Identification of a gene cluster encoding an arginine ATP

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Microbiology (2005), 151, 835–840
DOI 10.1099/mic.0.27591-0
Identification of a gene cluster encoding an
arginine ATP-binding-cassette transporter in
the genome of the thermophilic Gram-positive
bacterium Geobacillus stearothermophilus
strain DSMZ 13240
Rebecca Fleischer, Antje Wengner,3 Frank Scheffel, Heidi Landmesser
and Erwin Schneider
Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für
Biologie, Bakterienphysiologie, Chausseestr. 117, D-10115 Berlin, Germany
Correspondence
Erwin Schneider
[email protected]
Received 26 August 2004
Revised
17 November 2004
Accepted 22 November 2004
A single gene cluster encoding components of a putative ATP-binding cassette (ABC)
transporter for basic amino acids was identified in the incomplete genome sequence of the
thermophilic Gram-positive bacterium Geobacillus stearothermophilus by BLAST searches.
The cluster comprises three genes, and these were amplified from chromosomal DNA of
G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The
purified solute-binding protein (designated ArtJ) was demonstrated to bind L-arginine with high
affinity (Kd=0?39±0?06 mM). Competition experiments revealed only partial inhibition by excess
L-lysine (38 %) and L-ornithine (46 %), while no inhibition was observed with L-histidine or other
amino acids tested. The membrane-associated transport complex, composed of a permease
(designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli
membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP
complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus
lipids, displayed ATPase activity in the presence of ArtJ only. Addition of L-arginine further
stimulated the activity twofold. ATP hydrolysis was optimal at 60 6C and sensitive to the
specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP
hydrolysis of 0?71 mmol Pi min”1 (mg protein)”1 and a Km (ATP) of 1?59 mM. Together, these
results identify the ArtJMP complex as a high-affinity arginine ABC transporter.
INTRODUCTION
Amino acids are key intermediates in both carbon and
nitrogen metabolism in bacteria. Some are also implicated
in osmoregulation and pH homeostasis (Meng & Bennett,
1992; Sleator & Hill, 2001). Although bacteria are generally capable of synthesizing amino acids from metabolic
intermediates, the uptake of amino acids from the environment is energetically favoured. Thus, it is hardly surprising that due to the chemical diversity of the side chains, a
diverse number of transport systems have evolved. These
belong to either the amino acid/polyamine/organocation
(APC) superfamily or to the ATP-binding cassette (ABC)
superfamily (Saier, 2000). ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains that form the translocation
3Present address: Max-Delbrück-Center Berlin-Buch, Robert-RössleStr. 10, 13092 Berlin, Germany.
Abbreviation: ABC, ATP-binding cassette.
0002-7591 G 2005 SGM
pathway and two hydrophilic nucleotide-binding (ABC)
domains that hydrolyse ATP (Higgins, 1992). Members of
the subclass of ABC import systems that is confined to
prokaryotes require an additional extracellular substratebinding protein (or solute receptor) for function (Van der
Heide & Poolman, 2002).
ABC transporters mediating the uptake of amino acids
have been classified in three separate subfamilies designated PAAT (polar amino acid transporters), HAAT (hydrophobic amino acid transporters) and MUT (methionine
uptake transporters) (Hosie & Poole, 2001; Saier, 2000;
Zhang et al., 2003). The PAAT family comprises most of
the known transporters, including the well-studied histidine
permease of Salmonella enterica serovar Typhimurium (S.
typhimurium) (reviewed by Schneider, 2003). However,
very few of the other family members are functionally
characterized. In particular, this holds true for transport
systems from thermophilic micro-organisms. For example,
in the Gram-positive bacterium Bacillus (now Geobacillus)
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835
R. Fleischer and others
stearothermophilus NUB36, which grows best at 55–60 uC,
only two genes have been characterized so far, the products
of which are involved in glutamine uptake (Wu & Welker,
1991). The proteins displayed significant sequence identities
to the ABC subunits and solute-binding proteins, respectively, of glutamine, histidine or arginine transport systems
in Escherichia coli/S. typhimurium. This observation is consistent with the result of computer-aided analyses of complete genome sequences that group ABC transporters for
glutamine and basic amino acids in one subfamily (Dassa &
Bouige, 2001). Thus, in order to obtain a more complete
overview of the repertoire of ABC transporters for polar
amino acids in G. stearothermophilus, we searched the
incomplete genomic sequence of strain DSMZ 13240 (see
http://www.genome.ou.edu/bstearo.html) by BLAST (at
http://www.ncbi.nlm.nih.gov/) using the ATPase subunit
HisP of the histidine/lysine-arginine-ornithine transporter
(LAO/HisJ-QMP) of S. typhimurium (Schneider, 2003) as
the seed sequence. The data revealed highest identity (52 %
compared to 37 % for the next best hit) to a putative ABC
protein (27 kDa) encoded within contig 450 (the numbering is still subject to change). The corresponding gene is
part of a cluster that additionally includes ORFs for a
putative solute-binding protein (29 kDa) and a membraneintegral (permease) subunit (24 kDa). All three gene products also displayed similar identities to the corresponding
components of the arginine (ArtPIQMJ) (Wissenbach et al.,
1995) and glutamine (GlnHPQ) (Nohno et al., 1986) transporters of E. coli and to an arginine transporter (YqiXYZ)
of Bacillus subtilis (Sekowska et al., 2001). However,
attempts to more precisely predict the substrate by PSIBLAST searches gave contradicting results. While the ATPase
and membrane-integral subunits displayed highest similarities to glutamine transport components, the solutebinding protein was predicted to be most similar to the
arginine-binding protein (ArtJ) of E. coli (Wissenbach et al.,
1995). Thus, in order to clearly identify the physiological
substrate(s) we have cloned and overexpressed the genes
in E. coli and characterized the functions of their purified
products by binding assays and in a reconstituted system.
The results show that the gene cluster encodes a highaffinity arginine ABC transporter with low affinities for
lysine and ornithine. In accordance with the E. coli nomenclature and supported by sequence alignments the genes
are designated artJMP.
METHODS
Bacterial strains, plasmids and growth conditions. All bac-
terial strains and plasmids used in this study are listed in Table 1.
G. stearothermophilus was grown in peptone broth (medium 220,
DSMZ, Braunschweig, Germany) at 55 uC. E. coli strains were grown
in LB medium (Miller, 1972) at 37 uC. When required, ampicillin was
added at 100 mg ml21. Growth of all bacterial strains was followed by
measuring the OD650.
Standard DNA techniques. Isolation of genomic DNA of G. stearo-
thermophilus, preparation of plasmids, digestion by endonucleases,
ligation reactions and PCR were performed as described previously
(Hülsmann et al., 2000).
Cloning of artJ. The artJ gene was amplified by PCR using the Taq
platinum polymerase (Invitrogen), and G. stearothermophilus chromosomal DNA as the template. The primers (59-CGC TCC TCG AGG
CGG GCG GCA AGT CAA CGG AAA CAA G-39; 59-GAT CCA
GAA TTC TCA CCT TTC TCT ACG AAA AAT GCG TAA CTT
GC-39) were designed in such a way that the amplified fragment
lacked the 59 sequence encoding the signal peptide and that in the
translated mature polypeptide, alanine substituted for the N-terminal
cysteine residue. The amplified fragment was ligated with pGEM-T,
resulting in plasmid pAW10. To overexpress artJ in E. coli the gene
was further subcloned (as an XhoI–EcoRI fragment) into expression
vector pBAD/HisA. The resulting plasmid was designated pAW11.
Cloning of artMP. The artMP genes were co-amplified by PCR as
one fragment. The primers (59-AGA ACG GGA TCC GAT TTT
Table 1. Bacterial strains and plasmids
Strain or plasmid
Strains
G. stearothermophilus DSM13240
E. coli
JM109
TOP10
Plasmids
pBAD/HisA
pGEM-T
pQE60
pAW10
pAW11
pRF2
Relevant genotype or description
Source or reference
Wild-type
DSMZ*
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 D(lac–proAB) [F9 traD36 proAB
lacIq lacZDM15]
F9 mrcA D(mrr–hsdRMS–mcrBC) w80lacZDM15 DlacX74 deoR recA1 araD139
D(araA–leu)7697 galU galK rpsL endA1 nupG
Stratagene
ParaBAD, araC, histidine fusion vector, Apr
PT7, lacZ, Apr
PT5, histidine fusion vector, Apr
artJ (Cys1RAla) as PCR fragment on pGEM-T
artJ (Cys1RAla) as XhoI–EcoRI fragment on pBAD/HisA
artMP as BamHI–BglII fragment on pQE60
Invitrogen
Invitrogen
Promega
Qiagen
This study
This study
This study
*DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.
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Microbiology 151
Arginine-ABC transporter of G. stearothermophilus
CGT TTT GAT ATT ATT GTC-39; 59-GAG TGA GAT CTA AAT
ACT TTT GAC AAA AAC GCT TTT G-39) were designed in such a
way that the start codon of artM was replaced by a BamHI site and
that a BglII site substituted for the stop codon of artP. The resulting
1?4 kb fragment was cloned into expression vector pQE60, yielding
plasmid pRF2.
Purification of ArtJ. E. coli strain TOP10(pAW11) was grown in
LB, containing ampicillin, to an OD650 of 0?5 and supplemented
with 0?02 % arabinose to induce artJ expression, and growth was
continued for 4 h. Cells were harvested, resuspended in 50 mM
MOPS/KOH, pH 7, 100 mM NaCl, 0?1 mM PMSF and disintegrated by one passage through a French press at 14 000 p.s.i.
(96?6 MPa). After ultracentrifugation (45 min at 200 000 g), ArtJ
was recovered in the supernatant and subsequently purified by
immobilized metal-affinity chromatography using a Co2+-loaded
resin (TALON, Clontech).
Purification of ArtMP. E. coli strain JM109(pRF2) was grown
under the same conditions as TOP10(pAW11) but adding
0?5 mM IPTG for induction of gene expression. For purification of
ArtMP, the membrane fraction obtained after cell disruption and
ultracentrifugation (1 h at 200 000 g) was resuspended in 50 mM
Tris/HCl, pH 8, 5 % (v/v) glycerol, 0?1 mM PMSF and solubilized
with 1?2 % decanoylsucrose. After ultracentrifugation (30 min at
200 000 g) the supernatant was adjusted to 10 mM ATP and 20 mM
2-mercaptoethanol and purified on a Co2+-loaded affinity matrix in
the presence of 0?2 % decanoylsucrose.
Isolation of total lipids from G. stearothermophilus. Lipids
were extracted from cells of G. stearothermophilus by a published
method (Folch et al., 1957). Briefly, cells (10 g wet weight) were
washed once with 50 mM Tris/HCl, pH 8, homogenized in 20 vols
chloroform/methanol (2 : 1, v/v) and stirred for 18 h at room temperature. Undissolved material was removed by filtration and the
filtrate was extracted with 0?2 vols double-distilled water. After
separation into two phases, the lower (organic) phase was evaporated to dryness and washed with acetone for 1 h at room temperature. After removing acetone with a pipette, the sediment was dried
under a stream of nitrogen, dissolved to 20 mg ml21 in chloroform
and stored at 220 uC.
Preparation of proteoliposomes. The ArtMP complex was incor-
porated into liposomes essentially as described previously (Scheffel
et al., 2004). Lipids (20 mg) were dried under a stream of nitrogen,
and slowly redissolved in 50 mM MOPS/KOH, pH 7?5, containing
1 % octyl b-D-glucopyranoside. The mixture was then sonicated for
15 min, and then ArtMP (50 mg), ArtJ (120 mg) and L-arginine
(1 mM, final concentration) were added to the lipid/detergent mixture. These values correspond to a 7?6-fold molar excess of binding
protein over transport complex, which is in the range of what has
been proven optimal for the maltose and histidine ABC transporters
(Landmesser et al., 2002; Hall et al., 1998; Liu et al., 1997). Proteoliposomes were formed by removal of detergent by adsorption to
Biobeads (100 mg; Bio-Rad) at 4 uC overnight. After replacing the
beads with a new batch incubation was continued for 1 h. The mixture was subsequently centrifuged for 1 min at 300 g to pellet the
beads and the supernatant was assayed for ATPase activity. As controls proteoliposomes were prepared by the same procedure but
omitting ArtJ/L-arginine or L-arginine.
Binding assay. Binding of L-[14C]arginine to purified ArtJ was
performed by the method of Richarme & Kepes (1983). Standard
assay mixtures (100 ml) containing 50 mM MOPS/KOH, pH 7?5,
150 mM NaCl, 5 % (v/v) glycerol and purified ArtJ (3 mg) were preheated at 60 uC for 1 min prior to the addition of L-[U-14C]arginine
(Hartmann Analytic; 264 mCi mmol21, 9?77 GBq; 5 mM final concentration). After 1 min, the reaction was terminated by adding
http://mic.sgmjournals.org
2 ml of an ice-cold saturated (NH4)2SO4 solution and immediately
filtered through a nitrocellulose filter (0?45 mm). The filter was
washed once with the same solution, followed by distilled water, and
retained radioactivity was determined in a liquid scintillation counter.
In competition experiments, unlabelled L-amino acids (0?5 mM)
were added 1 min prior to the addition of L-[14C]arginine. To determine the binding constant, ArtJ was first subjected to a denaturation/renaturation procedure using guanidine hydrochloride (6 M) to
remove excess bound unlabelled substrate (Hall et al., 1998).
Analytical methods. Hydrolysis of ATP was assayed in microtitre
plates essentially as described by Landmesser et al., (2002). Protein
was assayed by using the BCA kit from Bio-Rad. SDS-PAGE and
immunoblot analyses were performed as described by Hülsmann
et al. (2000).
RESULTS AND DISCUSSION
Identification of the substrate specificity of ArtJ
Substrate specificity of an ABC importer is primarily
determined by the extracellular solute-binding protein. In
Gram-positive bacteria, binding proteins are anchored to
the cytoplasmic membrane by fatty acids that are covalently
linked to the N-terminal cysteine residue (Sutcliffe &
Russell, 1995). No evidence exists that the lipid modification is crucial for substrate binding or transport (Hülsmann
et al., 2000; Horlacher et al., 1998). However, when genes
encoding solute-binding proteins are expressed in E. coli,
overproduction is often poor (Horlacher et al., 1998) or
can have deleterious effects on the host cells (Sutcliffe &
Russell, 1995). Low protein yields were also observed when
genes encoding lipo-binding proteins were expressed with
their natural signal sequence, which is required for translocation across the cytoplasmic membrane via the Sec
apparatus (Horlacher et al., 1998; Kempf et al., 1997). Thus,
to avoid any of these potential problems and to assure the
availability of sufficient amounts of purified protein for the
intended binding and reconstitution studies, we changed
the N-terminal cysteine residue to alanine and deleted the
signal sequence (see Methods for details). As a consequence,
when overproduced as an N-terminal fusion with six consecutive histidine residues using strain TOP10(pAW11),
ArtJ was recovered with the cytosolic fraction. Purification
was achieved by using a Co2+-loaded affinity matrix and
was verified by SDS-PAGE (Fig. 1a).
In order to identify the substrate(s) of ArtJ, binding assays
using radiolabelled amino acids were performed at 60 uC.
Initial experiments identified L-arginine as the most likely
ligand. To further explore the arginine-binding activity the
protein was first subjected to a denaturation–renaturation
procedure using 6 M guanidine hydrochloride in order to
remove bound unlabelled amino acids. Subsequent binding
experiments at increasing concentrations of L-[14C]arginine
and analysis of the data by a Scatchard plot revealed a
dissociation constant (Kd) of 0?39±0?06 mM. This value is
in good agreement with the dissociation constant reported
for the arginine-binding protein ArtJ of E. coli (0?4 mM;
Wissenbach et al., 1995) but about 20 times higher than
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R. Fleischer and others
(a)
kDa
(b)
kDa
45
His6-ArtJ
31
31
ArtP-His6
ArtM
21
1
2
3
4
1
2
3
that of the lysine-arginine-ornithine-binding protein (LAO)
of S. typhimurium (Nikaido & Ames, 1992). When binding
assays were performed in the presence of 100-fold excess of
unlabelled amino acids, only L-lysine (38 %) and L-ornithine
(46 %) partially inhibited binding of L-[14C]arginine (5 mM)
to ArtJ. In contrast, histidine or glutamine had no effect
(Table 2). These data clearly identified ArtJ as a high-affinity
arginine-binding protein with low affinities for lysine and
ornithine, thereby suggesting that the complete gene cluster
encodes a transporter with preference for arginine. In order
to demonstrate this in vitro we also purified the ArtMP
proteins.
Functional characterization of the ArtJMP
transport system in proteoliposomes
The ArtMP proteins were overproduced from E. coli
strain JM109(pRF2). After solubilization of the membrane
Table 2. Arginine-binding activity of ArtJ in the presence of
competing substrates
The assays were performed with 5 mM L-[14C]arginine and competing amino acids were added in their L-forms at 500 mM. The data
represent the mean of two experiments.
Addition
None
Arginine
Asparagine
Aspartate
Cysteine
Glutamate
Glutamine
Histidine
Lysine
Ornithine
Binding of L-[14C]arginine (% of control)
100*
1?6
99?6
97?9
88?2
93?9
93?1
94?5
62
54?1
*The control value was 12?1 pmol arginine (mg ArtJ)21.
838
4
Fig. 1. Summary of purification of arginine
transport components. Protein samples were
run on an SDS gel and subsequently
stained with Coomassie blue R250. (a)
Purification of His6-ArtJ. Lanes: 1, standard
proteins; 2, cytosolic fraction of strain
TOP10(pAW11); 3, pooled fractions eluted
from TALON matrix with 100 mM imidazole
(4?5 mg); 4, denatured and renatured
ArtJ (3?7 mg). (b) Purification of ArtMP-His6.
Lanes: 1, standard proteins; 2, cytoplasmicmembrane fraction of strain JM109(pRF2);
3, proteins solubilized with 1?2 % decanoylsucrose; 4, pooled fractions eluted from
TALON matrix with 100 mM imidazole
(9 mg). The upper band was identified as
ArtP by immunoblotting using an anti-His-tag
antibody.
fraction with decanoylsucrose, purification was achieved
by affinity chromatography via a His6 extension at the
carboxy terminus of ArtP. SDS-PAGE of peak fractions
revealed two protein bands of the expected apparent
molecular mass (Fig. 1b). The upper band was identified
as ArtP by immunoblotting using an anti-His-tag antibody
(not shown). These results also demonstrate that ArtM
and ArtP form a stable complex in detergent solution.
ABC transporter functions of ArtJMP were subsequently
studied in proteoliposomes by assaying the following wellestablished criteria: (i) stimulation of ATPase activity of
the complex by the cognate binding protein (Davidson
et al., 1992; Liu et al., 1997; Landmesser et al., 2002; Scheffel
et al., 2004), and (ii) sensitivity of this activity to the
specific inhibitor orthovanadate (Landmesser et al., 2002;
Sharma & Davidson, 2000). To this end, proteoliposomes
were prepared from purified ArtMP and a crude total lipid
extract of G. stearothermophilus (mainly composed of
phosphatidylethanolamine, diphosphatidylglycerol and
phosphatidylglycerol; Jurado et al., 1991) in the presence
of L-arginine (1 mM) and/or ArtJ. By this procedure, substrate and binding protein were enclosed in the lumen of
the proteoliposomes. Assuming that incorporation of
complex molecules occurs randomly with respect to orientation, as has been demonstrated for example for the
maltose and histidine ABC transporters of S. typhimurium
(Landmesser et al., 2002; Liu & Ames, 1997), only proteoliposomes with the ArtP subunits facing the medium would
contribute to ATPase activity. As shown in Fig. 2, proteoliposomes prepared in the absence of ArtJ and arginine
exhibited only marginal ATPase activity; however, the
activity was markedly stimulated by ArtJ. Moreover, loading the proteoliposomes with ArtJ and L-arginine further
increased the initial rate of ATP hydrolysis by a factor of
1?6. Similar rates of substrate-dependent stimulation of
ATPase activity were reported for the maltose (Landmesser
et al., 2002) and histidine (Liu et al., 1997) transporters of
S. typhimurium. Consistent with the growth conditions of
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Microbiology 151
Arginine-ABC transporter of G. stearothermophilus
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sequence of ArtJ but disprove the results obtained with
ArtMP. Thus, the sequence of the cognate binding protein
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