepts the phosphoryl groups from the
IIAMan domain. All five invariant arginines are located in
the IIB domain. How does the substitution of the invariant arginines
affect phosphorylation at the two active sites? The arginine mutants of
IIAB were phosphorylated with [32P]PEP in the presence of
enzyme I and HPr. Thereafter the IIAMan and
IIBMan domains were cleaved by limited trypsinolysis and
separated by gel electrophoresis. All mutant proteins were
phosphorylated on IIAMan (as expected) and also on the
mutated IIBMan domains (Fig.
2A). The amount of
32P in the IIB band is lower than in the IIA band because
histidine N phosphates are more sensitive to hydrolysis than
histidine N
phosphates and because the IIB domain also is more
sensitive to incipient trypsinolysis than IIA (18). The ratio
32P-IIB to 32P-IIA after gel electrophoresis is
between 0.3 and 0.4 for wild-type, R190K, and R304Q and between 0.13 and 0.15 for the R168Q, R172Q and R204K mutants. This suggests that the
less active mutants are phosphorylated less efficiently or are less
stable toward hydrolysis during gel electrophoresis. The addition of
the IICMan-IIDMan complex and glucose in
excess of [32P]PEP results in the complete
dephosphorylation of all the phosphorylated proteins with the only
exception of the R172Q mutant, indicating that the R172Q mutant can
accept a phosphoryl group but is unable to donate it to glucose (Fig.
2B, + lanes). When glucose is omitted but the
IICMan-IIDMan complex is present, wild-type
IIABMan and the fully active R190K and R304Q are
phosphorylated to a lesser degree than the less active R168Q, R172Q and
R204K mutants (Fig. 2B,
lanes). This is
possibly due to IICMan/IIDMan-mediated
spontaneous hydrolysis, which is faster with active IIABMan
than with the less active IIABMan mutants. For a more
quantitative analysis, the time course of phosphorylation and
dephosphorylation was determined in solution. Purified
IIABMan was incubated with enzyme I, HPr, and
[32P]PEP, and aliquots were removed at the indicated time
points (Fig. 3A).
R168Q,R172Q,R190K are phosphorylated at 50-75% the rate of the
wild-type in the presence of a saturating concentration of HPr. The
remaining mutants are phosphorylated at more than 80% the rate. The
small difference vanishes when the HPr concentration is rate-limiting
(Fig. 3B). The dephosphorylation rates were measured in a
pulse-chase experiment. IIABMan was labeled with
32P in the presence of enzyme I and HPr. The chase was
triggered by the addition of an excess of
IICMan-IIDMan complex, glucose as P-acceptor
and unlabeled PEP. The dephosphorylation rate is maximal for wild-type
IIABMan. R204K, R190K, and R304Q dephosphorylate at an
intermediate rate whereas R168Q and R172Q are very slow (Fig. 3C). A
difference between R168Q and R172Q could be seen when the
IICMan/IIDMan concentration was increased. The
rate of dephosphorylation of R168Q as well as of wild-type
IIABMan increased with increasing
IICMan/IIDMan concentration, whereas R172Q
remained inactive irrespective of the
IICMan/IIDMan concentration (Fig.
3D).
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DISCUSSION |
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The IIABMan subunit together with the transmembrane
IICMan-IIDMan complex mediate transport and
phosphorylation of mannose and related hexoses. Arginines play a
specific role in many phosphoproteins where they stabilize the
phosphoryl group in the ground and/or in the transition state (6). The
IIB proteins of E. coli, B. subtilis, and
K. pneumoniae share five invariant arginines. To elucidate
the functional role of these conserved residues for phosphorylation and
transport, they were replaced one at a time with either Lys or Gln.
Substrate phosphorylation is completely blocked in the R172Q mutant,
strongly inhibited in the R168Q mutant, and three times slower than
wild-type for the other mutants. Arg-172 together with His-175 is at
the center of the active site loop (Fig.
4). The conformation of Arg-172 in the
crystal structure (of the nonphosphorylated IIBLev)
precludes a direct interaction between the guanidino group and a
phosphoryl group, which would be bound to N of His-175. However, already a minor conformational change would suffice to bring the guanidino group within van der Waals distance of P-His-175. The high
crystallographic B-factors for the loop and the complete absence of
electron density for the Arg-172 side chain indicate flexibility
allowing for such a move (20). Arg-168 and Arg-304 are components of a
hydrogen bonding network that links the active site loop to a second
large loop comprising residues 305-311. Other components of this
network are the invariant Asp-170 of the active site loop, the
invariant Asn-264 of the adjacent
-strand 5, residue 302 (Glu/Asp)
of
-strand 6, and the invariant Asp-309 in the loop between
-strands 6 and 7 (Fig. 4). The
6/
7 loop is accessible from the
protein surface and according to the docking model not involved in
contacts to the IIA domain (20).
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Arg-204 is located on the edge of the first half of the -sheet and
does not interact directly with the phosphorylation site. The R204K
mutant supports nonvectorial glucose phosphorylation but not glucose
translocation. Arg-204 of IIABMan therefore appears
important for solute translocation by the
IICMan-IIDMan complex. Mutation of the nearby
His-219 (H219Q) reduces the affinity of IIABMan for the
IICMan-IIDMan complex resulting in 20-fold
reduced phosphotransferase activity (25). Finally, these residues and
the region of the
-sheet comprising them yielded only weak signals
in the NMR experiments with the isolated IIBMan domain. The
high degree of conformational flexibility of this region could be due
to missing interactions with the IICMan-IIDMan
complex (33). Taken together these observations suggest that the first
half of the
-sheet of IIBMan might form the interface
with the IICMan-IIDMan complex.
While this work was in progress four new transporters belonging to the mannose family were discovered, one for glucose in Lactobacillus curvatus (34), one for glucose and mannose in Vibrio furnissii (35), and a system for N-acetylgalactosamine in E. coli comprising two IIB subunits (IIBAga and IIBAga') (36). All five arginines are conserved in L. curvatus. In the IIBs of V. furnissii and of the E. coli GalNAc system only the two arginines near the active site histidine (Arg-168 and Arg-172) are invariant, whereas Ile and Leu are found in the position of Arg-190 and Gln instead of Arg-204 and Arg-304.
Two Arg mutants (R426K and R428K) that have the same phenotype as R172Q
of IIABMan were found in IICBGlc of the
E. coli glucose transporter, which belongs to the glucose family and is structurally unrelated to the mannose transporter (12).
When the phosphorylation cascade (enzyme I-HPr-IIA-IIB) of the two
phosphotransferase systems for glucose and mannose are compared, the
following patterns of structural and functional properties can be
recognized. The active sites of enzyme I and IIA are on concave
surfaces, they do not contain arginines near the active site, and the
histidines are phosphorylated at N of the imidazole ring. The even
numbered components of HPr and IIB have active sites protruding from
the surface of the proteins, they are phosphorylated at N
of the
imidazole ring or at a cysteine, and they contain one or several
invariant and essential arginines. This periodicity and the structural
and functional complementarity between alternating active sites is
particularly striking between HPr and the IIB domain, where the
conformations of the
/
active site loops are very similar (20).
The fact that mutants can be phosphorylated but not donate the
phosphoryl group to the next component suggests that the structural
requirements for accepting a phosphoryl group are less exacting than
for donating a phosphoryl group. The transition state apparently is
stabilized by the phosphoryl donor rather than the phosphoryl acceptor
protein. This was demonstrated with short basic peptides containing
either a histidine or a cysteine that are rapidly phosphorylated by
enzyme I but cannot donate the phosphoryl group further to an acceptor
protein (37). The inverse mechanism has been observed with CheY, the
response regulator of the chemotactic signaling cascade. CheY, which is
phosphorylated at an aspartyl residue can autocatalyze the phosphoryl
transfer not only from the cognate phosphoryl donor, the sensor kinase CheA, but also from low molecular weight phosphates such as
phosphoramidate or acetylphosphate (38). Attempts to phosphorylate
IIABMan with phosphohistidine or phosphoramidate failed so
far. It remains to be seen in the future whether the phosphotransfer
mechanisms of the PTS and of the two component regulatory systems (39) can be characterized as catalysis by phospho-donor (kinase-like) and
catalysis by phospho-acceptor (phosphatase-like), respectively.
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ACKNOWLEDGEMENTS |
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We thank Stephan Schauder for helpful discussions, suggestions, and the preparation of Fig. 4.
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FOOTNOTES |
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* This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
41-031-631-43-46; Fax: 41-0-31-631-48-87;
E-mail:erni{at}ibc.unibe.ch.
1 The abbreviations used are: PTS, phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (EC 2.7.1.69); IIABMan, hydrophilic subunit of the mannose transporter; IICMan and IIDMan, transmembrane subunits of the mannose transporter; IICBGlc, transmembrane subunit of the glucose transporter of the PTS; HPr, histidine-containing phosphocarrier protein of the PTS; PEP, phosphoenolpyruvate.
2 R. Gutknecht, R. Lanz, and B. Erni, unpublished results.
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REFERENCES |
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