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J Biol Chem, Vol. 274, Issue 33, 22977-22984, August 13, 1999
From the Infectious Diseases Department, Parke-Davis Pharmaceutical
Research, Division of Warner-Lambert Company,
Ann Arbor, Michigan 48106-1047
A family of bacterial transporters, the SET
(sugar efflux transporter) family,
has been recently reported (Liu, J. Y., Miller, P. F.,
Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845-1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA,
SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad
substrate specificity, with preferences for glucosides or galactosides
with alkyl or aryl substituents. Consistent with the observed in
vitro substrate specificities, strains that hyperexpress SetA or
SetB are desensitized to lactose analogues as measured by induction of
the lac operon. In addition, strains that hyperexpress SetA
are resistant to the growth inhibitory sugar analogue
o-nitrophenyl- The recently described bacterial SET (sugar
efflux transporter) family of efflux pumps
shares amino acid sequence similarity with the Major Facilitator
Superfamily of transport proteins (1-3). Members of the Major
Facilitator Superfamily perform diverse functions including the uptake
of nutrients such as sugars and the secretion of noxious agents,
including antibiotics (2, 3). The SET proteins were first identified in
Escherichia coli, which encodes three members (SetA, SetB,
and SetC) that share a high degree (at least 70%) of amino acid
sequence similarity (1). Prior to this work, setA, setB, and
setC were given the generic names yabM, yeiO, and
yicK, respectively, for E. coli open reading
frames of unknown function. It was shown that two of the proteins (SetA and SetB) could catalyze the secretion of lactose (1). Two additional
members were identified as open reading frames in Deinococcus radiodurans and Yersinia pestis. A recent examination
of the sequenced microbial genomes data base yielded six additional,
more distantly related,
proteins.1 At present, the
transport properties of these new family members have not been
characterized. The SET proteins are not ubiquitously present in
bacteria, suggesting an ecologically specialized role for this family
of pumps.
Sugar efflux has been reported in many bacterial species (4, 5),
including E. coli (6-9). It was shown that sugar efflux is
an integral part of the metabolism of lactose in E. coli
(7). In a strain constitutive for the lac operon, the
addition of lactose led to the immediate and stoichiometeric appearance
of the products (glucose, galactose, and allolactose) of
Physiological evidence supports the hypothesis that efflux systems are
involved in the detoxification of nonmetabolizable sugars in E. coli (8-11). Methyl- Many nonmetabolizable lactose analogues such as
isopropyl- In this report, a role for the SET proteins (SetA, SetB, and SetC) in
the metabolism of lactose or the detoxification of nonmetabolizable sugar analogues is investigated. As we show in this study, the range of
sugars that are efflux substrates for the E. coli SET proteins include selective monosaccharides and disaccharides, in
addition to glycoside analogues such as IPTG. Because lactose and IPTG
are both substrates for Set protein-catalyzed efflux, we also generated
null mutations in setA, setB, and setC
and used these to help define the role of the Set proteins in E. coli sugar metabolism.
Bacterial Strains, Plasmids, Media, and Reagents--
The
bacterial strains and plasmids used in this study are listed in Table
1. Cultures were grown in L broth (10 g
tryptone/liter, 5 g yeast extract/liter, 5 g NaCl/liter) at
37 °C unless otherwise indicated. Plates contained 15 g
agar/liter and top agar contained 7.5 g agar/liter. Antibiotics
and arabinose, purchased from Sigma, were added to the following
concentrations: 50 µg/ml ampicillin, 10 µg/ml tetracycline, 12.5 µg/ml chloramphenicol, 15 µg/ml streptomycin, 35 µg/ml kanamycin,
15 mM arabinose. M63 medium contained 3.0 g
KH2PO4/liter, 7.0 g of
K2HPO4/liter, 2.0 g
(NH4)2SO4/liter, 0.5 mg
FeSO4/liter, 1 mM MgSO4/liter, 0.1 mg thiamine/liter, and 2.0 g glycerol/liter. Sugars, sugar
analogues, hexokinase, glucose-6-phosphate dehydrogenase, CCCP, and
NADP+ were purchased from Sigma. Ciprofloxacin was obtained
from the Parke-Davis chemical collection. Radiolabeled lactose (55.0 mCi/mmol) was purchased from Amersham Pharmacia Biotech, and
radiolabeled glucose (8.36 mCi/mmol) was purchased from NEN Life
Science Products.
Construction of Plasmids--
Standard DNA manipulation
techniques were used (15). DNA corresponding to each of the
set genes was obtained by the polymerase chain reaction. All
polymerase chain reaction products were sequenced, and except for three
silent mutations in yabM, the products were identical to the
sequences reported in the EBI/GenBankTM data base.
Construction of the plasmids pBAD-SetA, pBAD-SetB, and pBAD-SetC,
previously referred to as pBAD-YabM, pBAD-YeiO, and pBAD-YicK (1),
respectively, were described earlier (1). Plasmids pBR-SetA, pBR-SetB,
and pBR-SetC are derivatives of plasmid pBR-LacY (1) where the
lacY gene was replaced with the gene for either
setA, setB, or setC, respectively.
Growth Curves--
Overnight cultures of strains ML308/pBAD18,
ML308/pBAD-SetA, and ML308/pBAD-SetB grown in L broth containing
ampicillin were diluted 100-fold into the same medium or medium
containing 15 mM arabinose. After 1.6 h of growth,
ONPTG, dissolved at 20 times the final concentration in 60% dimethyl
sulfoxide, or solvent only, was added to the indicated concentrations.
The cultures were grown in a 96-well plate, and growth was followed
with a SpectraMax 96-well plate reader (Molecular Devices Corp,
Sunnyvale, CA).
Isolation of Inside-out Vesicles, Vesicle Transport Assays for
[14C]Lactose and [14C]Glucose, Kinetics of
SetA Catalyzed Lactose Transport, and Inhibitor Studies--
Cells
were grown in L broth with arabinose to an A600
of 0.6-1.0 and harvested for inside-out vesicles, essentially as
described previously (16). In the experiments described in Fig. 1
(A and C), the culture was grown in shake flasks.
In the experiments described in Fig. 1 (B and D),
the culture was grown in a fermentor with L broth containing ampicillin
to an A600 of 0.6-1.0. Subsequently, arabinose
was added to 15 mM, and the culture was harvested 2 h
later. Cell pellets were washed once with ice-cold lysis buffer (50 mM MOPS-KOH, pH 6.6, 180 mM NaCl, 10 mM EDTA), resuspended in lysis buffer to an
A600 of 40-80, and lysed in a French Press at
5000 p.s.i. The lysate was centrifuged at 27,000 × g for 10 min to remove unlysed cells and debris. The
supernatant was centrifuged for 1 h at 100,000 × g to pellet total membranes. Protein concentrations were
determined by the Bradford protein assay kit (Bio-Rad) using bovine
serum albumin as the standard. The transport assay was performed at
21 °C. A 1.5-µl solution of 50 mM ATP, pH 7.0, 50 mM MgSO4 was added to 13.5 µl of a suspension
of membrane vesicles (14.0 µg protein/ml) followed by incubation at
21 °C for 30 s. Transport was initiated by the addition of 60 µl of Buffer A (50 mM MOPS, pH 7.5, 10 mM
MgSO4, 192 mM NaCl) containing the labeled sugar ([14C]lactose: 0.181 mM, 55.0 mCi/mmol,
or [14C]glucose: 0.57 mM, 8.36 mCi/mmol). At
the specified times, the entire solution was mixed with 3 ml of
ice-cold stop buffer (50 mM MOPS-KOH, pH 7.5, 180 mM LiCl) followed by filtration through Whatman glass fiber
filters. The filters were washed twice with 3 ml of the same buffer,
and radioactivity was measured in a liquid scintillation counter. For
the CCCP-treated samples, CCCP was added to 80 µM at the
indicated time after the initiation of transport and allowed to
incubate further before stopping the assay with stop buffer. For the
kinetic studies of SetA catalyzed [14C]lactose transport,
the stock [14C]lactose solution was diluted with
unlabeled lactose so that the final concentrations in the assay were
0.33, 0.5, 1.0, 2.0, and 5.0 mM. In the assay for
inhibitors of SetA catalyzed transport of [14C]lactose,
Buffer A contained the tested inhibitor at 31.25 mM and
[14C]lactose at 0.181 mM (55.0 mCi/mmol). The
final concentrations of the test compound and lactose were 25 and 0.145 mM, respectively.
Induction of the lac Operon with IPTG and Lactose:
Gradient Plate Assay--
Gradient plates were prepared by the
method described previously (18). Antibiotics were used at the
following concentrations: 15 µg/ml kanamycin, 15 µg/ml neomycin,
0.008 µg/ml ciprofloxacin.
Lactose-dependent Glucose Efflux--
The
quantitative assay for glucose is essentially that described previously
(6). Log phase cells growing in M63 with 0.2% glycerol (6) were
pelleted and washed once with buffer containing 0.1 M
NaPO4, 1.0 mM MgSO4, pH 7.6. Cells
were resuspended in assay buffer (0.1 M NaPO4,
7.0 mM MgSO4, 6.0 mM ATP, 0.3 units/ml hexokinase, 2.5 µg/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml NADP+, pH 7.6) to an A600 of
0.25. The assay was initiated by the addition of lactose to 1.0 mM, and the absorbance at 340 nM was followed. To inhibit lactose uptake by LacY, cells were preincubated in assay
buffer with CCCP at 60 µM for 3.0 min before the addition of lactose. The glucose efflux activity was normalized to the Disruption of the set Genes and Construction of the Triple
Disruption Strain--
The disruption markers (19), referred to as
interposons (denoted by the symbol A study of the transport properties of the SET proteins was
undertaken as a first step toward elucidating the physiological function of this newly identified family of efflux proteins. Both setA, setB, and setC were cloned
downstream from the arabinose-inducible promoter, generating the
plasmids pBAD-SetA, pBAD-SetB, and pBAD-SetC, respectively, and the
resulting plasmids transformed into E. coli strain MC4100.
Inside-out membrane vesicles were prepared from cells that
hyperexpressed the plasmid-encoded Set proteins. (In this
configuration, an efflux pump would be expected to transport a
radiolabeled substrate into the inside of the vesicle, which can be
monitored as the accumulation of radioactivity in the vesicle interior.) It was shown previously and confirmed here that both SetA
and SetB transport [14C]lactose (Fig.
1, A and B).
Transport was sensitive to the addition of the protonophore CCCP, which
caused the release of the accumulated radioactivity to near basal
levels. The accumulation of the labeled lactose was also dependent on
the presence of either ATP or NADH, which gave equivalent rates of
labeled lactose transport (data not shown) and was not observed in
vesicles prepared from cells that harbored the control plasmid pBAD18.
Transport activity in the SetB-containing vesicles was higher than that
of SetA. The SetB and more recent preparations of SetA vesicles were
prepared from cells grown with increased aeration. The transport
activity of recent preparations of SetA vesicles is comparable with
SetB and is likely due to increased SetA protein expression (data not shown).
The ability of SetA and SetB to transport [14C]glucose
was also tested. In this assay, a higher concentration of
[14C]glucose, which was of a lower specific activity than
that of the [14C]lactose, was used. Both SetA and SetB
promoted the transport of [14C]glucose, which was
accumulated in the vesicle interior (Fig. 1, C and
D). The accumulation of the radiolabeled glucose was also
dependent on the presence of ATP, was sensitive to CCCP, and was not
observed in vesicles prepared from cells that harbored the control
plasmid pBAD18. We were unable to observe the transport of
[14C]galactose by SetA or SetB (data not shown). In
addition, vesicles prepared from the strain that harbored the
setC expression plasmid was unable to transport any of the
three sugars tested; however, the level of protein expressed was not determined.
Because our data from the SetA and SetB overexpression strains
indicated that SetA has a broader substrate specificity than SetB (see
below), an assay was developed to define the range of sugars and sugar
analogues that could serve as SetA substrates. To facilitate the design
of the assay, which was based on the inhibition of transport of
radiolabeled lactose into inside-out vesicles, a study of the kinetic
properties of lactose transport by SetA was conducted. Fig.
2A shows the kinetics of
lactose transport at varying substrate concentrations. The rate of
transport appears to be saturable (Fig. 2B) with an apparent
Km of 1.9 mM and
Vmax of 0.12 pmol lactose/µg protein/s (Fig.
2C). In the assay for possible substrates or inhibitors of
lactose transport, the accumulation of radiolabeled lactose into
inside-out vesicles was performed in the presence of unlabeled test
compounds at 25 mM and [14C]lactose at 0.14 mM. (Note that this assay cannot distinguish between
substrates that are effluxed by SetA and inhibitors that can compete
with lactose for binding but that themselves are not transported.) The
results of the competition assay are shown in Fig.
3 and are briefly summarized here. Of the
tested sugars, trioses, tetroses, pentoses, and heptoses are poor
inhibitors, whereas selective hexoses and disaccharides, the best being
D-glucose (72% inhibition; Fig. 3B) and
cellobiose (88% inhibition; Fig. 3C), respectively, show
inhibitory activity. Glucosides and galactosides with large alkyl or
aryl aglycone substituents are the most potent inhibitors of lactose
transport (91-100% inhibition; Fig. 3D). When the alkyl
substituent is a small methyl group, such as that found in the
methyl-galactosides, there is a significant reduction in the inhibitory
activity (methyl-
Functional and Biochemical Characterization of Escherichia
coli Sugar Efflux Transporters*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactoside. Strains
disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to
inducers of the lac operon and nonmetabolizable sugar
analogues. The data suggest that the set genes are either
poorly expressed under normal laboratory growth conditions or are
redundant with other cellular gene products.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase action in the medium (6). Consistent with this
hypothesis, mutants defective in the uptake of glucose and galactose
grow poorly on lactose as the sole carbon source (7).
-glucoside
(MG),2 a competitive
inhibitor of glucose utilization (12, 13), enters the cell mainly by
the transporter for glucose and mannose, the products of the genes
ptsG and pstM, respectively, and accumulates to
high levels as both the phosphorylated and the unmodified forms (9,
13). When glucose is the sole carbon source, growth inhibition by MG is
due to both decreased uptake of glucose and interference with the
utilization of intracellular glucose-6-phosphate, the latter because of
the accumulation of MG and MG-6-phosphate (9, 12). It was shown that
both MG and MG-6-phosphate are secreted from the cell by an
uncharacterized mechanism, the latter being first dephosphorylated
before secretion (9, 11, 12).
-D-thiogalactoside (IPTG) and
methyl--D-thiogalactoside are growth inhibitory when
lactose is the only carbon source (10). These compounds enter the cell
through the lactose permease, the product of the lacY gene
(14). To prevent the accumulation of IPTG and methyl--D-thiogalactoside, these sugar analogues are
first acetylated by the LacA transacetylase and then secreted from the
cell by an unknown transporter (8, 10). Re-entry into the cell is prevented because the acetylated sugar analogues are not substrates for
the permease (8).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E. coli strains and plasmids
-Galactosidase Activity Assays--
Log phase cultures
(A600 = 0.2-0.4) were grown at the indicated
concentration of IPTG or lactose for 1 h. Subsequently,
-galactosidase activity in the culture was determined as described
(17). To follow the kinetics of IPTG induction of the lac
operon, IPTG was added to early log phase cultures
(A600 = 0.1) to final concentrations of 2.5 and
5.0 µM. At the indicated times, samples of the culture were removed and assayed for -galactosidase activity.
-galactosidase activity (17) in the solution.
), which conferred
resistance to streptomycin, chloramphenicol, or tetracycline, were
inserted into setA (at MscI), setB (at
BsgI), and setC (at BsgI),
respectively, which were cloned in pBAD18. The disrupted alleles were
individually integrated into the chromosome by one of two independent
methods: 1) homologous recombination following transformation with
linearized DNA into a recD strain (20) and 2) cloning of the
disrupted alleles into the temperature sensitive replication plasmid
pMAK705 (21) to allow use of a two-step method (21) to obtain double cross-over recombinants. The disrupted alleles were confirmed both by
polymerase chain reaction analysis and by P1 mapping studies. The
triple mutant was assembled in both the W3110 and ML308 strain backgrounds by P1 transduction (22).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
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Fig. 1.
SetA and SetB both transport
[14C]lactose and [14C] glucose.
A and B, [14C]lactose transport.
C and D, [14C] glucose transport.
Inside-out membrane vesicles were prepared from strain MC4100/pBAD-SetA
(A and C, 
, 
, and 
), strain
MC4100/pBAD18 (A and C, 
), and strain
MC4100/pBAD-SetB (B and D) grown in the presence
of arabinose. ATP was used to generate a proton gradient to energize
transport. Partially filled square symbols () represent transport in
the absence of ATP. Uptake was initiated by the addition of
radiolabeled lactose or glucose and was followed for 1 min. At the
indicated time, CCCP was added to a final concentration of 80 µM (). Each time point represents the mean of
duplicate determinations ± range.
-galactoside 0%; methyl--galactoside 50%) in
addition to a pronounced preference for a -linkage of the aglycone
to the sugar (Fig. 3D). Interestingly, - and
-D-glucose-1-phosphate are both inactive inhibitors. The
inactivity is likely due to the presence of the negative charge and not
a steric effect of the phosphate group (Fig. 3D). The first
position can be replaced with the larger and uncharged phenyl
substituent found in phenyl- and
phenyl--D-glucosides, which are both potent inhibitors
(Fig. 3D).
View larger version (17K):
[in a new window]
Fig. 2.
Kinetic properties of SetA catalyzed
[14C]lactose transport. A,
SetA-dependent accumulation of [14C]lactose
as a function of time. Inside-out membrane vesicles prepared from
MC4100/pBAD-SetA were used for the transport studies. Transport was
assayed at 0.33, 0.50, 1.0, 2.0, and 5.0 mM
[14C]lactose. Transport assayed with 0.33 mM
[14C]lactose in the absence of ATP is also shown. For
clarity, the 5.0 mM substrate curve is omitted. Each time
point represents the mean of duplicate determinations ± range.
B, the relationship of the transport velocity
versus substrate concentration. The slopes of the lines from
A are plotted against the substrate concentration.
C, Lineweaver-Burk plot of the data from A.
Km = 1.9 mM, Vmax = 0.12 pmol lactose/µg protein/s. , 0.33 mM lactose;
, 0.5 mM lactose; , 1.0 mM lactose; 
,
2.0 mM lactose; 
, 0.33 mM lactose, no
ATP.
View larger version (62K):
[in a new window]
Fig. 3.
Inhibition of SetA catalyzed
[14C]lactose transport into inside-out vesicles. The
assay measured the amount of SetA-dependent
[14C]lactose taken up into inside-out vesicles in 30 s in the presence of the indicated inhibitor at 25 mM. The
amount of [14C]lactose transported in the presence of 25 mM NaCl is taken to be 100. The classes of compounds tested
for inhibitory activity were as follows: A, nonsugars, 3, 4, and 5 carbon monosaccharides. B, 6 and 7 carbon
monosaccharides. C, disaccharides and trisaccharides.
D, glucosides and galactosides. Each data point represents
the mean of triplicate determinations ± S.D.
A series of cell-based assays were designed to test whether SetA and
SetB transport the substrates identified by the in vitro transport assays. A set of pBR322 derived plasmids were constructed that constitutively expressed each of the set genes by
cloning them downstream from the promoter for the tetracycline
resistance gene. The plasmids were transformed individually into
E. coli strain W3110, which is wild type at the
lac locus, and the resulting strains were each expected to
hyperexpress one of the Set proteins. These strains were tested for the
ability of the individual Set proteins to transport either IPTG or
lactose. Because IPTG and lactose are both inducers of the
lac operon, the level of -galactosidase activity should
reflect the intracellular concentration of the inducer. If the inducer
is effluxed from the cell by a Set protein, the level of
-galactosidase activity would be expected to be lower. Strains
harboring individual set plasmids were titrated with either
lactose or IPTG, and the level of -galactosidase activity was
measured. Fig. 4A shows that
only SetA can efflux IPTG, because the level of -galactosidase
activity in the setA-containing plasmid was equal to that of
the background at 0.1 mM IPTG. The level of
-galactosidase activity increased as the concentrations of IPTG rose
from 1.0 to 100 mM, where it plateaued. This is the expected result if SetA is simply an efflux pump for the inducer. At
high extracellular concentrations of the inducer, the rate of entry of
the inducer exceeds the rate of efflux by SetA. In contrast, strains
with either the setB or the setC plasmid, as well
as the strain harboring the control plasmid pBR322, all showed high
levels of -galactosidase activity at 0.1 mM IPTG. The
plateau level of -galactosidase activity in the strain with the
setB plasmid was lower than that of the control strain with
pBR322, possibly reflecting the slow growth phenotype of this
strain.
|
,
pBR-SetB; Fig. 4B shows that both SetA and SetB can efflux lactose.
The -galactosidase activity of the strain with the setA
plasmid remained at background levels when titrated with lactose at up to 10 mM. Growth of this strain was not affected by the
addition of lactose at the concentrations used. The -galactosidase
activity of the strain with the setB plasmid was equal to
that of the background level at 0.1 mM lactose and was 22%
of the level present in the control strain with pBR322 at 1 and 10 mM lactose. In contrast, the level of -galactosidase
activity in the strain with the setC plasmid was similar to
that of the control strain with pBR322. Comparison of the lactose and
IPTG titration curves indicates that IPTG is a better inducer of the
lac operon, which is due to the fact that lactose is not
active as an inducer until it is rearranged to allolactose by the
action of -galactosidase (23).
Because the in vitro transport studies indicated that
aryl-glycosides may possibly serve as substrates for SetA, a cell-based assay was therefore designed to test this. It was previously reported that the intracellular accumulation of the toxic -galactoside analogue, ONPTG, inhibits growth (24). Growth inhibition is dependent
on the expression of lacY, because ONPTG is a substrate for
the LacY permease (25). If ONPTG is also a substrate for SetA efflux,
it would be expected that strains that hyperexpress SetA would be more
resistant to the sugar analogue. A strain constitutive for the
lac operon (ML308) was transformed with plasmids pBAD-SetA, pBAD-SetB or the control plasmid pBAD18. Transformants were tested for
resistance to ONPTG by following the growth curves of cultures in the
absence or presence of arabinose; the latter condition is expected to
induce expression of SetA or SetB in the corresponding transformants.
In the pBAD18 transformant, the presence of ONPTG caused a decrease in
the growth rate (Fig. 5A). The
severity of the growth inhibition was dependent upon the concentration
of ONPTG in the medium. The presence of arabinose slightly decreased the growth yield (compare Fig. 5, A and B without
ONPTG) but did not change the pattern of growth inhibition seen with
increasing concentrations of ONPTG (Fig. 5B). Similarly, the
pBAD-SetB transformant was as sensitive to ONPTG as the control pBAD18
transformed strain in medium with or without arabinose (Fig. 5,
E and F). In contrast, the growth of the
pBAD-SetA transformant was uninhibited in arabinose medium at ONPTG
concentrations up to 1.71 mM (Fig. 5D). However, in the absence of arabinose, this strain was as sensitive to ONPTG as
the pBAD18 transformant (Fig. 5C). These results indicate
that ONPTG and likely other aryl- or alkyl--glycosides are
substrates for SetA efflux.
|
It was shown above with the in vitro SetA lactose transport
assay that positively charged sugars such as glucosamine and
aminophenyl-glucosides are good inhibitors of lactose accumulation
(Fig. 3, B and D). This feature is reminiscent of
the aminoglycoside family of antibiotics such as kanamycin and
neomycin. A cell-based assay was used to determine whether kanamycin
and neomycin are substrates for SetA efflux, which would become
apparent as resistance to the antibiotics. An E. coli strain
deleted for acrAB, which encodes a multiple antibiotic
efflux pump, was transformed with either plasmid pBAD-SetA or control
plasmid pBAD18. The resulting transformants were tested on antibiotic
gradient plates with or without arabinose. Again, arabinose is expected
to induce expression of setA. In arabinose-containing medium, the strain with the setA plasmid was only slightly
more resistant to kanamycin (31% growth across the gradient plate for pBAD-SetA versus 25% growth for pBAD18) and neomycin (63%
growth for pBAD-SetA versus 53% growth for pBAD18) than the
control strain with pBAD18 (Fig. 6). No
difference in resistance to the tested antibiotics was observed in the
absence of arabinose. As an additional control, both strains with
either pBAD-SetA or pBAD18 showed identical sensitivity to the
fluoroquinolone ciprofloxacin with or without arabinose in the medium.
The data suggest that kanamycin and neomycin are poor substrates for
SetA efflux.
|
It can be concluded from the in vitro and cell-based
transport assays that SetA and SetB each can efflux glucose and
lactose. Because the secretion of glucose was observed in the
metabolism of lactose (6), the question emerged as to whether the
set genes are involved in lactose utilization. To answer
this question, all of the set genes (setA,
setB, and setC) were disrupted in E. coli strains W3110 and ML308 by the insertion of different antibiotic resistance cassettes (see "Materials and Methods"). The
mutant alleles were confirmed by polymerase chain reaction as well as
by P1 mapping (data not shown). The growth rate of the triple mutant
was identical to the parental strain in LB or minimal medium with
lactose, glucose, glycerol, succinate, or lactate as the sole carbon
source (data not shown). Also, the addition of lactose to log phase
cells that were previously growing on glycerol did not cause any
noticeable growth inhibition in the triple mutant compared with the
parental strain. The triple mutant in the ML308 genetic background was
further tested for glucose efflux in the presence of lactose. In this
assay, washed log phase cells were placed in the coupled assay
solution, which contained the enzymes hexokinase and
glucose-6-phosphate dehydrogenase. The appearance of glucose was
monitored as an increase in the absorbance at 340 nm because of the
accumulation of NADPH as one of the final products of the sequential
action of the two enzymes on glucose. In both the triple mutant and the
parental strain, the addition of lactose led to the immediate
appearance of glucose in the medium at a rate that reached steady state
within 1.5 min (data not shown). No difference in the steady state rate
of glucose efflux between the triple mutant and parental strains was
observed (Fig. 7). As a control to show
that the detected glucose was due to its secretion from the cell and
not to the action of free -galactosidase in the medium on
extracellular lactose, preincubation of both the triple mutant and
parental strains with the protonophore CCCP at 60 µM
completely abolished the signal (Fig. 7). The complete inhibition of
glucose efflux by treatment with CCCP indicated that there was no free
-galactosidase in the medium.
|
, parent with CCCP; SetA and SetB can secrete inducers of the lac operon. The
presence of efflux proteins for lactose or IPTG would be expected to
lower the intracellular concentration of the inducer. To determine whether the triple mutant is more sensitive than the parental strain to
induction of the lac operon by lactose or IPTG, the triple
mutant in the W3110 strain background, which is wild type for the
lac operon, along with the parental strain were titrated with IPTG or lactose and the levels of -galactosidase activity in
the cells after 1 h of exposure to the inducer was measured. No
difference was observed between the triple mutant and the parental strain in this set of experiments (Figs.
8, A and B). Given
that the setA hyperexpressing strain does not respond at all
to lactose and is desensitized to IPTG, this finding suggested that the
level of SetA, and possibly SetB, is low in wild type cells. In an
effort to employ a more sensitive assay for SetA activity, the kinetics of lac operon induction at low concentrations of IPTG (2.5 and 5.0 µM) were examined in both the triple mutant and
the parental strain. It was shown previously that induction of the
lac operon, as measured by the level of -galactosidase
activity, is bi-phasic at low concentrations of IPTG (14). This is
manifest as a lag in the rate of synthesis of -galactosidase for
2-3 h before a high steady state rate is attained. This bi-phasic
behavior is dependent on the lacY gene and can be explained
as a positive feedback loop; leaky expression of the lac
operon results in a low basal level of LacY permease activity, which
catalyzes the uptake of the inducer IPTG, leading to higher rates of
synthesis of the lac operon. If the SetA protein is present
at an appreciable amount in the wild type cell, the triple mutant might
be expected to reach the high steady state rate of -galactosidase
synthesis sooner, because there would be no efflux protein to
counteract the initial low levels of LacY. As shown in Fig.
8C, the kinetics of lac operon induction, with
2.5 and 5.0 µM IPTG, is the same for both the triple
mutant and the parental strain. This result further supports the
hypothesis that the expression of setA is low in the wild
type cell. This conclusion is also consistent with our observation that
the triple mutant is not more sensitive than the parental strain to
growth inhibition by toxic sugar analogues such as IPTG, ONPTG, or
methyl--glucoside (data not shown).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Biochemical characterization of the newly discovered SET proteins
showed that SetA and SetB can transport selective monosaccharides and
disaccharides. Using an inside-out vesicle system, glucose and lactose
were demonstrated to be substrates for SetA and SetB efflux. In
addition, as judged by the set of substrates considered here, SetA has
broader substrate specificity than SetB. SetA also transports
glucosides and galactosides with alkyl or aryl substituents. The
results of these in vitro transport studies were confirmed using a series of cell-based assays for transport. Overexpression of
SetA or SetB desensitized cells to the induction of the lac operon with lactose as the inducer but only SetA overexpression desensitized cells to IPTG. Transport of aryl--glycosides by SetA
was confirmed by a growth sensitivity assay, because cells that
overexpress SetA were more resistant to the toxic sugar analogue ONPTG.
Although vesicles or cells with the setC expression
constructs were unable to transport glucose, galactose, or lactose, the
high degree of protein sequence identity of SetC to SetB (70%)
suggests that the function is also conserved. It remains possible that setC may be poorly expressed in our plasmid constructs.
However, each of the set genes, when present on a high copy
number plasmid and expressed from the strong lac promoter,
inhibited growth when induced with IPTG (data not shown), suggesting
that a protein product was expressed in all cases. Additional transport
studies with other sugars are warranted to identify substrates for SetC.
The range of substrates that are transported by SetA and SetB suggested
possible roles for the SET proteins in lactose metabolism and in the
secretion of nonmetabolizable sugar analogues. However, a mutant
disrupted in all three set genes showed no defect in lactose
utilization; growth was normal in minimal medium supplemented with
lactose as the sole carbon source, and glucose efflux was normal. In
addition, no increase in sensitivity to the sugar analogues IPTG,
methyl--D-thiogalactoside, and ONPTG was observed. These results indicate that other transporters, possibly one of the newly
identified E. coli members of the SET family,1
are responsible for the observed efflux of glucose in wild type cells.
Interestingly, it was recently reported that the E. coli multidrug efflux pump cmlA may have IPTG efflux activity
(26).
Our results suggest that the set genes are poorly expressed
in E. coli under normal laboratory growth conditions.
Consistent with this hypothesis, the triple setABC mutant
has no observable phenotype. Because the expression of many bacterial
efflux systems are induced by their substrates (3), it is likely that
the true inducers or inducing conditions for the set genes
have yet to be found. It is quite possible that the set
genes function in a protective role, because hyperexpression of SetA
was shown to protect cells against ONPTG. However, conditions that
induce bacterial gene expression are frequently not obvious. Operons that were once thought to be poorly expressed (e.g. the
-glucoside operon (27, 28) and the chitose operon (29)) have been
shown to be induced under conditions that were not apparent when these systems were first discovered. Indeed, methyl--glucoside and methyl--glucoside can modestly induce (about 4-fold) setA
expression.3
The growth inhibitory effects of toxic sugar analogues may result from
either the inhibition of uptake of a bona fide sugar substrate or the interference with normal metabolism by the
intracellularly accumulated sugar analogues. The effectiveness of an
efflux system to protect the cell from a toxic sugar analogue would be
dependent on the rate of entry of the analogue into the cell and the
activity of the efflux system. A sugar analogue that would otherwise be poisonous but have no pathway to enter the cell is harmless. As exemplified by ONPTG, sensitivity is dependent on the expression of
lacY, the transporter for -galactosides (24, 25). On the other hand, it would be difficult to protect a cell from a toxic sugar
analogue that enters the cell through a highly efficient and
constitutively expressed uptake system. It is likely that the
physiological substrates for Set protein efflux would also be expected
to be substrates for E. coli uptake systems. Under this
scenario, the role of the Set proteins would be to secrete toxic sugar
analogues that are taken up by mistake. However, it remains possible
that the Set proteins function to remove toxic sugar-like by products
that are produced by normal E. coli metabolism.
High level expression of SetA alone would be expected to be sufficient to protect cells from selective sugar analogues that are not modified upon entry. Among the known sugar transporters in bacteria, one family of transporters, the PTS (phosphoenolpyruvate-sugar phosphotransferase system) family, modifies the sugars upon entry by the addition of a phosphate group (30, 31). Our biochemical studies showed that negatively charged sugars are not substrates for SetA efflux. This is reminiscent of an unidentified transporter for the removal of MG (9, 11, 12). In the case of MG, entry into the cells is mainly through ptsG and ptsM, the PTS uptake proteins for glucose and mannose, respectively (13). MG accumulates in the cell predominately in the phosphorylated form as MG-6-phosphate (9). Presumably a phosphatase converts MG-6-phosphate to MG before secretion by the unidentified transporter. This detoxification system would rely on the unidentified phosphatase to discriminate between the phosphorylated sugar analogue and the bona fide phosphorylated sugar, because only the uncharged sugar would be a substrate for efflux. We have not tested the set mutants for defects in MG secretion, although MG inhibits SetA catalyzed in vitro lactose transport (data not shown). However, the triple set mutant was not more sensitive to the growth inhibitory effect of MG as compared with the parental strain (data not shown).
The elucidation of the physiological function of the set
genes would depend on finding growth conditions where the
set mutants display a defective phenotype. Because the data
suggest that the set genes are poorly expressed under normal
laboratory growth conditions, a first step would be to identify
conditions that induce set gene expression. Reporter strains
have been constructed that carry chromosomal set fusions to
lacZ or other reporter enzymes to screen for conditions or
sugars that induce set gene expression. It may also be
possible to design a genetic selection for chromosomal mutants that are
up-regulated for setA expression based on its glycoside
(such as IPTG) efflux activity.
| |
ACKNOWLEDGEMENTS |
|---|
We thank J. Li and T.-F. Wang for helpful comments on our manuscript. In addition, we are grateful for the encouragement and assistance received from members of our research group.
| |
FOOTNOTES |
|---|
* 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.
Present address: Infectious Diseases Department, Pfizer Central
Research, B118 Bin 2, Groton, CT 06340.
§ To whom correspondence should be addressed. Tel.: 734-622-5961; Fax: 734-622-7158; E-mail: Eric.Olson@wl.com.
1 M. Saier, manuscript in preparation.
3 J. Y. Liu, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
MG, methyl--glucoside;
IPTG, isopropyl--D-thiogalactoside;
ONPTG, o-nitrophenyl--D-thiogalactoside;
CCCP, carbonyl cyanide
m-chlorophenylhydrazone;
MOPS, 4-morpholinepropanesulfonic
acid.
| |
REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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