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Previous Article | Next Article 
J Bacteriol, May 1998, p. 2630-2635, Vol. 180, No. 10
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Periplasmic Cyclodextrin Binding Protein CymE
from Klebsiella oxytoca and Its Role in Maltodextrin and
Cyclodextrin Transport
Markus
Pajatsch,1
Maria
Gerhart,1
Ralf
Peist,2
Reinhold
Horlacher,2
Winfried
Boos,2 and
August
Böck1,*
Lehrstuhl für Mikrobiologie der
Universität München, D-80638
München,1 and
Lehrstuhl für
Mikrobiologie der Universität Konstanz, D-78434
Konstanz,2 Germany
Received 6 November 1997/Accepted 9 March 1998
 |
ABSTRACT |
Klebsiella oxytoca M5a1 has the capacity to transport
and to metabolize 
-, 
- and 
-cyclodextrins. Cyclodextrin
transport is mediated by the products of the cymE,
cymF, cymG, cymD, and cymA genes, which are functionally homologous to the
malE, malF, malG, malK,
and lamB gene products of Escherichia coli.
CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding
of ligands was analyzed. CymE bound -cyclodextrin, -cyclodextrin, and -cyclodextrin, with dissociation constants
(Kd) of 0.02, 0.14 and 0.30 µM, respectively,
and linear maltoheptaose, with a Kd of 70 µM.
In transport experiments, -cyclodextrin was taken up by the
cym system of K. oxytoca three to five times
less efficiently than maltohexaose by the E. coli maltose
system. Besides -cyclodextrin, maltohexaose was also taken up by the
K. oxytoca cym system, but because of the inability of
maltodextrins to induce the cym system, growth of E. coli mal mutants on linear maltodextrin was not observed when the
cells harbored only the cym uptake system. Strains which gained this capacity by mutation could easily be selected, however.
| |
INTRODUCTION |
Cyclodextrins (CDs) are
-1,4-glycosidically linked, cyclic maltooligosaccharides. The main
forms are -, -, and -CDs, which have 6, 7, and 8 glucose
residues, respectively (5, 24). Their three-dimensional
structure is torus shaped, with a hydrophilic outside ring and a
interior hydrophobic cavity. Dependent on the size of the hydrophobic
cavity of the respective CD, inclusion complexes with a variety of
guest molecules can be formed, which is the basis of broad applications
in industry (5, 24). CDs are formed enzymically from starch
by CD-glucanotransferases (CGTases) a subgroup of the -amylase class
of enzymes (27, 35). One of the producers of CGTases is
Klebsiella oxytoca M5a1 (1).
K. oxytoca can utilize starch as the sole carbon and energy
source via two metabolic routes. The first one involves the
extracellular degradation into linear maltodextrins by hydrolysis of
the -1,6-glycosidic bonds via the cell surface-associated
pullulanase (1, 32) and the subsequent cleavage of the
-1,4-glycosidic linkages by the disproportionation activity of the
extracellular -CGTase (1). Escherichia coli
(11, 15, 16, 36) and Klebsiella pneumoniae
(7, 44) can then take up these linear maltodextrins (maltose
up to maltoheptaose) via a binding protein-dependent ABC transporter
(12) consisting of maltoporin (LamB), the
maltodextrin-binding protein (MalE), the cytoplasmatic membrane
proteins (MalF and MalG), and the ATP-binding protein (MalK).
Intracellularly, the linear maltodextrins are degraded into glucose and
glucose-1-phosphate by the enzymes amylomaltase (MalQ) (31),
the maltodextrin phosphorylase (MalP) (43), and maltodextrin
glucosidase (MalZ) (30).
In the second pathway, present only in K. oxytoca, starch is
converted extracellularly into CDs by the cyclization activity of the
-CGTase, forming first and predominately -CD, which later on is
transformed into the thermodynamically favored -CD (3, 35). The growth of K. oxytoca with CD as its sole
carbon and energy source (2) is based on the uptake of the
- and -CD via the cym system (17).
Intracellularly, CDs are linearized by a cyclodextrinase (CymH) into
linear maltooligosaccharides (14) which enter the maltose
degradation pathway (17).
The genes responsible for CD metabolism were localized at the 5' side
of the gene coding for the -CGTase (6, 17). Sequence analysis demonstrated the homology of the gene products of
cymE, cymF, cymG, and cymD
to MalE, MalF, MalG, and MalK, respectively (17). In
addition, a functional homology could be shown between the gene product
of cymA and the maltoporin LamB (29). A
mutational analysis showed that cymE, cymF,
cymD, and cymA were essential for growth at the
expense of CDs (17, 29).
The existence of a specific transport system for CDs, which are rigid
molecules of considerable size (ranging from 1.37 nm for -CD to 1.69 nm for -CD [outer diameter]), was unexpected. Since their transfer
through the outer membrane and the cytoplasmic membrane may involve
novel mechanisms, we have initiated the biochemical analysis. Here we
report on the biochemical characterization of CymE as a periplasmic CD
binding protein as well as the kinetic analysis of the cym
transport system.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids, media, and growth
conditions.
The bacterial strains and the plasmids used in this
study are listed in Table 1, together
with their source or derivation.
The rich medium used was Luria-Bertani (LB) medium (26).
mal and cym mutants were discriminated on
MacConkey agar plates enriched with 0.5% (wt/vol) maltose or -CD.
The minimal medium employed was salt solution P supplemented with 12 mM
ammonium sulfate (18). If not indicated otherwise,
particular carbon sources were added at concentrations of 0.2 or 0.5%.
The concentrations of antibiotics added and growth conditions were
described previously (17).
Standard genetic procedures.
Standard genetic procedures
were adopted from Miller (26) and Sambrook et al.
(33). Enzymes for recombinant DNA techniques were used
according to the recommendations of the manufacturer.
Immunoblotting analysis.
Proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
(23) and subjected to immunoblotting analysis
(34) with rat anti-CymH monoclonal antibodies at a dilution
of 1:10,000 as described previously (14).
Construction of a K. oxytoca cymE-deletion
mutant.
The wild-type K. oxytoca strain was transformed
with the plasmid pCYM
E carrying an in-frame deletion in
cymE (17). The transformants were grown overnight
in LB medium without antibiotics, and the resulting cultures were
diluted to cell densities of 1 × 103 to 2 × 103 cells/ml and plated on MacConkey plates supplemented
with 0.5% -CD. Colorless colonies (about 1%) were purified and
tested for the inability to grow with -CD as the sole carbon and
energy source as well as for their sensitivity to the antibiotic marker of the plasmid used. The presence of the deletion was verified by PCR
employing oligonucleotides priming up- and downstream of the deletion
(MC7, 5'-CCCATTTCCGGAATAGAT-3'; MC15,
5'-CAGCCAGGTAAGATTTAC-3'). Finally, the strains were tested
for complementation of the cym phenotype by transformation
with a plasmid carrying a wild-type copy of the gene.
Construction of plasmid pCYME2.
Plasmid pCYME2 is a
derivative of the plasmid pT7-H6-TRXFUS (22). It carries a
translational fusion of the gene coding for a His-tagged thioredoxin,
followed by an enterokinase cleavage site and the cymE gene
lacking its signal sequence coding region (bp 1 to 60). For
construction, the 'cymE part was amplified via PCR with the
oligonucleotide primers CYME6 (5'-GCTTGGGGAGAGAGTATT-3') and
CYME7 (5'-TTTCCCGTCGACAATAACATAGTTACTCCT-3'). The resulting fragment was phosphorylated and cleaved with SalI. The
pT7-H6-TRXFUS vector part was restricted with KpnI, treated
with Klenow fragment, subsequently cleaved by SalI, and
dephosphorylated. Finally, both fragments were ligated. All fusion
joints and the entire 'cymE gene region were verified by
sequencing.
Overproduction of the His-tag-thioredoxin-CymE fusion
protein.
For overproduction and purification of the
His-tag-thioredoxin-CymE fusion protein, strain BL21 (DE-3) was
transformed with plasmid pCYME2. Cells were grown aerobically at 37°C
in 200 ml of LB medium and inoculated 1:100 with an overnight culture.
At an A600 of 1.0, cells were induced with
isopropyl--D-thiogalactopyranoside (IPTG; 0.5 mM final
concentration), harvested 2 h later by centrifugation, and stored
at 
80°C.
Purification of CymE.
The purification procedure was
conducted at 4°C. The protein concentration was assayed during
purification according to the method of Whitaker and Granum
(45). The CymE content of the fractions was analyzed by
SDS-PAGE (23) following the distribution of the 55-kDa band
of the His-tag-thioredoxin-CymE fusion protein. After the
enterokinase treatment, the appearance of the 39-kDa band of the mature
form of CymE was monitored.
Cells were suspended in a 20-fold volume of buffer A (50 mM sodium
phosphate [pH 7.4], 500 mM NaCl, 1 mM dithiothreitol) containing 1 mM
phenylmethylsulfonyl fluoride and broken by three consecutive passages
through a French pressure cell at 16,000 lb/in2. The
extract was clarified by centrifugation for 10 min at 30,000 × g, resulting in the S30 fraction.
The S30 fraction was applied to a Zn2+-chelating Sepharose
FF column (1-ml bed volume) that had been equilibrated in buffer A. After loading, the column was rinsed with buffer A and developed with a
30-ml linear gradient ranging from 10 to 200 mM imidazol. The flow rate
was 0.5 ml/min. Fractions containing the His-tag-thioredoxin-CymE fusion protein were pooled and dialyzed against buffer B (20 mM Tris-HCl [pH 7.6], 0.5 mM EDTA).
For the cleavage of the fusion protein, the dialysate was incubated
with enterokinase (Boehringer Mannheim GmbH) for 6 h at 37°C at
a ratio between fusion protein and enterokinase of 1:20 (wt/wt). To
separate the mature form of CymE from the His-tag-thioredoxin part of
the fusion protein and from the noncleaved fusion protein and minor
impurities, ion-exchange chromatography through a Mono Q-HR 5/5 column
(1-ml bed volume) was performed. The column was equilibrated with
buffer B; after the sample was loaded, the column was washed with 5 ml
of buffer B, developed with a 20-ml linear gradient reaching from 0 to
1 M KCl, and finally washed with 5 ml (each) of buffer B and buffer C
(buffer B plus 1 M KCl). The flow rate was 0.5 ml/min. Fractions
containing the mature form of CymE were pooled and dialyzed against
buffer B. The solution was frozen in liquid nitrogen and stored at
80°C.
Fluorescence spectroscopy.
Fluorescence spectroscopy was
performed to measure the binding of substrates by the purified CymE
(42). Fluorescence measurements were carried out in an LS50B
Luminescence spectrometer (Perkin-Elmer, Norwalk, Conn.) with
excitation and emission slits between 5 and 10 nm and at an excitation
wavelength of 280 nm. The emission spectrum was monitored between 300 and 400 nm. Changes in the fluorescence intensity as a function of
ligand concentrations were recorded at an emission wavelength of 355 nm. All measurements were performed at room temperature in 1 ml of 50 mM Tris-HCl (pH 7.6), which also served as a reference. Protein
concentrations ranged from 0.8 to 8 µg/ml, and the substrates were
added in small volumes. Changes in the intensity of fluorescence
emission due to dilutions were corrected with a control containing the
same concentration of protein and receiving buffer instead of
substrate. For determination of the respective
Kd values, a series of substrate concentrations
were examined for the degree of quenching of fluorescence. The results
reported are the mean values of three consecutive measurements for 5 min each. Maximal quenching was set at 1. The substrate concentration
resulting in half-maximal quenching was defined as the
Kd and was derived from Lineweaver-Burk
plots.
Chromatographic techniques.
Linear and cyclic
maltooligosaccharides were analyzed qualitatively by thin-layer
chromatography (TLC) as described previously (14).
Transport assays.
In order to measure transport, the
bacteria were first adapted in a preculture to the carbon source
employed. For the characterization of CD uptake, 0.5% -CD or a
mixture of 1% succinate plus 0.1% -CD was used as the carbon
source, whereas for studying the maltose system, 0.2% maltose was used
as the carbon source. After inoculation of the main culture, cells were
grown to the late exponential growth phase, harvested by
centrifugation, washed three times with minimal medium without a carbon
source, resuspended in the same medium, and adjusted in terms of their
cell density. For transport measurements, samples of 3 or 6 ml of this
stock solution were adjusted to 25°C. To start the uptake reaction, a
small volume of the labeled substrate was added (time zero). At
different time intervals, samples of 500 or 1,000 µl were withdrawn,
filtrated through Schleicher & Schuell (Dassel) NC45 membrane filters
(0.45-µm pore size), washed with 10 ml of minimal medium and dried.
Their radioactivity was determined in a TRI-CARB 2100 TR liquid
scintillation analyzer (Packard, Dreieich, Germany) with the Ultima
Gold scintillation cocktail (Packard). The rate of uptake was taken
from the linear portion of the resulting curve (initial rate of
uptake).
For the Vmax and Km
determination of -CD and maltohexaose uptake,
[-14C]CD (1,890 mCi/mmol) and
[14C]maltohexaose (2 mCi/mmol) were synthesized as
described by Pajatsch et al. (28) [-14C]CD
of low specific radioactivity (2 mCi/mmol) obtained from Wacker
Chemicals (Munich, Germany) was also used. Km
and Vmax values were deduced from
Lineweaver-Burk plots. Competition of [-14C]CD uptake
by unlabeled cyclodextrins or linear maltodextrins was measured with a
saturating concentration (20 µM) of the [-14C]CD of
the specific radioactivity (2 mCi/mmol) mixed with the indicated molar
excess of unlabeled compounds.
To observe the metabolism of the -CD taken up by the cells,
chromatographic analysis of the intracellular low-molecular-weight compounds was performed. To this end, cells were incubated with a
sufficient amount of the [-14C]CD (2 mCi/mmol) to
ensure maximal uptake rates over the entire incubation time. At the
times indicated, samples were harvested by filtration, washed, frozen
immediately in liquid nitrogen, and stored at 80°C. The cells were
broken by resuspension in 1 ml of a solution containing 2.5% SDS and
1% chloroform. The solution was clarified by centrifugation, and the
supernatant was desalted by the mixed bed ion-exchange material
Serdolit MB-1 (Serva, Heidelberg, Germany). Finally, the solution was
lyophilized, dissolved in a small volume of water, and separated by
TLC. The TLC plate was then autoradiographed and developed.
Special chemicals.
The unlabeled -, -, and -CDs, as
well as the [-14C]CD of low specific radioactivity (2 mCi/mmol), were gifts from Wacker Chemicals. [-14C]CD
of high specific radioactivity (1,890 mCi/mmol) and
[14C]maltohexaose (2 mCi/mmol) were synthesized as
described previously (28). Nonradioactively labeled linear
maltodextrins were purchased from Sigma Chemicals (Deisenhofen,
Germany). [35S]dATP used for sequencing was delivered by
NEN/DuPont (Dreieich, Germany). Oligonucleotide primers were
synthesized by MWG (Ebersberg, Germany). Molecular biological reagents
were from Boehringer Mannheim GmbH, Pharmacia (Freiburg, Germany). or
New England Biolabs (Schwalbach, Germany).
| |
RESULTS AND DISCUSSION |
Overproduction and purification of CymE.
A fragment containing
the part of the cymE gene corresponding to its mature
(leaderless) form (17) plus 22 bp downstream of the coding
region was amplified via PCR and cloned into the vector pT7-H6-TRXFUS
to generate a translational fusion with the gene coding for a
His-tagged thioredoxin, which at its C-terminus contains the cleavage
site for enterokinase. Considerable attempts to overproduce native CymE
from other vector systems and under many growth conditions only led to
insoluble protein. For overproduction of the CymE fusion protein, the
resulting plasmid, pCYME2, was transformed into E. coli BL21
(DE-3), and the expression was induced in LB medium at 37°C by the
addition of IPTG. SDS-PAGE of a crude cellular extract showed that a
protein of the expected molecular mass was overproduced (Fig.
1, lane 1), the major part in soluble form (data not shown). The purification involved the preparation of a
30,000 × g supernatant, affinity chromatography on a
Zn2+-chelating Sepharose FF column, cleavage of the
His-tag-thioredoxin-CymE fusion protein by enterokinase, and
ion-exchange chromatography through a Mono-Q-HR5/5 column. A
symmetrical peak consisting solely of the mature form of CymE was
eluted from the final column (data not shown). Figure 1 demonstrates
the course of purification as analyzed by SDS-PAGE of the pooled
fractions from each step. From 200 ml of culture, 1 mg of purified CymE
was obtained.
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FIG. 1.
Course of purification of the mature form of CymE
followed by SDS-PAGE of the respective fractions. Lanes: 1, crude
extract; 2, S30 fraction; 3, eluate of the Zn2+-chelating
Sepharose FF column; 4, eluate of the Mono-Q column; 5, molecular mass
standard (97, 85, 55, 39, 33, 27, 19, and 14 kDa). Proteins were
stained with Coomassie brilliant blue.
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Specificity and affinity of substrate binding by CymE.
The
CymE protein contains six tryptophan residues, which, when excited at a
wavelength of 280 nm in the absence of ligand, induce a fluorescence
emission spectrum with a maximum of 338 nm (Fig.
2). The addition of 0.1 mM substrate such
as -CD (Fig. 2A) decreased the fluorescence intensity and caused a
shift of the emission spectrum to shorter wavelengths, in the case of
-CD by about 4 nm. Addition of 0.1 mM - and -CD and of 0.1 mM
maltodextrins also quenched fluorescence, with a shift of the emission
peak to shorter wavelengths, although this shift becomes less prominent with increasing size of the CDs or decreasing chain length of the
maltodextrins, respectively (data not shown). Addition of maltose (Fig.
2B) was without effect. Since the substrate-induced quenching was
maximal at 355 nm (Fig. 2A), this wavelength was used in further
experiments.
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FIG. 2.
Fluorescence quenching of CymE induced by -CD (A) and
maltose (B). Excitation was at 280 nm. The emission spectrum of CymE
(A, 0.8 µg/ml; B, 4 µg/ml) before (solid lines) and after (dotted
lines) the addition of 0.1 mM substrate is shown.
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The substrate specificity of CymE was analyzed by measuring the effect
of addition of 0.1 mM cyclic and linear maltodextrins and of several
mono- and disaccharides (Fig. 3). The
mono- and disaccharides, including maltose, only marginally quenched
fluorescence, which may be due to maltodextrin impurities. Linear
maltodextrins of a chain length of 3 glucose units and longer, however,
had a significant effect. The CDs displayed the highest degree of quenching.
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FIG. 3.
Decrease in fluorescence of CymE (4 µg/ml) induced by
different substrates at a concentration of 0.1 mM. The value exhibited
by -CD is set at 100%.
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The Kd values of CymE for the three CDs and for
maltoheptaose were then determined by titrating the protein with a
series of concentrations of the ligands and measuring the effect on
fluorescence. As measured by fluorescence quenching, the affinity of
CymE for the different sugars decreased with increasing size of the CDs as follows. The Kd values for -CD, -CD,
and -CD were 0.02, 0.14, and 0.30 µM, respectively. The affinity
for maltoheptaose (Kd, 70 µM) was much lower
than those for the CDs.
CymE dependence of transport of CDs and maltodextrins into K. oxytoca and recombinant E. coli cells.
To gain
information on whether the binding affinity of CymE determines the in
vivo affinity for cellular uptake, transport experiments with
radioactively labeled -CD and maltohexaose were conducted at
different substrate concentrations. Several strains were used: K. oxytoca (wild type and a cymE mutant), E. coli GM15/pGM200 harboring a plasmid that encodes the
cym genes except that for the cyclodextrin
glucanotransferase (cgt), and E. coli
GM15/pCYME with a deletion in cymE of pGM200 (both
strains lack the E. coli maltose transport system because of
a chromosomal mutation in malE). E. coli MC4100
served as control strain for the determination of the uptake of
maltohexaose by the mal system. All strains, with the
exception of MC4100, were grown under conditions used to induce the
cym system. Table 2 gives the
apparent Km and Vmax values for -CD and maltohexaose. The results allow the following conclusions. (i) Under Vmax conditions, the
recombinant E. coli strain transports the two substrates at
nearly identical rates. (ii) The apparent Km for
-CD transport in K. oxytoca as well as in the recombinant
E. coli strain is higher than the Kd
of the binding protein (1 versus 0.02 µM). (iii) In the recombinant E. coli strain, the Km for -CD is
nearly 10-fold higher than that for maltohexaose. Also, transport of
maltohexaose via the E. coli mal system is more efficient
than that via the K. oxytoca cym system.
Since only the initial rate of uptake was used, the kinetic constants
of Table 2 reflect the properties of the uptake system and are not
influenced by subsequent metabolism or by a possible feedback
inhibition due to the accumulating substrate. We also monitored the
intracellular fate of radioactive -CD during the course of an uptake
experiment with the K. oxytoca wild type. Figure
4 shows that -CD is accumulated more
rapidly than it is hydrolyzed, which indicates that it is taken up in
an unaltered form.
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FIG. 4.
TLC analysis of the intracellular compounds of the
K. oxytoca wild type during the uptake of
[-14C]CD. (A) Scanned autoradiograph. Lanes: 1, authentic [-14C]CD applied as standard; 2 to 5, accumulated radioactivity after 20 s, 1 min, 2 min, and 5 min of
incubation with [-14C]CD, respectively. (B) Comparison
of increases in spot intensities for -CD, G2, and G1. Arrows
correspond to the respective lanes of the autoradiograph. G1 and G2 to
G8 denote glucose and maltose to maltooctaose, respectively. The spot
for -CD may be contaminated by a minor amount of G4.
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Finally, we have carried out -CD uptake competition experiments in
the absence and presence of competing substrates (Fig. 5). Competition decreased with increasing
size of CDs and with decreasing chain length of linear
maltooligosaccharides; maltose, again, was without any effect. This
pattern roughly reflects the binding affinities of CymE towards these
substrates.
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FIG. 5.
Competition of the uptake of [-14C]CD
(20 µM) by the addition of unlabeled substrates. The % v
(Vmax) values are relative to the rate of uptake
of [-14C]CD. G2 and G3 to G7 denote maltose and
maltotriose to maltoheptaose, respectively. The competing substrates
were added at the indicated fold concentration.
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Can the cym system support growth at the expense of
maltooligosaccharides?
The results described above have shown that
CymE binds maltooligosaccharides, that maltooligosaccharides can be
transported in a CymE-dependent fashion, and that they compete with the
uptake of -CD. Therefore, one would assume that the cym
system would allow the cells to grow on these linear
maltooligosaccharides. This assumption was tested with strain
GM15/pGM200 (Fig. 6A). It grows well in
-CD-containing minimal medium, with a mean doubling time of about
2 h. When subcultured into medium with linear maltodextrins from
maltotetraose to maltoheptaose, growth continued with doubling times at
least for maltohexaose and maltoheptaose comparable to those with
-CD, but decreased thereafter and finally stopped. However, after a
considerable lag, growth resumed and a resubculture grew without a lag.
For maltohexaose and maltoheptaose, this lag phase lasted at
least 36 h, and it was even longer on maltopentaose and
maltotetraose. No growth took place at all on maltose and maltotriose (not shown). Growth on maltodextrins from maltotetraose to
maltoheptaose was completely blocked when a mutation was present in
cymE (not shown).
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FIG. 6.
(A) cym system-dependent growth of strain
GM15/pGM200 with maltoheptaose (G7) as the carbon source. The
preculture was grown with -CD as the carbon source. Before being
subcultured, the cells were harvested, washed, and resuspended in
minimal medium. I and II indicate the time points at which samples were
withdrawn from the culture to retrieve single colonies which
consecutively were characterized. (B) Immunoblotting analysis of the
expression of the cym system in strain GM15/pGM200 during
growth with maltoheptaose (G7) as the carbon source. At the time points
indicated, samples of the respective cultures were withdrawn, and a
crude extract of the harvested cells was used for immunoblotting with
an anti-CymH monoclonal antibody (14). G1 and G2 denote
glucose and maltose, respectively. The amount of purified CymH
(14) used was 40 ng.
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To test whether the resumption of growth is the consequence of a
selection of mutants, we have taken samples at the two time points
indicated in Fig. 6. Only 20% of the colonies retrieved from the
culture at time 1 were able to grow on -CD and on maltoheptaose without a lag phase. This proportion increased to 80% at time 2.
Figure 6B shows that the ability to grow on -CD correlates precisely
with the expression pattern of cymH, an enzyme essential for
CD metabolism (14, 17). The results of the experiment therefore show that the cym system can be used for the
uptake of linear maltodextrins, but linear maltodextrins are unable to induce cym gene expression. Mutations, however, can be
selected which lead to expression. The underlying mutations will be
characterized in the future.
In conclusion, the results from previous work
that cym
genes code for products of a transport system specific for CDshave been established now by the in vitro analysis of CymE, the periplasmic binding protein, as well as by transport studies. Meanwhile, the gene
product of cymA also has been identified as the functional homolog of the maltoporin LamB (29). CymE binds CDs,
especially -CD, with high affinity. It also binds linear
maltooligosaccharides with a chain length of 3 and higher. However,
binding appears to be productive and competent for transport only for
-CD and -CD and for the longer linear maltooligosaccharides. The
metabolism of -CD requires the activity of CGTase (17),
which allows the conclusion that -CD has to be converted into -
or -CD or linearized (3, 4, 29) by this enzyme to be
transported. The fact that -CD does not support growth
(17) could reside in the fact that its complex with CymE
does not acquire the conformation required for docking to the membrane
CymFG partner (37, 39, 40).
A similar situation has been described for MalE. MalE binds -CD and
-CD with high affinity (Kd values of 4 and
1.8 µM, respectively) (38), but in the form of an
unproductive complex (17, 19-21, 37, 38).
The Kd of CymE for -CD binding is about
30-fold lower than the Km in the uptake
reaction. Since it appears that the outer membrane does not limit the
diffusion (10), the uptake process must be limited at some
other step, most probably at the interaction of the substrate-loaded
CymE with the membrane complex. Possibly, the amount of CymE is not
large enough to ensure equality of Kd with
Km (8, 9).
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ACKNOWLEDGMENTS |
We are greatly indebted to G. Wich from Wacker Chemicals, Munich,
Germany, for the generous gift of CDs.
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (SFB156) to W.B. and the Fonds der Chemischen
Industrie to A.B. and W.B.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Genetics and Microbiology, Maria-Ward-Straße 1a, D-80638
München, Germany. Phone: 89-17919856. Fax: 89-17919862. E-mail:
august.boeck{at}rz.uni-muenchen.de.
| |
REFERENCES |
| 1.
|
Bender, H.
1977.
Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae. 1. Synthese, Reinigung und Eigenschaften des Enzyms von Klebsiella pneumoniae M5a1.
Arch. Microbiol.
111:271-282[Medline].
|
| 2.
|
Bender, H.
1977.
Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae. 2. Bedeutung des Enzyms für den Metabolismus der Cyclodextrine bei Klebsiella pneumoniae M5a1.
Arch. Microbiol.
113:49-66[Medline].
|
| 3.
|
Bender, H.
1980.
Kinetische Untersuchungen der durch die Cyclodextrin-Glycosyltransferase katalysierten (1 4)--D-Glucopyranosyltransferreaktionen, insbesondere der Zyklisierungreaktion, mit (14)--D-Glucopyranosylketten (durchnittlicher Polymerisationsgrad von 16) als Substrat.
Carbohydr. Res.
78:133-145[Medline].
|
| 4.
|
Bender, H.
1981.
Enzymology of the cyclodextrins, p. 77-88.
In
Proceedings of the First International Symposium on Cyclodextrins. D. Reichel Publishing Company, Dordrecht, The Netherlands.
|
| 5.
|
Bender, H.
1986.
Production, characterization, and application of cyclodextrins.
Adv. Biotechnol. Proc.
6:31-71.
|
| 6.
|
Binder, F.,
O. Huber, and A. Böck.
1986.
Cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5a1: cloning, nucleotide sequence and expression.
Gene
47:269-277[Medline].
|
| 7.
|
Bloch, M.-A., and O. Raibaud.
1986.
Comparison of the malA regions of Escherichia coli and Klebsiella pneumoniae.
J. Bacteriol.
168:1220-1227[Abstract/Free Full Text].
|
| 8.
|
Bohl, E.,
H. A. Shuman, and W. Boos.
1995.
Mathematical treatment of the kinetics of binding protein-dependent transport systems reveals that both the substrate loaded and unloaded binding proteins interact with the membrane components.
J. Theor. Biol.
172:83-94[Medline].
|
| 9.
|
Bohl, E., and W. Boos.
1997.
Quantitative analysis of binding protein-mediated ABC transport systems.
J. Theor. Biol.
186:65-74[Medline].
|
| 10.
| Bohl, E., M. Pajatsch, and W. Boos. Unpublished
data.
|
| 11.
|
Boos, W.,
R. Peist,
K. Decker, and E. Zdych.
1996.
The maltose system, p. 201-229.
In
E. C. C. Lin, and A. S. Lynch (ed.), Regulation of gene expression in Escherichia coli. R. G. Landes Company, Georgetown, Tex.
|
| 12.
|
Boos, W., and J. M. Lucht.
1996.
Periplasmic binding protein-dependent ABC transporters, p. 1175-1209.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: Cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C.
|
| 13.
|
Casadaban, M. J., and S. N. Cohen.
1979.
Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences.
Proc. Natl. Acad. Sci. USA
76:4530-4533[Abstract/Free Full Text].
|
| 14.
|
Feederle, R.,
M. Pajatsch,
E. Kremmer, and A. Böck.
1996.
Metabolism of cyclodextrins by Klebsiella oxytoca M5a1: purification and characterisation of a cytoplasmically located cyclodextrinase.
Arch. Microbiol.
165:206-212[Medline].
|
| 15.
|
Ferenci, T.
1980.
The recognition of maltodextrins by Escherichia coli.
Eur. J. Biochem.
108:631-636[Medline].
|
| 16.
|
Ferenci, T.,
M. Muir,
K.-S. Lee, and D. Maris.
1986.
Substrate specificity of the Escherichia coli maltodextrin transport system and its component proteins.
Biochim. Biophys. Acta
860:44-50[Medline].
|
| 17.
|
Fiedler, G.,
M. Pajatsch, and A. Böck.
1996.
Genetics of a novel starch utilisation pathway in Klebsiella oxytoca.
J. Mol. Biol.
256:279-291[Medline].
|
| 18.
|
Fraenkel, D. G., and F. C. Neidhardt.
1961.
Use of chloramphenicol to study control of RNA synthesis in bacteria.
Biochim. Biophys. Acta
53:96-100[Medline].
|
| 19.
|
Hall, J. A.,
K. Gehring, and H. Nikaido.
1997.
Two modes of ligand binding in maltose-binding protein of Escherichia coli: correlation with the structure of ligands and the structure of binding protein.
J. Biol. Chem.
272:17605-17609[Abstract/Free Full Text].
|
| 20.
|
Hall, J. A.,
T. E. Thorgeirsson,
J. Liu,
Y.-K. Shin, and H. Nikaido.
1997.
Two modes of ligand binding in maltose-binding protein of Escherichia coli: electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling.
J. Biol. Chem.
272:17610-17614[Abstract/Free Full Text].
|
| 21.
|
Hall, J. A.,
A. K. Ganesan,
J. Chen, and H. Nikaido.
1997.
Two modes of ligand binding in maltose-binding protein of Escherichia coli: functional significance in active transport.
J. Biol. Chem.
272:17615-17622[Abstract/Free Full Text].
|
| 22.
|
Kromayer, M.,
R. Wilting,
P. Tormay, and A. Böck.
1996.
Domain structure of the procaryotic selenocysteine-specific elongation factor SelB.
J. Mol. Biol.
262:413-420[Medline].
|
| 23.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of the bacteriophage T4.
Nature (London)
227:680-685[Medline].
|
| 24.
|
Loftsson, T., and M. E. Brewster.
1996.
Pharmaceutical applications of cyclodextrins. 1. Drug solubilisation and stabilization.
J. Pharm. Sci.
85:1017-1025[Medline].
|
| 25.
|
Martinez, E.,
B. Bartolomé, and F. de la Cruz.
1988.
pACYC184-derived cloning vectors containing the multiple cloning site and lacZa reporter gene of pUC8/9 and pUC18/19 plasmids.
Gene
68:159-162[Medline].
|
| 26.
|
Miller, J. H.
1992.
In
A short course in bacterial genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 27.
|
Nakamura, A.,
K. Haga, and K. Yamane.
1993.
Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of the replacement on pH dependence and transition-state stabilisation.
Biochemistry
32:6624-6631[Medline].
|
| 28.
| Pajatsch, M., A. Böck, and W. Boos. Enzymatic
preparation of radioactively labeled linear maltodextrins and
cyclodextrins of high specific activity from [14C]maltose
using amylomaltase, cyclodextrin glucanotransferase and
cyclodextrinase. Carbohydr. Res., in press.
|
| 29.
| Pajatsch, M., G. Fiedler, and A. Böck.
Unpublished data.
|
| 30.
|
Peist, R.,
C. Schneider-Fresenius, and W. Boos.
1996.
The MalT-dependent and malZ-encoded maltodextrin glucosidase of Escherichia coli can be converted into a dextrinyltransferase by a single mutation.
J. Biol. Chem.
271:10681-10689[Abstract/Free Full Text].
|
| 31.
|
Pugsley, A. P., and C. Dubreuil.
1988.
Molecular characterisation of malQ, the structural gene for the Escherichia coli enzyme amylomaltase.
Mol. Microbiol.
2:473-479[Medline].
|
| 32.
|
Pugsley, A. P.
1993.
The complete general secretory pathway in gram-negative bacteria.
Microbiol. Rev.
57:50-108[Abstract/Free Full Text].
|
| 33.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
In
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 34.
|
Schmid, G., and A. Böck.
1984.
Immunoblotting analysis of ribosomal proteins from archaebacteria.
Syst. Appl. Microbiol.
5:1-10.
|
| 35.
|
Schmid, G.
1996.
Enzymology of cyclodextrins, p. 615-626.
In
J. L. Atwood, J. E. D. Davies, D. D. MacNicol, and F. Vögtle (ed.), Comprehensive supramolecular chemistry, vol. 3. Cyclodextrins. Elsevier Science, Ltd., Oxford, United Kingdom.
|
| 36.
|
Schwartz, M.
1987.
The Maltose regulon, p. 1482-1502.
In
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C.
|
| 37.
|
Sharff, A. J.,
L. E. Rodseth,
J. C. Spurlino, and F. A. Quiocho.
1992.
Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.
Biochemistry
31:10657-10663[Medline].
|
| 38.
|
Sharff, A. J.,
L. E. Rodseth, and F. A. Quiocho.
1993.
Refined 1.8 Å structure reveals the mode of binding of -cyclodextrin to the maltodextrin binding protein.
Biochemistry
32:10553-10559[Medline].
|
| 39.
|
Shilton, B. H.,
H. A. Shuman, and S. L. Mowbray.
1996.
Crystal structures and solution conformations of a dominant-negative mutant of Escherichia coli maltose-binding protein.
J. Mol. Biol.
264:364-376[Medline].
|
| 40.
|
Spurlino, J. C.,
G.-Y. Lu, and F. A. Quiocho.
1991.
The 2.3-Å resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis.
J. Biol. Chem.
266:5202-5219[Abstract/Free Full Text].
|
| 41.
|
Studier, F. W., and B. A. Moffatt.
1986.
Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes.
J. Mol. Biol.
189:113-130[Medline].
|
| 42.
|
Szmelcman, S.,
M. Schwartz,
T. J. Silhavy, and W. Boos.
1976.
Maltose transport in Escherichia coli K12. A comparison of transport kinetics in wild-type and  -resistant mutants with the dissociation constants of the maltose binding protein as measured by fluorescence quenching.
Eur. J. Biochem.
65:13-19[Medline].
|
| 43.
|
Watson, K. A.,
R. Schinzel,
D. Palm, and L. N. Johnson.
1997.
The structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.
EMBO J.
16:1-14[Medline].
|
| 44.
|
Werts, C.,
A. Charbit,
S. Bachellier, and M. Hofnung.
1992.
DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.
Mol. Gen. Genet.
233:372-378[Medline].
|
| 45.
|
Whitaker, J. R., and P. E. Granum.
1980.
An absolute method for protein determination based on difference in absorbance at 235 and 280 nm.
Anal. Biochem.
109:156-159[Medline].
|
| 46.
|
Yanisch-Perron, C.,
J. Vieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene
133:103-119.
|
J Bacteriol, May 1998, p. 2630-2635, Vol. 180, No. 10
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Abstract
Introduction
