Reduced Na?Affinity Increases Turnover of Salmonella enterica
Serovar Typhimurium MelB
S. Vivek Jakkula and Lan Guan
Department of Cell Physiology & Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, USA
The melibiose permease of Salmonella enterica serovar Typhimurium (MelBSt) catalyzes symport of melibiose with Na?, Li?, or
port in Escherichia coli DW2 and greatly decrease Na?affinity, indicating that intracellular release of Na?is facilitated. Rapid
the primary sequence of its Escherichia coli orthologue (MelBEc)
(45, 15). Like MelBEc(1, 8, 12–14, 23–25, 27, 29, 35), MeBStcata-
ing the free energy from the downhill translocation of one cosub-
strate to drive uphill translocation of the other (3, 39, 40, 42, 44),
26, 31). A threading model of MelB (45) based on the crystal
the major facilitator superfamily; thus, the protein is likely orga-
a long middle loop surrounding an internal cavity facing the cy-
within the internal cavity (Fig. 1). This model is consistent with
and biophysical results, as well as with low-resolution electron
microscopy (EM) structures of MelBEc(20, 37).
The proposed Na?-binding site lies between helices II and IV,
and the carboxyl groups of conserved Asp55 and Asp59 (helix II)
(14, 21, 28, 34, 36, 46, 47) and the carbonyl oxygen of Gly117
two cytoplasmic loops (loop4-5in the N-terminal domain and
loop10-11in the C-terminal domain) contain highly conserved
charged and polar residues (45), some of which are functionally
loop4-5and loop10-11play an important role(s) in ligand recogni-
tion and/or conformational switching between functional states
during the turnover (45).
or Arg (15), and the effects of these mutations on cosubstrate
erties of the side chain. Compared to wild-type (WT) MelBSt, the
ing or Na?- or Li?-coupled melibiose transport; the other three
mutations reduce melibiose active transport and decrease the ap-
parent affinity for cations, with a stronger effect on Na?. Among
he melibiose permease of Salmonella enterica serovar Typhi-
murium (MelBSt) shares 86% identity and 96% similarity to
these mutations, a bulky Trp at position 117 causes the greatest
inhibition of melibiose binding. Remarkably, the G117R mutant
catalyzes melibiose exchange in the presence of Na?or Li?but
does not catalyze translocation reactions that involve net flux of
the coupling cation. The data support a kinetic model in which
melibiose is released prior to release of the coupling cation. The
findings also support the conclusion that Gly117 plays an impor-
tant role in cation binding and translocation. Further mutational
analyses of Gly117 are reported in this communication.
MATERIALS AND METHODS
Materials. [1-3H]melibiose was custom synthesized by PerkinElmer
(Boston, MA). 2=-(N-Dansyl)minoalkyl-1-thio-?-D-galactopyranoside
(D2G) was kindly provided by H. Ronald Kaback and Gérard Leblanc.
Oligodeoxynucleotides were synthesized by Integrated DNA Technolo-
gies. MacConkey agar medium (lactose free) was from Difco. All other
materials were reagent grade and obtained from commercial sources.
Bacterial strains and plasmids. E. coli strain DW2 (melA??melB
?lacZY) was used for the functional characterization. E. coli XL1-Blue
cells were used for DNA manipulations. The expression plasmid pK95
?AH/MelBSt/CHis10(18, 35), which encodes the full-length MelBStwith
as the template. All mutants were constructed by a QuikChange site-
directed mutagenesis kit from Stratagene and confirmed by DNA se-
Protein overexpression. E. coli DW2 cells containing a given plas-
mid were grown in Luria-Bertani (LB) broth (5 g yeast extract and 10
g tryptone per liter with 171 mM NaCl) with 100 mg/liter of ampicillin
in a 37°C shaker. The overnight cultures were diluted by 5% with LB
broth supplemented with 0.5% glycerol (LB-G) and 100 mg/liter of
30°C for another 5 h.
Received 6 July 2012 Accepted 31 July 2012
Published ahead of print 3 August 2012
Address correspondence to Lan Guan, Lan.Guan@ttuhsc.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jb.asm.orgJournal of Bacteriologyp. 5538–5544October 2012 Volume 194 Number 20
Preparation of crude membranes, SDS-12% PAGE, and Western
out as described previously (15). After a protein assay using a Micro-
bicinchoninic acid protein assay kit (Pierce), 25 ?g of crude membranes
a polyvinylidene difluoride (PVDF) membrane by the Trans-Blot Turbo
transfer system (Bio-Rad), the PVDF membrane was reacted with the
penta-His horseradish peroxidase conjugate (Qiagen). MelBStproteins
were detected using the SuperSignal West Pico chemiluminescent sub-
strate (Thermo Scientific) by the ImageQuant LAS 4000 Biomolecular
Imager (GE Health Care Life Science).
Melibiose effect on cell growth. Overnight cultures in the absence of
absence or presence of melibiose at 0.4, 10, or 30 mM, respectively, and
brane vesicles were prepared from E. coli DW2 cells by osmotic lysis (18,
10 mM MgSO4at a protein concentration of 25 to 30 mg/ml, frozen in
liquid N2, and stored at ?80°C.
Melibiose fermentation and acidification. TheDW2cellsweretrans-
melibiose at a range of concentrations between 0.01 and 30 mM (the sole
carbohydrate source) and 100 mg/liter ampicillin, and incubated at 37°C
were washed with 100 mM KPi(pH 7.5; so-called Na?-free buffer) as
KPi, pH 7.5, 10 mM MgSO4and adjusted to an A420of 10 (?0.7 mg
protein/ml). Intracellular melibiose was assayed by fast filtration as de-
scribed previously (18).
a range of melibiose concentrations between 0.05 and 2.5 mM were ob-
tained by a linear fitting of the melibiose uptake at 0, 3, 4, 6, 8, and 10 s,
corrected by the rates obtained from nontransformed DW2 cells, and
plotted as a function of melibiose concentration. The melibiose concen-
hyperbolic function to the data (OriginPro 8.6).
Steady-state measurements were performed with an AMINCO-Bowman
series 2 spectrometer with RSO membrane vesicles at a protein concen-
tration of ? 0.5 mg/ml in 100 mM KPi, pH 7.5 (15). With an excitation
Na?for D2G fluorescence resonance energy transfer (FRET).
FIG 1 Putative cosubstrate-binding sites of MelB viewed from the cytoplas-
mic side. The helices are colored with the colors of the rainbow from N (blue)
to C termini (red) and are numbered with Roman numerals. Side chains es-
sential (D55 and D59) for Na?binding and important for melibiose binding/
transport (D19, D124, R52, R149, and K377) are shown as sticks. Gly117 is
and Loop10-11. Pro132, Pro146, and Pro148 are shown as sticks. Positions for
Arg141 and Glu142 in loop4-5and Asp351, Asp 354, and Arg363 in loop10-11
are indicated by blue or red dots. A melibiose molecule and a sodium ion are
shown as green and yellow spheres, respectively (45).
each well for SDS-12% PAGE. After being transferred onto a PVDF mem-
brane, MelBstproteins were detected by anti-His tag antibody.
FIG3 Melibiose fermentation. E. coli DW2 (?lacY ?lacZ melA??melB) cells
were transformed with a plasmid encoding WT or mutant MelBSt, plated on
MacConkey agar (lactose free) containing melibiose at 0.4 to 30 mM, and
incubated at 37°C for 18 h before photography. The black circle indicates the
October 2012 Volume 194 Number 20jb.asm.org 5539
wavelength at 290 nm, the emission intensity was recorded at 500 nm.
After the addition of 10 ?M D2G (the KD[equilibrium dissociation con-
stant] for the WT), NaCl was consecutively added until no change in
fluorescence emission occurred. An identical volume of water was used
for the control. Increase in intensity (?I, the difference before [I0] and
by a dilution effect, and then plotted as a function of Na?concentration.
The apparent Na?stimulation constant (K0.5
by fitting a hyperbolic function to the data (OriginPro 8.6).
Melibiose concentration for the half-maximal displacement of
bound D2G (IC50). Applying the same experimental setup, melibiose
was added stepwise to the samples containing the RSO vesicles supple-
Na?) value was determined
mented with D2G (10 ?M) and NaCl (20 or 200 mM) until no change
in fluorescence emission occurred. An identical volume of water was
added as a negative control. The decrease in intensity after each addi-
tion of melibiose (?F) was corrected by the dilution effect and plotted
as a function of melibiose concentration. The 50% inhibitory concen-
tration (IC50) was determined by fitting a hyperbolic function to the
data (OriginPro 8.6).
Gly117 in MelBStwas mutated to Cys, Ser, and Asn by site-di-
FIG4 Growth curves. E. coli DW2 cells with or without a given expression plasmid were incubated in LB broth at 37°C. Overnight cultures were diluted by 5%
Cell optical density was monitored hourly at A600and averaged from 2 to 5 tests; error bars represent standard deviations.
Jakkula and Guan
jb.asm.org Journal of Bacteriology
Melibiose fermentation. After entry into the cell, melibiose is
hydrolyzed into glucose and galactose by ?-galactosidase, fol-
lowed by glycolysis with acidification of the surroundings, which
is detected by dark red colonies when the cells are grown on Mac-
Conkey agar containing melibiose at 10 mM or higher. The rate-
limiting step is the entry of melibiose (41). DW2 cells (melA?
?melB) overexpressing WT MelBStform dark red colonies when
the melibiose concentration is 10 mM or greater (Fig. 3), indicat-
ing that melibiose is transported into the cell and metabolized. At
decreasing concentrations of melibiose, the colonies change to
lighter shades, implying weaker acidification. Nontransformed E.
coli DW2 cells form pale/white colonies, denoting no melibiose
are seen, but the DNA sequences of the mutants are unchanged.
Notably, G117C, G117S, and G117N mutants form pale/white
colonies of normal size on the plates containing melibiose at a
0.1 mM melibiose not shown).
Second-site revertants. A red colony was found with the
days, and DNA sequencing analysis revealed a Pro148¡Leu mu-
tation with the G117C mutation unchanged. DW2 cells trans-
size independent of melibiose concentration and ferment melibi-
ose similarly to WT MelBSt. Accordingly, G117C/P148L, G117S/
P148L, and G117N/P148L double mutants, as well as the P148L
mutant, were generated, and all show membrane expression sim-
ilar to that of the WT (Fig. 2), form normal-size colonies, and
ferment melibiose indistinguishably from the WT (Fig. 3).
the LB-G broth, the nontransformed E. coli DW2 cells reach sta-
tionary phase in 6 h, and the growth was not affected by addition
of melibiose to the medium (Fig. 4A). With cells containing the
log phase with declining cell densities is observed when diluted
into melibiose-containing fresh medium. The lag phase is pro-
longed with up to 10 mM melibiose; however, there is no further
change when the melibiose concentration is increased to 30 mM.
G117C and G117S mutants manifest lag phases that are about 1 h
tion of 10 mM or higher. The G117N mutant exhibits a growth
rate similar to that of the WT with 0.4 mM melibiose but shows a
significantly longer lag phase of 7 h with melibiose at 10 mM or
higher. A decrease in optical density occurred within a few min-
utes of mixing the melibiose-free overnight cultures with fresh
medium containing melibiose. Viable cells during the lag phase
were dramatically decreased, as indicated by CFU assay (data not
shown). It is noteworthy that the rate of growth during log phase
under all conditions is similar.
To test osmotic effects, overnight cultures in LB-G medium
NaCl was removed. Nontransformed DW2 cells exhibit a slightly
reduced growth rate in the lag phase with no effect on log phase;
again, there is no melibiose effect on growth (Fig. 4B). The WT
shows a lag phase that is 1 h longer in the NaCl-removed LB-G
medium at each melibiose concentration. Strikingly, G117C and
G117S mutants grown in the presence of melibiose at 10 mM or
higher show a lag phase significantly longer than that in the LB-G
media, approaching a 7-h delay that is similar to that observed in
the G117N mutant. Even at a lower concentration (0.4 mM), the
G117S mutant has a 7-h lag phase, which is drastically different
from its growth in LB-G media.
For all three double mutants (C117C/P148L, C117S/P148L,
and C117N/P148L), as well as the P148L mutant, melibiose has
little or no effect on cell growth in the LB-G (Fig. 4C) or the
tant (15) that neither binds nor transports melibiose (Fig. 5, bot-
tom) behaves like the nontransformed DW2 cells (Fig. 4).
Melibiose transport in intact cells. In a nominally Na?-free
buffer, the WT catalyzes H?-coupled melibiose accumulation at
0.4 mM to a steady state of about 110 nmol/mg in 5 min (Fig. 5,
upper); Na?significantly increases melibiose transport (18, 32).
Previously, we demonstrated that all of the accumulated [1-
3H]melibiose molecules are completely exchanged with extracel-
lular melibiose within 10 min, indicating there is little or no hy-
drolysis or chemical alteration of the accumulated intracellular
melibiose (15). G117C and G117S mutants catalyze H?-coupled
with about a 10-fold increase. The G117N mutant catalyzes H?-
FIG 5 Melibiose transport in intact cells. E. coli DW2 cells were washed and
resuspended with 100 mM KPi, pH 7.5, 10 mM MgSO4and adjusted to 0.7
mg/ml of protein. Transport was initiated by adding melibiose (0.4 mM, 10
is plotted as a function of time.
October 2012 Volume 194 Number 20jb.asm.org 5541
that observed in the G117C and G117S mutants. The P148L mu-
WT level. All three double mutants containing the P148L muta-
melibiose transport kinetics were determined with intact cells
a Kmof 0.44 mM and Vmaxof 262 nmol/mg/min. The G117C and
little change in Km, whereas the G117N mutant exhibits 16- and
3-fold increases in Kmand Vmax, respectively. The single-site
P148L mutant shows a 2-fold decrease in both Kmand Vmax. All
three of the G117C/P148L, G117S/P148L, and G117N/P148L
double mutants show little change in Kmbut dramatically de-
creased Vmaxvalues to levels less than 180 nmol/mg/min, with 4-,
3-, and 8-fold changes compared to the parents, respectively.
of binding affinity for cosubstrates (Na?and melibiose) using
FRET from endogenous Trp residues to a fluorescent sugar sub-
strate, D2G, has been well documented (5, 15, 18, 27). With the
WT, the Na?stimulation constant (K0.5
(Fig. 6B) and the IC50for melibiose displacement of bound D2G
(Fig. 6C) are about 1 and 2 to 3 mM, respectively (Table 1). The
G117C, G117S, and G117N mutants exhibit a K0.5
about 20, 7, and 125 mM (19-, 7-, and 114-fold increase), respec-
tively. The IC50is little affected in G117C and G117S mutants but
increased 7-fold in the G117N mutant.
The P148L mutant alone exhibits less than a 3-fold increase in
mutants show little change in K0.5
signal with the G117N/P148L mutant is insufficient for the deter-
mination of these constants.
Na?) for D2G FRET
Na?and IC50, and G117C/P148L and G117S/P148L double
Na?and IC50relative to the
The G117A mutation of MelBSthas little effect, but a bulky Trp
iose and Na?, as well as their coupled symport (15). In this study,
were individually placed at position Gly117. Cells carrying these
mutants form tiny red colonies on MacConkey agar supple-
mented with melibiose at a concentration of 2.5 mM or higher.
Furthermore, cell lysis occurs after a dilution of the overnight
an elevated Vmaxfor melibiose transport (Table 1) with little
change in protein expression (Fig. 2). The higher the Vmax, the
more severe the cell lysis and the longer the lag phase. Moreover,
the osmotic stress in the NaCl-removed LB-G broth prolongs the
values smaller than that observed with the G117N mutant. The
osmotic lysis in the lag phase.
FIG 6 Transport kinetics and cosubstrate-binding affinity. (A) Kmand Vmax
for melibiose transport. Cell preparation and transport assay were performed
as described in Materials and Methods. Fitted initial rates of melibiose trans-
port at a given melibiose concentration between 0.05 and 2.5 mM (specific
activity, 3.2 to 10 mCi/mmol) were corrected by the rates obtained from non-
binding. Determination of K0.5
mutant at excitation and emission wavelengths of 290 and 500 nm, respec-
were plotted as a function of Na?concentration. (C) Apparent affinity for
melibiose binding. Determination of IC50for melibiose displacement of
Na?for the D2G FRET was carried out with
bound D2G was performed with RSO vesicles containing 10 ?M D2G and 200
with SE and/or data from 2 to 3 tests were plotted as a function of melibiose
fitting a hyperbolic function to the data (OriginPro 8.6).
Jakkula and Guan
jb.asm.orgJournal of Bacteriology
Growth curves with melibiose at a concentration of 10 and 30
is depleted during the log phase; otherwise, the lag phase should
last longer at 30 mM. The data suggest that the recovered cells are
no longer affected by the presence of melibiose. Consistent with
this interpretation, the growth rates during log phase for all con-
ditions are similar. It is possible that one or more mechanosensor
channel(s) can be activated, releasing the accumulated melibiose
and Na?. The red, normal-size colonies formed in 18 h of incu-
bation by the G117C mutant might be those adapted cells (Fig. 3,
Consistent with these findings, the inactive G117W mutant
shows growth curves identical to those of the nontransformed
DW2 cells in the absence or presence of melibiose (Fig. 4 and 5);
mutants show melibiose-independent growth and have a Vmax
value of less than 180 nmol/mg/min. Although it is not clear why
the spontaneous G117C/P148L mutant was identified on a plate
with a melibiose concentration of 0.01 mM, the P148L mutation
inhibits transport Vmaxand thereby blocks the lethal effect of ele-
vated melibiose transport in the Gly117 mutants. It is interesting
that the Vmaxvalue of WT MelBStapproaches a lethal level. In
addition, the G117N mutant has higher Kmand Vmaxvalues,
which explain why the melibiose uptake at a concentration of 0.4
mM is low and why a higher concentration of melibiose is re-
quired to trigger cell lysis.
All three G117C, G117S, and G117N mutants have a signifi-
cantly decreased K0.5
increased Vmaxfor melibiose transport results from the decreased
transport in MelBEcis limited by intracellular release of Na?(3,
intracellular release. The results support the previous conclusion
that Gly117 is a component of the cation-binding site (15, 45).
the important helix V (Fig. 1). Helix V contains a likely sugar-
binding residue, Arg149 (adjacent to P148) (1, 45), and loop4-5
(29). Although the role of loop4-5has not been studied in
MelBSt, a sugar-induced conformational change in loop4-5of
MelBEc(19, 29) has been well recognized; moreover, it was also
proposed to be important for coordinating the two cosub-
strate-binding sites (1). Our three-dimensional model also
Na?for D2G FRET; thus, it is likely that the
suggests that both loop4-5and loop10-11participate in opening
and closing the cytoplasmic cavity that is probably essential for
the alternating access mechanism (45). Pro146 and Pro148
(Fig. 1) may form kinks or hinges important for the transport-
required rearrangement or conformational change in loop4-5.
Compared to the parents, the mutation of Pro148 to Leu has
little or no change in the affinity for cosubstrate binding but
specifically decreases the transport velocity. The kinetic effect
is consistent with the notion that loop4-5involves a large con-
formational rearrangement during melibiose transport. The
ing a rate-limiting step that significantly decreases the turnover
of the permease.
master’s student intern of the Center for Biotechnology and Genomics
This work was supported by Texas Norman Hackerman Advanced
Research Program 010674-0034-2009 and National Science Foundation
MCB-1158085 to L.G.
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TABLE 1 Melibiose transport kinetics and cosubstrate affinitya
0.44 ? 0.03
0.52 ? 0.07
0.70 ? 0.08
7.22 ? 1.80
0.55 ? 0.12
0.56 ? 0.13
4.53 ? 2.01
0.17 ? 0.03
262.68 ? 5.77
576.48 ? 25.65
583.86 ? 26.38
811.22 ? 160.22
142.17 ? 10.50
178.01 ? 13.02
99.86 ? 31.51
105.68 ? 4.93
1.11 ? 0.12
20.62 ? 2.47
7.43 ? 2.39
125.1 ? 19.8
31.83 ? 4.30
15.99 ? 4.02
1.35 ? 0.11
2.22 ? 0.18 (3.53 ? 0.56)
3.92 ? 0.34 (4.0 ? 0.86)
5.43 ? 0.6 (10.9 ? 2.64)
14.66 ? 4.42
4.87 ? 0.37 (14.70 ? 2.35)
4.47 ? 1.12
5.92 ? 0.57 (8.63 ? 1.91)
aThe Kmand Vmaxvalues for melibiose transport were determined with intact DW2 cells. The Na?stimulation constant for D2G FRET (K0.5Na?) and the melibiose concentration
for the half-maximal displacement of bound D2G (IC50) were determined with RSO membrane vesicles prepared from DW2 cells. Methods are described in Materials and Methods
and the legend to Fig. 6. Data are means ? standard errors.
bThe IC50s were measured in 200 mM NaCl; IC50s given in parentheses were measured in 20 mM NaCl.
c–, the FRET signal is insufficient for the determination.
October 2012 Volume 194 Number 20jb.asm.org 5543
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