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Journal of Bacteriology, May 2003, p . 3218-3222, Vol . 185,
No . 10
Identification of the L-Aspartate Transporter in
Bacillus subtilis
Graciela Lorca, Brit Winnen, and Milton H . Saier Jr.*
Division of Biological Sciences, University of California at San Diego, La
Jolla, California 92093-0116
Received 8 January 2003/ Accepted 3 March 2003
YveA of Bacillus subtilis, a putative transporter of the amino
acid/polyamine/organocation (APC) superfamily, is shown to mediate
uptake of both L-aspartate and L-glutamate
as well as having sensitivity to L-aspartate
hydroxamate . This 14 TMS protein is the primary aspartate uptake
system in B . subtilis and serves as the prototype for a new
family within the APC superfamily .
Many families of transport proteins mediate the uptake of amino acids
and their derivatives (6, 7) . The largest
such family is the amino acid/polyamine/organocation (APC)
superfamily (4) . This ubiquitous superfamily
includes 10 well-defined families with numerous paralogues in a
single organism . For example, Escherichia coli and Bacillus
subtilis encode within their genomes 24 and 21 recognized
paralogues of this superfamily in five and four assigned APC
families, respectively (4) .
Very few of the proteins of the APC superfamily are functionally
characterized, and 2 of the 10 previously defined families within the
APC superfamily do not include even one functionally characterized
member . We have (i) cloned, (ii) overexpressed, and (iii) knocked out
the gene encoding a member of a novel family within the APC
superfamily from B . subtilis, yveA . We show that this protein
(i) catalyzes uptake of L-aspartate, (ii) mediates
sensitivity to aspartate hydroxamate, (iii) probably exhibits
specificity for L-aspartate, L-glutamate,
L-aspartic hydroxamate, and possibly
L-asparagine and L-glutamine, (iv)
allows enhanced growth with L-aspartate as the
sole nitrogen source, and (v) exhibits maximal activity after growth
in the presence of L-aspartate . The results
show that YveA is the principal aspartate transporter in B .
subtilis .
The B . subtilis yveA gene was amplified by PCR using the Pfx
platinum polymerase and B . subtilis chromosomal DNA as the
template . To overexpress yveA in B . subtilis, the
XbaI-PstI-amplified fragment was fused to the B .
subtilis promoter Pspac present on plasmid pAG58 (3) .
The yveA gene combined with Pspac was further
subcloned (as an EcoRI-PstI DNA fragment) into the E .
coli-B . subtilis shuttle vector pMK4, which encodes a chloramphenicol
resistance marker (9) . The Pspac promoter is
known to be a weak isopropyl-ß-D-thiogalactopyranoside-inducible
promoter, and pMK4 is a low-copy-number plasmid (3,
9) . These facts are in agreement with the results
reported below (see Fig . 2) .
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FIG . 2 . Time courses for the uptake of L-[14C]aspartate
(A) and L-[14C]glutamate (B) into
B . subtilis cells . M168 (wild type) ( ),
knockout mutant (M168 yveA) (•), M168 cells expressing the
plasmid-encoded yveA gene (M168 with Pspac-yveA)
( ),
and the chromosomal yveA knockout mutant expressing the yveA-bearing
plasmid ( ) .
Cells were grown for 24 h at 37°C in minimal SM medium with 0.1%
D-glucose and 10 mM aspartate . Cells were
harvested in the exponential growth phase, washed twice, and resuspended
in 50 mM Tris-maleate-5 mM MgCl2 (TM buffer) (pH 7.0) to an
optical density of 0.1 . The energy source (8 mM glucose) and either
L-aspartate or L-glutamate
(20 µM; 5 µCi/µmol) were added to a temperature-equilibrated 1-ml cell
suspension at 0 min . Transport assays were conducted at 37°C . Samples
(0.1 ml) were removed at appropriate times as shown, filtered (25-mm
membrane filters; 45-µm pore size), washed three times with cold TM
buffer, dried, and then transferred to vials containing 10 ml of
scintillation fluid for determination of radioactivity . Values reported
represent the averages of two independent assays.
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The bacterial strains and plasmids used in this study are described
in Table 1 . Recombinant plasmid was recovered in E . coli
DH5
cells and transformed in B . subtilis M168 (wild-type) cells
by natural competence . To construct the knockout mutant, the
yveA gene was amplified by PCR and inserted into the cloning
vector pPCR-Script Amp in the SrfI site . An internal fragment
(500 bp) of the cloned gene was deleted by cutting with MunI .
The 4-kb fragment of the cut plasmid was recovered from the gel,
purified, and blunted with the Klenow enzyme . The fragment was
further dephosphorylated and ligated to the kanamycin gene from pER82
(kindly provided by K . Pogliano, University of California at San
Diego) . The presence of the inserted gene was confirmed by PCR .
| TABLE 1 . Bacterial strains and plasmids used in this study
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Figure 1 shows growth curves for (i) wild-type B . subtilis,
(ii) a yveA knockout mutant, and (iii) the wild-type strain
expressing the yveA gene from a plasmid . All cells were grown
under conditions where L-aspartate served as the
sole source of nitrogen . The extent of growth of the yveA
knockout mutant was poor, and the estimated doubling time (dt) was
 21
h . Growth enhancement relative to that of the wild-type strain (both
the extent of growth and apparent growth rate) (dt =
10.4
h) was observed for the yveA plasmid expression strain (dt =
7.3
h) . Similar behavior was observed when 0.1% Casamino Acids was
included in the growth medium, although the differences were less
pronounced (data not shown) . These results suggested that YveA
mediates uptake of L-aspartate but that it is
not the sole transporter providing this function .
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FIG . 1 . Growth curves for B . subtilis strain M168 (wild type) (),
a yveA knockout mutant (M168 yveA) (•), and M168 cells
overexpressing the yveA gene (M168 + Pspac-yveA)
( ) .
Cells were grown for 48 h at 37°C in minimal SM medium (80 mM K2HPO4,
44 mM KH2PO4, 3.4 mM trisodium citrate, 2 mM MgSO4,
6.7 mM KCl, 0.5 mM CaCl2, 5 µM MnCl2, 0.5 µM FeSO4)
with 0.1% D-glucose and 10 mM aspartate . The
optical density was measured at 600 nm.
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Sensitivities of the same three strains used in the growth experiment
shown in Fig . 1 to the toxic aspartate-asparagine analogue
D,L-aspartic acid
ß-hydroxamate were measured . In these experiments, the bacteria in
soft agar (0.7% agar in Luria-Bertani [LB] medium) were plated over
hard agar (1.5% agar in LB), and a disk containing 50, 100, or 150 µg
of D,L-aspartic acid ß-hydroxamate
was placed on the agar plates prior to incubation at 37°C . In
all cases, the knockout mutant was less sensitive and the
overexpressing strain was more sensitive than the wild-type strain to
the aspartate analogue . For example, after a growth period of 24 h,
the radius of the zone of killing on the plate with 150 µg of
D,L-aspartic acid ß-hydroxamate
was 15 mm for the wild type, 19 mm for the overexpressing strain,
and 0.8 mm for the knockout mutant . These results suggest that
YveA is the major uptake system for this analogue .
Figure 2 shows the uptake of L-[14C]aspartate
(Fig . 2A) and L-[14C]glutamate
(Fig . 2B), both at a concentration of 20 µM, as a
function of time for the wild type and yveA knockout mutant
with and without the overexpressing plasmid . The knockout mutant
barely took up L-[14C]aspartate, while the
overproducing strain took up more than the wild type . The same was
observed for L-[14C]glutamate
uptake, although the background activity of the yveA knockout
mutant was substantially higher (over twofold higher than the
wild-type aspartate uptake rate), and the increase, due to the
reintroduction of the yveA gene, was less . The L-[14C]aspartate
incorporation rate by the wild-type strain showed saturation at
100 µM aspartate, with a Vmax of 1.1 ± 0.1 nmol min-1
mg-1 (dry weight) and an apparent Km of 25 ± 3
µM .
Using the same conditions described in Table 2, the
energetics of L-aspartate uptake were studied .
Carbonyl cyanide m-chlorophenyl hydrazone and carbonyl cyanide
4-trifluoromethoxyphenylhydrazone, both at 5 µM, blocked uptake >95% .
Substitution of Na+ for K+ in the uptake buffer
did not result in decreased uptake (±5%) . It is therefore likely that
L-aspartate uptake is a proton-motive
force—rather than a sodium-motive force-driven process—and that the
mechanism of transport is amino acid-H+ symport .
| TABLE 2 . Inhibition of the initial L-[14C]aspartate
uptake rate by the L- and D-isomers
of several amino acids
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Several amino acids were used to estimate substrate recognition by
measuring the percent inhibition of L-aspartate uptake
(Table 2) . Surprisingly, D,L-aspartate
ß-hydroxamate was more inhibitory than L-aspartate .
When a 10-fold excess of nonradioactive L-aspartate
(200 µM) was added uptake of L-[14C]aspartate
was reduced by 80%, while L-asparagine at 200 µM
inhibited 58% . L-Glutamate and
L-glutamine inhibited L-[14C]aspartate
uptake 41 and 35%, respectively . Additionally, L-threonine
and L-serine inhibited about 50%, although
other L-amino acids tested inhibited less than
33% . D-Aspartate and D-asparagine
were about equally inhibitory (45%),
showing that substrate recognition is not strictly stereospecific .
These results, together with the growth and aspartic hydroxamate
inhibition results, suggest that YveA transports amino acids with
relative affinities in the order of aspartate hydroxamate >
L-aspartate > L-glutamate,
but it recognizes a much broader range of amino acids .
Uptake of several L-amino acids was measured in the
wild-type and yveA mutant strains . The cells were grown with
either aspartate or proline as the sole source of nitrogen . Table
3 shows that the yveA mutant strain grown
with aspartate took up less aspartate and glutamate but more
asparagine, glutamine, alanine, and serine compared to the wild-type
control strain . These results indicate that poor growth observed in
the yveA mutant strain might have caused increased activity of
other permeases . This suggestion was substantiated by the fact that
proline-grown mutant cells showed decreased uptake of aspartate and
glutamate but a lesser differential for most of the other amino acids
when compared with the wild-type strain . The large reproducible
differential observed for L-asparagine and
L-glutamine is unexplained .
| TABLE 3 . Uptake of14C-labeled L-amino
acids in cells grown in SM medium with either aspartate or proline (10
mM) as the sole nitrogen sourcea
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A yveA-lacZ fusion was constructed using the pJF751 plasmid (11) .
This vector allows the construction of a translational fusion with
codon 8 of the promoterless ß-galactosidase gene and inserts into the
chromosome by homologous recombination . The N-terminal fragment of
the yveA gene (positions -229 to +14) was amplified by PCR,
cut with EcoRI and BamHI, and ligated into pJF751 which
had been cleaved with the same enzymes . The recombinant plasmid was
transferred to B . subtilis M168 by natural competence, and the
effects of different compounds included in the growth medium on
ß-galactosidase production were measured . Cells were grown in minimal
salts medium with the added amino acid present at 10 mM . The presence
of L-aspartate resulted in a threefold
increase in ß-galactosidase activity compared with cells grown with
(NH4)2SO4 or any one of most other
amino acids (Table 4) . Proline, asparagine, and
glutamine induced activity to lesser extents (about twofold), while
all other amino acids induced minimally .
| TABLE 4 . ß-Galactosidase activity of a yveA-lacZ translational
fusion after growth with different amino acids as the sole nitrogen
sourcea
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Immediately downstream of the yveA gene and transcribed in the
same direction is the yvdT gene encoding a transcriptional regulator
of the TctR/AcrR family . YvdT is the most likely mediator of
yveA induction by L-aspartate . Downstream of
yvdT are two small genes, yvdS and yvdR, probably
sequence-divergent members of the small multidrug resistance family
within the drug/metabolite transporter superfamily (TC 2.A.7) (5) .
Preliminary evidence suggests that YvdR may mediate ammonium efflux
(reference 1 and Y . J . Chung and M . H . Saier, Jr.,
unpublished observations) .
In this communication we have demonstrated that YveA is the
primary L-aspartate transporter of B . subtilis
following growth under standard laboratory conditions . Based on the
inhibition studies with aspartate hydroxamate, it must also transport
this aspartate analogue . The system also transports L-glutamate
but with lower affinity and efficiency . The main glutamate
transporter, GltP (P39817), has been described previously (2,
10) .
YveA is the first member of a new family within the APC superfamily
to be characterized functionally . In an earlier communication,
10 families were described for the APC superfamily (4) . Figure
3 shows a phylogenetic tree for this superfamily,
including representative members of the 10 previously defined
families (families 1 to 10 in Fig . 3) . As can be
seen in the figure, members of these 10 families cluster in
accordance with the expectation based on the results of Jack et al . (4) .
However, the tree shown in Fig . 3 defines a new
family that we have called the aspartate/glutamate transporter (AGT)
family (family 11; TC 2.A.3.11) . YveA of B . subtilis is the
only characterized member of the AGT family, and it is therefore the
prototype for this family . In contrast to all other prokaryotic
members of the APC superfamily (4), all of the
members of this family exhibit 14 rather than 12 putative
transmembrane segments (TMSs) . The two extra TMSs are found
C-terminal to the 12 TMSs that are common to other members of the
superfamily . It should be noted that YveA was included as a highly
divergent member of the ABT family by Jack et al . (4) .
The expansion of this family due to the increased sequence data now
available allows segregation of the previously specified ABT family
into two families of different topological protein types (Fig.
3) .
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FIG . 3 . Phylogenetic tree for representative members of the APC
superfamily . The 10 previously identified families (labeled 1 to 10 in
parentheses following the family abbreviation) were as described by Jack
et al . (4) . Abbreviations of the proteins in these
families were as defined therein . The new family to which YveA belongs
is the AGT family (TC 2.A.3.11) . The tree was derived from a multiple
alignment obtained using the ClustalX program as detailed previously (4) .
Members of the AGT family include YveA Bsu (B . subtilis
gi1945680); YbeC Bsu (B . subtilis gi2632498); Orf1 Sto (Sulfolobus
tokodaii gi15922067); Orf1 Tvo (Thermoplasma volcanium
gi13542093); and Orf1 Tac (Thermoplasma acidophilum gi16081310).
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Two B . subtilis proteins are found within the AGT family, the
other being YbeC . Three other proteins, all from archaea, two from
two different species of Thermoplasma and one from Sulfolobus
tokodaii, are also included in this family . As revealed by the
tree shown in Fig . 3, these five proteins form a clear
cluster, although they are all distant homologues of each other . The
two Bacillus paralogues cluster loosely together . Further studies
will be required to determine the range of substrates transported
by members of the AGT family .
We thank Y . J . Chung for helpful discussions and Mary Beth Hiller for
assisting in the preparation of the manuscript .
G . Lorca and B . Winnen contributed equally to the work reported .
This work was supported by NIH grant GM64368 . G.L.L . was supported
by a fellowship from the Pew Latin American Program in the Biomedical
Sciences .
* Corresponding author . Mailing address: Division of Biological
Sciences, University of California at San Diego, La Jolla, CA 92093-0116 . Phone:
(858) 534-4084 . Fax: (858) 534-7108 . E-mail:
msaier@ucsd.edu .
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The amino acid/polyamine/organocation (APC) superfamily of transporters
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- Jack, D . L., N . M . Yang, and M . H . Saier, Jr. 2001 . The
drug/metabolite transporter superfamily . Eur . J . Biochem . 268:3620-3639 .
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