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Laboratory of Enzymology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555,1 Microbiology Group, Research and Development Division, Kikkoman Corporation, Noda 278-0022,2 Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, 305-8566, Japan,3 Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 212054
Received 31 October 2001/ Accepted 10 February 2002
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ABSTRACT |
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aspD aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-ß-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-ß-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyrate (6, 10, 24), malate to lactate (20), citrate to lactate (17, 18), and histidine to histamine (19). Some aspartate-decarboxylating strains (AspD+) of T. halophila become decarboxylation-defective strains (AspD-) after treatment with curing agents, such as ethidium bromide or acridine orange (11). In one such AspD+ strain, T. halophila D10, we previously found a 25-kb plasmid encoding the aspartate decarboxylation trait (11). In the present study, we report the cloning, sequencing, and expression of the asp operon consisting of two genes, which we designated aspD and aspT, encoding an aspartate ß-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively. In addition, we used proteoliposomes to demonstrate the electrogenic character of the aspartate-alanine exchange catalyzed by AspT. This is the first discovery of a proton motive metabolic cycle encoded on a plasmid and the first report of the aspT gene coding for the aspartate-alanine antiporter.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Growth of cells and plasmid preparation. T. halophila D10 from which plasmid pD1 was to be extracted was grown for 72 h in lactobacillus MRS broth (Difco) with 5% (wt/vol) NaCl at 30°C. Plasmids were prepared by the method of Anderson and McKay (5), except that the lysis broth was supplemented with 5% NaCl because T. halophila requires NaCl for optimal growth. The plasmid samples were electrophoresed on horizontal 0.7% agarose gels. Plasmids were electrophoretically fractionated from the gels and purified by standard DNA recombinant techniques. E. coli cells were grown aerobically or anaerobically at 30 or 37°C in Luria broth with antibiotics as required (100 µg of ampicillin per ml, 50 µg of kanamycin per ml, and 50 µg of spectinomycin per ml).
Subcloning of asp operon from pD1. All basic molecular biology procedures were carried out as described by Sambrook et al. (23). SalI digestion of plasmid pDI gave three fragments of DNA (12.2, 10.8, and 2.2 kb). The 10.8-kb SalI fragment was ligated to pBluescript II KS(-), giving pAspS, and a nested deletion series was prepared for sequencing. After identification of the genes encoding AspD and AspT, the DNA sequence in this region was confirmed by second-strand sequencing using plasmids selected from the same deletion series. The 12.2- and 2.2-kb SalI fragments were also ligated to pBluescript II KS(-), and the nucleotide sequences of both strands were determined with a nested deletion series and/or primer walking.
A new NcoI site was introduced at the initiation codon of the aspD gene in the asp operon on pAspS, producing pAsp(Nco). Subsequently, pAspS(Nco) was digested with NcoI plus SalI to give a 4.3-kb fragment containing the complete asp operon. This 4.3-kb fragment was ligated into the NcoI-SalI site of pTrc99A to generate pTrcAsp, in which expression of the asp operon was regulated by the trc promoter. The asp operon was translated from the ATG codon in the NcoI site. As a final step, pTrcAsp was placed in E. coli XL3 for functional tests.
To generate histidine-tagged AspD (AspDHis), a histidine tag (6x histidine) and a new XhoI site were introduced at the C-terminal end of aspD in pTrcAsp by using a QuikChange site-directed mutagenesis kit (Stratagene) with oligonucleotides AspD6His+ (5' C TAT GAT AAA TTC CAA CAA AAA TAA CAC CAC CAC CAC CAC CAC CCA TGG AAT TCG AGC TCG AGA CCC GGG GAT CCT CTA GAG TCG ACC; the underlining indicates the new XhoI site) and AspD6His- (5' GGT CGA CTC TAG AGG ATC CCC GGG TCT CGA GCT CGA ATT CCA TGG GTG GTG GTG GTG GTG GTG TTA TTT TTG TTG GAA TTT ATC ATA G). Then the mutated plasmid was digested with XhoI to excise a fragment of aspT and ligated to generate the resulting plasmid, pTrcAspDHis.
Sequencing. Double-stranded DNA was sequenced with an ABI Prism BigDye terminator cycle sequencing reaction kit (PE Applied Biosystems) by automated DNA sequencing using an ABI Prism 377 DNA sequencer (PE Applied Biosystems). A nested deletion series was sequenced by using universal primers for pBluescript II KS(-); as primers for sequencing the asp operon opposite strand, we designed appropriate complementary synthetic oligonucleotides.
RNA isolation and primer extension. Cells of T. halophila D10 grown for 72 h in lactobacillus MRS broth with 5% (wt/vol) NaCl and 10 mM L-aspartate at 30°C were harvested and washed once in 50 mM Tris-Cl (pH 7.5), and RNA was isolated with Sepasol-RNA I Super kits (Nacalai Tesque, Kyoto, Japan) used according to the manufacturer's instructions. Primer extension analysis was performed by using oligonucleotides AspDL48 (5'-TCC AAG AAA AGG GCA GCA ACT) and AspDL229 (5'-GCC TTC TTT TTC AAC ATA GCC). The primers end labeled with IRD800 infrared dye were purchased from Aloka (Tokyo, Japan). Each primer (10 pmol) was annealed to 15 µg of total RNA, and primer extension reactions were performed with Ready-To-Go RT-PCR beads (Amersham Pharmacia Biotech) as recommended by the manufacturer's protocol. The cDNAs, which were synthesized along with sequence ladders generated with the same primers, were electrophoresed on an 8% polyacrylamide gel with 7 M urea by using a Li-Cor automated DNA sequencer 4200G (Aloka). The sequencing reaction for the ladders was performed with Thermo Sequenase cycle sequencing kits (Amersham Pharmacia Biotech).
Expression of asp operon in E. coli. An overnight preculture of E. coli XL3 carrying pTrcAsp or pTrc99A was diluted 100-fold in fresh Luria broth containing 10 mM aspartate and 2 mM pyridoxal 5'-phosphate (PLP). The cell suspension was incubated statically for 48 h at 30°C. At 12 h prior to cell harvest, 200 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the cultures. IPTG-induced cells and uninduced control cells were washed with and resuspended in 50 mM potassium morpholineethanesulfonic acid (MES) buffer (pH 6). The aspartate decarboxylation reaction was started by adding 10 mM aspartate to the cell suspension (80 mg [dry weight] of cells/ml) and incubating the reaction mixture at 30°C for 20 min. Concentrations of aspartate and alanine in the reaction mixtures were measured with a Hitachi L8500 amino acid analyzer.
Assay of AspD activity. IPTG-induced E. coli cells harboring pTrcAspDHis or pTrc99A were disrupted in lysis buffer (50 mM sodium phosphate buffer [pH 8], 300 mM NaCl, 10 mM imidazole) by sonication or high-pressure lysis as described below, and cell extracts were prepared by centrifugation (15,000 x g) for 10 min. Aspartate decarboxylation was monitored by the appearance of the product alanine as follows. The reaction mixture contained 200 mM sodium acetate buffer (pH 5.3), 5 mM pyruvate, 0.1 mM PLP, and 0.003% (wt/vol) hydroxypropyl cellulose (Wako, Tokyo, Japan). After incubation of cell extracts in the mixture, the reaction was terminated by boiling for 5 min. The solution was then clarified by centrifugation (15,000 x g) for 5 min, and the concentration of L-alanine in the supernatant was determined by using alanine dehydrogenase and the method described previously (28). Aspartate decarboxylase activities were calculated by determining the rate of alanine appearance.
Purification of AspDHis. Crude cell extract (684 µg of protein) containing AspDHis was loaded on a Qiagen Ni2+-nitrilotriacetic acid (NTA) column, washed, and eluted according to the manufacturer's instructions.
Electrophoresis and Western blotting. Samples of fractions obtained during purification of AspDHis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide, as described previously (13), and protein content was evaluated by staining with Coomassie brilliant blue. The position of AspDHis was verified by Western blotting of a duplicate gel in which proteins were transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan) and exposed to anti-6His mouse monoclonal antibodies (Covance Inc., Princeton, N.J.). Binding of the primary antibody was visualized by using anti-mouse immunoglobulin G (heavy and light chains) horse polyclonal antibody (Vector Laboratories Inc., Burlingame, Calif.) and an AP detection kit (Bio-Rad) according to the manufacturer's instructions.
Preparation of membrane vesicles, solubilization, and reconstitution of AspT. IPTG-induced cells and uninduced control cells (5 mg of protein each) were harvested by centrifugation and washed with 100 mM potassium phosphate (pH 7); membrane vesicles were prepared by high-pressure lysis in the presence of 100 mM potassium phosphate (pH 7), as previously described (3), and the vesicles were stored at -70°C as concentrated stock preparations (10 to 20 mg of protein per ml).
Membrane vesicles (1 to 2 mg of protein) were solubilized (3) by using 1.25% (wt/vol) octylglucoside in the presence of 0.4% (wt/vol) acetone-ether-washed E. coli phospholipid, 100 mM potassium phosphate (pH 7), 4 mM magnesium sulfate, 1 mM dithiothreitol, 0.75 mM phenylmethylsulfonyl fluoride, and 20% glycerol. Control extracts were prepared in the same way but without added protein.
Reconstitution was in a final volume of 1 ml by using 400 µl of detergent extract (or control lipid extract), 130 µl of bath-sonicated liposomes (5.9 mg of E. coli phospholipid), and 18 µl of 15% octylglucoside, with the balance comprised of 1 mM dithiothreitol and 100 mM phosphate (pH 7) as either the potassium or N-methylglucamine (NMG) salt. After incubation for 20 min on ice, proteoliposomes (or control liposomes) were formed at 23°C by rapid injection into 20 ml of a loading buffer containing 100 mM potassium NMG phosphate (pH 7) and 1 mM dithiothreitol along with 100 mM aspartate (potassium or NMG salt, as specified below). After an additional 20 min, the substrate-loaded proteoliposomes (or liposomes) were recovered by centrifugation and washing (3), with resuspension in 100 mM potassium sulfate or 100 mM NMG sulfate plus 100 mM potassium phosphate or 100 mM NMG phosphate (pH 7) and 1 mM dithiothreitol (resuspension buffers). The final resuspension volume was usually 300 µl, which resulted in protein and lipid concentrations of approximately 50 to 250 µg/ml and 13 mg/ml, respectively (3). When proteoliposomes (liposomes) were loaded with alanine, the same procedure was used, except that the buffer for loading, washing, and resuspension contained 200 mM alanine instead of aspartate and the resuspension volume for proteoliposomes was 20 µl.
Assays of transport. To assay for [3H]aspartate transport by aspartate-loaded particles, proteoliposomes were diluted 20-fold from the concentrated stock preparations into an appropriate volume of assay buffer (resuspension buffer lacking dithiothreitol) along with other required materials. After 1 to 3 min of preincubation at 23°C, 3H-labeled substrate was added to a normal concentration of 100 µM, and at different times 50- to 100-µl aliquots were removed for membrane filtration (0.22-µm pore-size GSTF Millipore filters), followed by two washes with 5 ml of assay buffer (3). For transport assays of alanine-loaded proteoliposomes, proteoliposomes were diluted 100-fold with the assay buffer containing 200 µM [3H]aspartate with or without 1 µM valinomycin. In this way, it was possible to generate a membrane potential whose polarity was either interior positive (potassium outside, NMG inside) or interior negative (NMG outside, potassium inside). As an additional basis for comparison, proteoliposomes were loaded with NMG-alanine and tested by using the NMG-based assay buffer.
Protein estimation. Protein content was measured by using a modification of the method of Schaffner and Weissman (25), as described elsewhere (3).
Chemicals. L-[2,3-3H]Aspartic acid (1.07 GBq/mmol) was purchased from Amersham-Pharmacia Biotech. Octyl-ß-D-glucoside was obtained from Dojin (Kumamoto, Japan). E. coli phospholipid was provided by Avanti Polar Lipids, Inc. (Alabaster, Ala.) (3).
Nucleotide sequence accession number. The nucleotide sequence of the asp operon has been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession number AB072729.
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RESULTS |
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G = -10 kcal/mol) is located 73 bp downstream from the translation end of aspT. Primary amino acid sequences of the asp gene products. We searched current protein databases with the BLAST network service and the United States patent database. The aspD gene product (AspD) was predicted to be composed of 532 amino acids and to have a molecular mass of 60,073 Da. On the basis of the database searches, known aspartate ß-decarboxylase genes have been isolated only from Alcaligenes faecalis (7) and Pseudomonas dacunhae (22). Although the products of these two genes exhibited significant relatedness to each other (93% identity, 96% similarity), the tetragenococcal aspD gene product (AspD) was less related to them (37 and 35% identity, respectively; 59 and 58% similarity, respectively). Figure 2 shows an alignment of the amino acid sequences of the three aspartate ß-decarboxylases. PLP attachment motifs (7) were conserved in all three aspartate ß-decarboxylases.
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Expression of asp operon in E. coli. To determine whether the genes tentatively identified as the asp operon specify the aspartate decarboxylase AspD and the AspT transport protein, we constructed a vector (pTrcAsp) in which aspD and aspT were cotranscribed in the authentic order (Fig. 1) under control of the trc promoter. Expression of the asp operon and assays of its function were then carried out in E. coli XL3. This strain also harbored middle-copy compatible plasmid pMS421 encoding the gene for LacIq, which strongly repressed expression of the asp operon in the absence of IPTG. This enabled pTrcAsp to be propagated without the selective pressures that might accompany leaky protein expression. The results shown in Fig. 3 document that the cloned asp operon encoded the proteins catalyzing aspartate decarboxylation in washed cells of E. coli. IPTG-induced cells with pTrcAsp or pTrc99A were harvested and washed as described above. The washed cell suspensions were incubated with 10 mM aspartate. Cells harboring pTrcAsp metabolized aspartate with nearly stoichiometric release of alanine. By contrast, neither aspartate metabolism nor alanine production was observed in E. coli cells harboring the pTrc99A vector. Aspartate decarboxylase activities were detected intracellularly but not extracellularly in E. coli cells containing pTrcAsp (data not shown). These results suggest that gene products of the asp operon are able to catalyze inward transport of aspartate, intracellular decarboxylation of aspartate, and subsequent outward excretion of alanine.
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We initially focused on studying aspartate-loaded proteoliposomes, with the expectation that AspT would catalyze an aspartate self-exchange. Proteoliposomes prepared from membranes of E. coli harboring pTrcAsp were loaded with 100 mM potassium aspartate and 0.1 M potassium phosphate buffer (pH 7) and suspended in an NMG-based medium (0.1 M NMG sulfate, 0.1 M NMG phosphate; pH 7) to which 100 µM external [3H]aspartate was added. [3H]aspartate was loaded into proteoliposomes by aspartate self-exchange. In the experiment whose results are shown in Fig. 5A, the steady-state incorporation of [3H]aspartate was approximately 200 nmol/mg of protein. Moreover, the incorporated material was readily expelled after a later addition of excess unlabeled aspartate or alanine, as if [3H]aspartate had been taken up by an exchange reaction and alanine had been the countersubstrate for aspartate. Since accumulated [3H]aspartate was released by either aspartate or alanine (Fig. 5A), it seems feasible that both compounds served as substrates. As expected, a membrane potential (outside positive) generated by addition of valinomycin had no effect on the release of [3H]aspartate induced by unlabeled aspartate (Fig. 5A), because this release was attributable to the electroneutral property of aspartate self-exchange (Fig. 5A). On the other hand, the steady-state level of aspartate self-exchange observed in the control proteoliposomes prepared from E. coli cells with the pTrc99A vector was less than 15% of the level observed in the proteoliposomes from E. coli cells with pTrcAsp, and the incorporated [3H]aspartate in the control proteoliposomes (pTrc99A) was released after a later addition of unlabeled aspartate but was not expelled after a subsequent addition of unlabeled alanine even in the presence or absence of valinomycin (Fig. 5A). The results for aspartate transport observed with the control proteoliposomes suggested that E. coli cells had weak background activities of aspartate self-exchange in the assay conditions used but did not have activities of heterologous exchange of aspartate with alanine. Hence, the major activities of aspartate transport observed in the proteoliposomes from E. coli with pTrcAsp are attributable to the function of expressed AspT proteins.
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The electrogenic nature of the aspartate-alanine exchange catalyzed by AspT was further supported by the results of experiments shown in Fig. 5B. A detergent extract from IPTG-induced cells with pTrcAsp was also used to prepare proteoliposomes loaded with the potassium or NMG salt of alanine to monitor the exchange of aspartate with alanine (Fig. 5B). The proteoliposomes were diluted into media containing NMG phosphate or potassium phosphate, so that addition of valinomycin negatively or positively polarized the membranes. Imposition of an internally positive electrical potential strongly stimulated aspartate transport, whereas imposition of an internally negative potential significantly inhibited the transport reaction. In such experiments (Fig. 5B), control proteoliposomes were prepared and assayed by using NMG as the internal and external cations (Fig. 5B). These controls, whose behavior was largely unaffected by valinomycin, showed aspartate transport virtually identical to that found for the potassium- or NMG-loaded proteoliposomes not exposed to valinomycin. On the other hand, proteoliposomes prepared from membranes of E. coli harboring the pTrc99A vector were also loaded with alanine and examined; however, the proteoliposomes of pTrc99A scarcely showed aspartate transport activity even in the presence of an inside-positive membrane potential (Fig. 5B). Therefore, the activities of aspartate-alanine exchange observed in the proteoliposomes from E. coli with pTrcAsp are attributable to the function of AspT. The pattern of responses indicates that the aspartate-alanine exchange is electrogenic, with a negative charge moving in parallel with aspartate.
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DISCUSSION |
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Since the intergenic region between aspD and aspT did not seem to form any secondary structure, the probable gene organization of the asp operon is promoter aspD aspT, and the two genes are likely to be cotranscribed by the predicted promoter upstream of the 5' end of aspD.
At this time, only two aspartate ß-decarboxylases, one originating from P. dacunhae (22) and one originating from A. faecalis (7), can be found in the United States patent and protein databases, respectively. It is noteworthy that these two decarboxylases have been used industrially for enzymatic or fermentative production of L-alanine or D-aspartate. The deduced amino acid sequence of tetragenococcal AspD is only moderately related to those of aspartate ß-decarboxylases from P. dacunhae and A. faecalis (37 to 38% identity); however, the relatedness between the two decarboxylases from P. dacunhae and A. faecalis is significant (93% identity). Therefore, tetragenococcal AspD seems to be evolutionarily distant from the two other decarboxylases, although the PLP attachment sites are well conserved in the three decarboxylases (Fig. 2). The activities of AspD were localized in cytoplasmic fractions prepared from both the authentic T. halophila D10 cells and E. coli cells in which the tetragenococcal asp operon was expressed with pTrcAsp (data not shown).
Analysis of the AspT amino acid sequence revealed a hydrophobic membrane protein (data not shown), and the biochemical features of AspT transport demonstrated by using proteoliposomes (Fig. 5) are sufficient to classify AspT as a conventional secondary transport protein, in particular, an electrogenic antiporter similar to OxlT from O. formigenes (2, 4, 14, 15, 16). However, there is no apparent sequence homology between AspT and known membrane carriers, including PMF-generating antiporters (2, 17, 24), suggesting that the structures of PMF-generating antiporters are divergent. We previously found an electrogenic aspartate-alanine antiporter in Lactobacillus subspecies M3 by employing a proteoliposome reconstitution system (1); however, the predicted gene encoding AspT in Lactobacillus subspecies M3 has not yet been cloned. Consequently, at this time we are not able to compare the deduced structures of AspTs from T. halophila and Lactobacillus subspecies M3. Cloning of the lactobacillus aspT gene is also in progress. Based on the features of the tetragenococcal asp operon products described above, we expect that the cytoplasmic aspartate decarboxylase (AspD) and the electrogenic aspartate-alanine antiporter (AspT) compose a proton motive metabolic cycle similar to the oxalate-formate exchange system observed in O. formigenes (4) and to the aspartate-alanine exchange system observed in Lactobacillus subspecies M3 (1).
T. halophila naturally grows from a neutral pH (pH 7) toward an acidic pH (pH 4 to 5) due to lactic acid production in the presence of a large amount of sodium chloride (17%, wt/vol) (11, 27). The presence of a large amount of extracellular cations (H+, Na+) might force tetragenococcal cells to pump out the cations. The proton motive metabolic cycle encoded in the asp operon may contribute directly to the extrusion of protons from cells or indirectly to sodium extrusion (Na+/H+ exchange) coupled with a PMF generated from the cycle, because T. halophila is an anaerobe and is thought to lack oxidative proton pumps. Similar bacterial proton motive metabolic cycles coupled with glutamate decarboxylation have recently been recognized as cycles that are involved in acid tolerance (6, 8, 10, 24).
To our knowledge, this is the first report of plasmid-encoded genes involving a proton motive metabolic cycle and the first report of an aspT gene coding for an aspartate-alanine antiporter. The entire sequence of plasmid pD1 contains transposon-like sequences and probable genes encoding machinery required for conjugal transfer (Abe and Higuchi, unpublished data), showing the possibility of mobility of the asp operon not only among strains but also among genomes and plasmids. Studies of the origin of the mobile asp operon would also be interesting from an evolutionary point of view.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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-aminobutyrate in a Lactobacillus strain. J. Bacteriol. 179:3362-3364.
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and 
, pTrcAsp; 
and 
, pTrc99A). The vector without inserts was pTrc99A. The error bars indicate standard errors.
and 
, pTrc99A), 10 mM NMG-alanine (
). Samples were taken and used for filtration and washing at the times indicated. Control liposomes (
) were also examined without the addition of unlabeled substrates. Error bars are omitted in the case of pTrc99A (
, pTrcAsp; 
) without protein loaded with the NMG-based buffer containing 200 mM alanine were also diluted into the potassium-based buffer with 200 µM [3H]aspartate plus 1 µM valinomycin. Error bars are omitted for Kin[+Val] (