INTRODUCTION

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Periodontal diseases are complex, bacterial-associated inflammatory diseases of the supporting tissue of the teeth. The change from a periodontally healthy site to one undergoing destruction is accompanied by a large increase in the relative abundance of a small number of opportunistic pathogens, including Porphorymonas gingivalis (9, 31). To understand this shift in the species composition of subgingival plaque during disease initiation and progression, information on the ability of periodontopathogenic bacteria to compete for, acquire, and utilize nutrients, especially energy sources, is crucial. P. gingivalis is a black-pigmented, gram-negative, asaccharolytic anaerobe that relies on the fermentation of amino acids for the production of metabolic energy.

To date, the majority of work investigating the utilization of amino acids by P. gingivalis has involved growth studies in a range of media supplied with different amino acid sources. While early reports suggested that P. gingivalis was capable of at least limited growth in defined media with amino acids supplied only in the free form (25, 36), later studies have indicated that P. gingivalis requires a source of peptides for sustained growth (14, 17). The Arg- and Lys-specific extracellular cysteine proteinases of P. gingivalis, RgpA, RgpB, and Kgp, have been demonstrated to be essential for the hydrolysis of proteins and the production of peptides of the desired size to support bacterial growth (28).

The lack of growth of P. gingivalis in defined media with amino acids supplied only in the free form has been taken to indicate that P. gingivalis may not be able to utilize free amino acids due to a lack of suitable transport systems (17). Tang-Larsen et al. (34) have shown, by an indirect method, that P. gingivalis does not utilize the sulfur-containing amino acids cysteine and methionine when supplied in the free form but could metabolize these amino acids when supplied in peptide form. Similar results have also been reported by Takahashi et al. (33), who concluded that the amino acids glutamate and aspartate were metabolized when supplied in peptide form but not when supplied as free amino acids. Although Shah and Gharbia (27) reported uptake of free amino acids by P. gingivalis, this was by the indirect and nonspecific method of measuring changes in conductance due to the disappearance of metabolites from the suspension buffer.

Here we report on the assimilation of free amino acids by P. gingivalis grown in continuous culture and on the first direct study of an amino acid transport system in this bacterium. By characterizing uptake of radiolabeled serine we have identified a sodium ion-driven serine/threonine transporter in P. gingivalis.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. Lyophilized cultures of P. gingivalis W50 (ATCC 53978) were obtained from the culture collection of the School of Dental Science, the University of Melbourne. P. gingivalis was grown anaerobically and maintained on blood agar (Oxoid Blood Agar Base 2 supplemented with 10% defibrinated horse blood) with weekly subculturing. Batch cultures of P. gingivalis were grown at 37°C in 3.7% brain heart infusion (BHI) broth (Oxoid) supplemented with 0.5 g of cysteine-HCl per liter and 5 mg of bovine hemin (Sigma, St. Louis, Mo.) per liter. Cells were harvested by centrifugation (5,500 × g, 20 min, 4°C) during exponential growth phase, as determined by monitoring optical density at a wavelength of 650 nm. Escherichia coli JM109 cells were grown in Luria-Bertani broth or on Luria-Bertani agar plates at 37°C. When required, tetracycline (1.0 µg/ml [for plates] and 0.5 µg/ml [for broth culture]) was added to P. gingivalis cultures and ampicillin (100 µg/ml) was added to E. coli cultures. Culture purity was assessed regularly by Gram stain and colony morphology.

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Amino acid analysis. Free and peptide amino acid concentrations in uninoculated and cell-free spent media from P. gingivalis continuous culture were determined. Liquid phase hydrolysis was carried out according to the method of Barkholt and Jensen (1), and amino acid analysis was carried out on an Amino Quant II amino acid analyzer (Hewlett-Packard, Palo Alto, Calif.). Amino acids were derivatized according to the methods of Blankenship et al. (3). The derivatized amino acids were then separated on a C₁₈ reversed-phase high-performance liquid chromatography column, detected by UV absorbance, and identified by retention time. The amino acid calibration standard used was from Pierce (Rockford, Ill.).

Characterization of serine transport. All transport assays with P. gingivalis W50 cells were conducted in an anaerobe chamber (MK3 Anaerobic workstation; Don Whitley Scientific Ltd., Adelaide, South Australia, Australia) in an atmosphere of 5% hydrogen, 10% carbon dioxide, and 85% nitrogen. Working at 4°C, P. gingivalis cells grown in batch culture were harvested by centrifugation, washed, and suspended in Pga buffer (10.0 mM NaH₂PO₄, 10.0 mM KCl, 2.0 mM citric acid, 1.25 mM MgCl₂, 20.0 µM CaCl₂, 25.0 µM ZnCl₂, 50.0 µM MnCl₂, 5.0 µM CuCl₂, 10.0 µM CoCl₂, 5.0 µM H₃BO₃, 0.1 µM Na₂MoO₄, 10 mM cysteine-HCl with the pH adjusted to 7.5 with 5 M NaOH at 37°C), which was based on the chemically defined medium of Milner et al. (17). The P. gingivalis cell suspension was divided into 2-ml aliquots and stored on ice until use.

Role of cysteine in serine transport. The effect of cysteine concentration on P. gingivalis serine transport was determined by varying the cysteine concentration in the Pga buffer (1 to 20 mM). To determine the role of cysteine it was replaced with either an alternative reducing agent (10 mM dithiothreitol [DTT] or 10 mM -mercaptoethanol) or a combination of both (5 mM cysteine and 5 mM DTT or 10 mM cysteine and 10 mM DTT). The rate of [¹⁴C]serine (25 µM, 20 Ci/mol) transport by P. gingivalis was determined in triplicate in each buffer. Cysteine transport experiments were conducted by adding [¹⁴C]cysteine (1 mM, 5 Ci/mol) {prepared by reducing L-[¹⁴C(U)]cystine with 10 mM DTT} to a P. gingivalis cell suspension in Pga buffer without cysteine but containing 10 mM DTT, essentially as described above.

Construction of a serine transport mutant. A gene potentially encoding the serine/threonine transporter in P. gingivalis was identified by a BLAST search of the unfinished P. gingivalis W83 genome on The Institute for Genomic Research website at http://www.tigr.org using the sequence of an E. coli serine/ threonine transporter (sstT) (open reading frame ygjU, accession number AE000391) (4, 18). The TopPred 2 program (35), provided by Stokholm University and accessed through the ExPASy molecular biology server (http://www.expasy.ch), was used for hydropathy analysis. Sequence alignments were prepared using the CLUSTALW program accessed through the European Bioinformatics Institute website at http://www.ebi.ac.uk.

	RESULTS

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Amino acid assimilation by P. gingivalis in continuous culture. Amino acid analysis was performed on nonhydrolyzed and hydrolyzed samples of uninoculated and cell-free spent growth medium from P. gingivalis continuous culture (Table 1). The medium contained BHI, BSA, and a 4 mM concentration of (each) free serine, threonine, and arginine. These free amino acids were included because initial experiments revealed the disappearance of serine (95%), threonine (100%), and arginine (82%) from P. gingivalis growth medium when these amino acids were present in relatively low concentrations (data not shown). At the higher concentrations effectively all of the free serine (99%), threonine (100%), and arginine (98%) disappeared from the medium (Table 1). The concentration of a number of other free amino acids also decreased, most notably phenylalanine, although the decrease in concentration only represented 53% disappearance of the total available phenylalanine, and the initial concentration was relatively low (Table 1). There was a decrease in the concentration of all amino acids in peptide form from the uninoculated to cell-free spent medium (Table 1). The concentration of free proline markedly increased, even though there was an overall decrease in the amount of proline present in the medium. This could indicate that this residue is removed prior to peptide assimilation or, more likely, that excess proline produced within the cell by peptide hydrolysis is excreted into the medium.

Characterization of serine transport. The disappearance of free serine, threonine, and arginine from the growth medium strongly suggested the presence of transport systems for these amino acids. The transport of the amino acid serine was therefore selected for characterization. P. gingivalis W50 grown in complex media (BHI) was washed and suspended in Pga buffer, and transport experiments were conducted under strictly anaerobic conditions in the presence of the tetrapeptide Gly-Gly-Tyr-Arg as an energy source. The addition of the tetrapeptide Asp-Cys-Glu-Cys as an energy source under identical conditions gave a similar rate of serine uptake (data not shown). In the absence of the tetrapeptide, the accumulation of serine by P. gingivalis was markedly slower, indicating that these cells are able to transport and utilize tetrapeptides as an energy source (Fig. 1). The kinetics of serine transport in P. gingivalis were determined under the same conditions by measuring the uptake rate of [¹⁴C]serine at initial extracellular concentrations between 3 and 200 µM. A double reciprocal plot gave a straight line (R = 0.988) that indicated a single transport system, with an affinity constant for transport (K_t) of 23.8 µM and a V_max of 1.24 nmol/mg (dry weight)/min (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   [14C]serine (25 µM) accumulation by washed cells of P. gingivalis W50 () and the serine transport mutant W50ST () that were energized by tetrapeptide Gly-Gly-Tyr-Arg (100 µM) addition 5 min prior to radiolabeled serine addition. The effect of gramicidin addition () on serine accumulation by P. gingivalis W50 is shown (). Serine accumulation by washed P. gingivalis W50 cells with no tetrapeptide addition is also shown (). All experiments included 10 mM cysteine in the assay buffer. The data points represent the mean values from three separate experiments.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Lineweaver-Burk double reciprocal plot of initial serine uptake rate versus initial serine concentration. Initial uptake rates were determined in washed P. gingivalis cells energized by addition of the tetrapeptide Gly-Gly-Tyr-Arg 5 min prior to [14C]serine addition at pH 7.5, 37°C. Data points represent the average of three to six determinations.

Role of cysteine in serine transport. In all of the experimental data presented above, 10 mM cysteine was present in the Pga buffer. Lowering the concentration of cysteine in the buffer markedly decreased the rate of serine transport. At 5 mM cysteine the rate of serine transport was 50% ± 1% of the control value, and a further lowering of the cysteine concentration to 1 mM effectively abolished serine transport (2% ± 1% of control). Raising the cysteine concentration in the buffer from 10 to 20 mM had little effect on the serine uptake rate. When cysteine was replaced in the Pga buffer with the same concentration (10 mM) of an alternative reducing agent, either DTT or -mercaptoethanol, the rate of serine transport by P. gingivalis was less than 5% of that in the presence of cysteine. These alternative reducing agents did not inhibit the rate of serine uptake, as in the presence of 10 mM DTT and 10 mM cysteine the rate was the same as the rate in the presence of 10 mM cysteine alone; furthermore, the rate with 5 mM DTT and 5 mM cysteine was similar to that with 5 mM cysteine alone.

Identification and insertional inactivation of the P. gingivalis gene, sstT, encoding the serine transporter. A BLAST search of the unfinished P. gingivalis genome using the amino acid sequence of SstT, an E. coli sodium ion-driven serine and threonine transport protein (18), identified a single match. This open reading frame encoded a putative protein containing 412 amino acids with a predicted molecular mass of 44 kDa and an amino acid sequence that was 27% (112 out of 412 amino acids) identical and 64% (262 out of 412 amino acids) similar to the E. coli SstT (Fig. 3). Hydropathy plots of the deduced amino acid sequences of the P. gingivalis and E. coli proteins had similar profiles with nine predicted transmembrane domains. Based on this similarity the P. gingivalis gene was identified as potentially encoding a serine/threonine transporter.

View larger version (60K): [in this window] [in a new window]   FIG. 3.   Alignment of the deduced amino acid sequences of SstT from P. gingivalis (Pg) and E. coli (Ec). Shading indicates identical residues and bold indicates conservative substitutions. Conserved motifs found in the glutamate transport family (30) are boxed.

View larger version (89K): [in this window] [in a new window]   FIG. 4.   Southern blot analysis of P. gingivalis genomic DNA. The DNA of the wild-type W50 (lane 1) and W50ST (lane 2) were digested with BamHI and PstI, and the resulting DNA fragments were subjected to agarose gel electrophoresis and Southern blot hybridization with a tetQ (a) or sstT (b) probe.

	DISCUSSION

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When P. gingivalis was grown in continuous culture in a complex medium containing free amino acids, peptides, and protein, the most highly consumed amino acids (not differentiating between free and peptide forms) were glutamate/glutamine, serine, threonine, aspartate/asparagine, and leucine (Table 1). This is consistent with other studies that collectively show that P. gingivalis preferentially utilizes aspartate/asparagine, glutamate/glutamine, threonine, serine, and possibly arginine and leucine from the culture medium (16, 26, 33). The disappearance of small amounts of free serine, threonine, and arginine has previously been shown for P. gingivalis incubated with tryptone, although those authors concluded that this bacterium did not transport free amino acids (33). We found that nearly all the free serine, threonine, and arginine, even when initially present at relatively high concentrations, disappeared from the P. gingivalis continuous culture growth medium. This led us to investigate the transportation of serine by P. gingivalis.

The ability of P. gingivalis to transport [¹⁴C]serine was determined with washed cells, energized with the tetrapeptide Gly-Gly-Tyr-Arg, under strictly anaerobic conditions. Serine was transported and rapidly accumulated within the cell to levels 8 to 16 times that of the extracellular concentration. Competition assays indicated that the P. gingivalis serine transporter also transported threonine, but none of the other amino acids that we tested were transported by the serine transporter. The transport of these two free amino acids by washed cells of P. gingivalis is consistent with their disappearance from the continuous culture medium. The removal of relatively high concentrations of serine and threonine from the P. gingivalis continuous culture medium indicates that these amino acids may also be being fermented for energy production.

The abolition of serine transport by P. gingivalis in the presence of the ionophore gramicidin indicated that the transport is driven by a transmembrane ion gradient. This was confirmed by the efflux of accumulated serine from P. gingivalis upon the addition of gramicidin. Ion-motive force-driven transporters are reversible and facilitate the transmembrane movement of substrate in either direction dependent on the transmembrane ion-motive force and the transmembrane substrate concentration gradient, as opposed to ATP-driven transport systems that are essentially unidirectional (21). The relatively low affinity of the transporter for serine (K_t = 24 µM) and the low level of intracellular accumulation are both consistent with an ion-motive force-driven transport system. The accumulation of serine within the cell indicates that the magnitude of the ion-motive force driving uptake was 56 to 77 mV under the experimental conditions (7).

A reduction of sodium ion concentration in the cell suspension caused a decrease in the rate of serine transport, indicating that transport was driven by a sodium ion-motive force (Table 2). The transport of amino acids in a number of gram-negative bacteria has been shown to be dependent on a transmembrane sodium gradient (21, 23). Like some amino acid transporters in E. coli and Clostridium fervidum, the serine/ threonine transporter in P. gingivalis is inhibited by high concentrations of sodium under experimental conditions (12, 32).

Insertional inactivation of the P. gingivalis sstT gene prevented serine uptake, confirming the identity of SstT as the P. gingivalis serine transporter. The P. gingivalis SstT appears to belong to the glutamate transport family (30), also known as the DAACS (dicarboxylate/amino acid-Na⁺/H⁺ symporter) family of amino acid transporters (23). This is based on the size of the putative protein, hydropathy profile, sequence similarity, and presence of some of the conserved sequence motifs. Four conserved motifs have been recognized in the glutamate transport family (30) (Fig. 3). Motif A [(ST)(STARK)S(ST)] and motif B [PxGx(TS)xN(ML)DGxx(LI)(FY)] are found in SstT from P. gingivalis, although in motif A the second amino acid is Q in the P. gingivalis protein. There are also slight differences in motif B found across the bacterial serine transporters. A third motif found in many members of the glutamate transport family, motif C, is not present in any of the putative bacterial serine transporters. Motif D is believed to be part of the translocation channel and is generally conserved throughout the family of glutamate transporters, but the exact amino acid sequence depends on substrate specificity (30). Motif D GhhQDSxETALNSSTD (where h is any hydrophobic residue) is found in SstT from E. coli, along with the putative serine transporters (YgjU) from Haemophilus influenzae, Neisseria meningitidis, and Campylobacter jejuni (10, 19, 20). However, in P. gingivalis SstT, the sequence in this region (TMDNFGTACNVTGD) is considerably different from the proposed motif, with only 5 of the proposed 13 amino acyl residues conserved (Fig. 3). Therefore a more generalized consensus sequence may be required to describe this motif across a broader range of bacterial ion-motive force-driven serine transporters.

The transportation of free serine by P. gingivalis was dependent on the concentration of cysteine in the cell suspension buffer. The lack of serine transport in the presence of alternative reducing agents indicates that the effect of cysteine is specific and dependent on the free amino acid. P. gingivalis did not transport free cysteine, indicating that its effects were extracellular. This is consistent with the work of Tang-Larsen et al. (34), who found that P. gingivalis did not metabolize free cysteine. Interestingly, free cysteine has been shown to specifically activate the extracellular proteinases RgpA, RgpB, and Kgp of P. gingivalis, and this activation could not be replicated using alternative reducing agents (2). Serine transport by E. coli SstT has not been reported to be cysteine dependent (18).

In conclusion, we have characterized a cysteine-activated, sodium ion-motive force-driven serine/threonine transporter in P. gingivalis. This is the first direct demonstration of amino acid transport in this asaccharolytic bacterium.