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Applied and Environmental Microbiology, September 2006, p. 6018-6026, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00733-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Halotolerant Cyanobacterium Aphanothece halophytica Contains a Betaine Transporter Active at Alkaline pH and High Salinity

Surasak Laloknam,1 Kimihiro Tanaka,2 Teerapong Buaboocha,1 Rungaroon Waditee,3 Aran Incharoensakdi,1 Takashi Hibino,2 Yoshito Tanaka,2 and Teruhiro Takabe2,3*

Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand,1 Graduate School of Environmental and Human Sciences, Meijo University, Nagoya 468-8502, Japan,2 Research Institute of Meijo University, Nagoya 468-8502, Japan3

Received 30 March 2006/ Accepted 13 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetTA. halophytica) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis, is basic, 9.54, that of BetTA. halophytica is acidic, 4.58. BetTA. halophytica specifically catalyzed the transport of betaine. Choline, {gamma}-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetTA. halophytica. Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetTA. halophytica is a Na+-betaine symporter. Betaine uptake activities of BetTA. halophytica were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetTA. halophytica showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetTA. halophytica were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na+-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetTA. halophytica is the first identified transporter for compatible solutes in cyanobacteria.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Salinity causes a detrimental effect on soil microorganisms and, in general, results in a decreased productivity of crop plants (20, 31). Organisms that thrive in hypersaline environments possess specific mechanisms to adjust their internal osmotic status (1, 12, 19, 30). One such mechanism is the ability to accumulate compatible low-molecular-weight organic solutes such as glycine betaine (12, 19). Another mechanism for adaptation to high salinity is the exclusion of Na+ ions from the cells (1, 29).

Aphanothece halophytica is a halotolerant cyanobacterium which can grow in a wide range of salinity from 0.25 to 3.0 M NaCl and in extreme alkaline conditions up to an external pH of 11.0 (9, 26). Na+/H+ antiporters of alkaliphilic A. halophytica may play a crucial role of Na+ efflux and of cytoplasmic pH homeostasis. Previous studies showed that A. halophytica contains NhaP- and NapA-type Na+/H+ antiporters with novel ion specificities (25, 29), and the overexpression of the NhaP antiporter significantly improved the salt tolerance of the freshwater cyanobacterium Synechococcus, making it capable of growth in seawater (23). Moreover, A. halophytica synthesizes betaine. Although almost all known biosynthetic pathways of betaine are two-step oxidations of choline, A. halophytica synthesizes betaine from glycine by three-step methylation reactions with S-adenosylmethionine acting as the methyl donor (26, 27). Overexpression of glycine methylation genes could confer tolerance for salt stress on both plant and freshwater cyanobacteria (27).

Hitherto, betaine transporters from heterotrophic bacteria had been extensively studied at the molecular level (5, 12, 18). However, information on betaine transporters from photosynthetic organisms is still limited (23, 28). We previously reported the isolation and functional characterization of betaine transporters from a betaine-accumulating mangrove plants (24). Betaine transporters from mangrove catalyze the uptake of betaine and proline. The uptake rates were faster at acidic pH, suggesting that they are H+-betaine symporters. Both NaCl and KCl markedly enhanced betaine uptake rates, with optimum concentrations at 0.5 M. However, a betaine transporter has not yet been reported for cyanobacteria. Here, we isolated a betaine transporter from A. halophytica. It was found that a betaine transporter from A. halophytica specifically catalyzes the uptake of betaine and that uptake activities are very high at alkaline pH. It was also found that the expression of BetTA. halophytica in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942, which normally cannot take up betaine, significantly enhanced the salt tolerance of the cells to the extent that they could grow in seawater supplemented with betaine.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and culture conditions.
A. halophytica cells were grown photoautotrophically in BG11 liquid medium plus 18 mM NaNO3 and Turk Island salt solution at 28°C as previously described (9). Synechococcus sp. strain PCC 7942 cells were grown at 30°C under continuous fluorescent white light (40 µE m–2 s–1) in BG11 liquid medium supplemented with 10 mM HEPES-KOH and bubbled with 3% CO2 (23). Escherichia coli DH5{alpha} cells were grown at 37°C in Luria-Bertani (LB) medium. E. coli MKH13 cells deficient in betT, putPA, proP, and proU genes (7) were grown at 37°C in minimal medium A (MMA) containing 0.2% glucose and ampicillin (50 µg/ml). Radiolabeled [1-14C]betaine (55 mCi/mmol) and L-[U-14C]proline (50 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis) and Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom), respectively.

Construction of expression plasmids.
The betTA. halophytica gene was amplified by PCR using the primer set ApBetTNcoI-F/ApBetTSalI-R. The sequences of ApBetTNcoI-F and ApBetTSalI-R are 5'-TTCCATGGTTAAACAATCAAAACGT-3' and 5'-CAGTCGACTTCATCTTGGGCAAATCG-3', respectively. The amplified fragment was ligated into the EcoRV restriction site of pBSK+ (Stratagene, California) and sequenced. Next, the insert was transferred into the NcoI/SalI sites of pTrcHis2C (Invitrogen, California). The resulting plasmid, pApBetT encoding BetTA. halophytica, was fused in frame to six histidines at the C terminal and transferred first to E. coli DH5{alpha} and then to MKH13 (7) cells.

Complementation test.
For the complementation test on the agar plate, E. coli MKH13 cells transformed with pTrcHis2C and pApBetT were grown overnight at 37°C in MMA (pH 7.0) containing 0.2% glucose and ampicillin (50 µg/ml). Cells were then spread on a 1.4% agar plate containing various concentrations of NaCl, 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside), and 1 mM betaine or 1 mM proline and incubated at 37°C for the indicated times.

Transport assays.
Transport assays were carried out as previously described (24). Briefly, E. coli MKH13 cells transformed with pTrcHis2C and pApBetT were grown overnight at 37°C in MMA (pH 7.0) containing 0.2% glucose and ampicillin (50 µg/ml) and were inoculated into the same fresh medium with an optical density at 620 nm (OD620) of 0.05. IPTG (1 mM) was added to the mid-log-phase cells. After 3 h of incubation, cells were harvested, washed twice, and suspended to an OD620 of 1.0 in the same medium. Subsequently, the cells were incubated with shaking for 5 min at 37°C, and transport was initiated by the addition of 0.1 mM [1-14C]betaine or L-[U-14C]proline. For Km and Vmax determinations, the concentrations of betaine were varied from 0.01 to 5 mM. Glucose was added to a final concentration of 5 mM to energize the cells, and where indicated, salt (NaCl or KCl), sucrose, or sorbitol was added to the indicated concentrations. Cells were collected on 0.2-µm-pore-size cellulose nitrate filters (Advantec MFS, Chiba, Japan). Filters were washed with 3 ml of buffer (the same salinity as assay buffer), and the radioactivity trapped in the cells was measured with a liquid scintillation counter (model 3200C; Aloka Instruments Co., Tokyo, Japan). Competitions for betaine uptake were performed in the presence of a 100-fold molar excess (10 mM) of competitors.

For the growth experiments with E. coli, the E. coli MKH13 cells at the late logarithmic phase were transferred into fresh medium with a starting OD620 of 0.02 and containing various concentrations of NaCl at pH 7.0. For determination of the pH effects on growth, the cells were incubated with MMA containing 0.5 M NaCl at the indicated pHs. Growth of the cells was determined from the OD620.

Overexpression of BetTA. halophytica in a freshwater cyanobacterium.
The expression plasmid for betTA. halophytica, which contains its own promoter, was constructed as previously described (23). The promoter region of betTA. halophytica, 500 bp, was amplified from the genomic DNA of A. halophytica by use of the primer set ApBetTProNcoI-F and ApBetTProNcoI-R. The sequences of ApBetTProNcoI-F and ApBetTProNcoI-R are 5'-AGCCATGGAAGCGGTGCATTACATG-3' and 5'-AACCATGGAATATTTTCTTTGAAAAGA-3', respectively. The amplified fragment was ligated into the NcoI site of pApBetT. After the orientation of the promoter was checked by sequencing, the full length of betTA. halophytica (containing the promoter and His tag) was amplified by the primer set ApBetTProNcoI-F/HisBamHI-R, blunt ended, and ligated into the BamHI-digested site of the E. coli/Synechococcus shuttle vector pUC303-Bm (23). The sequence of HisBamHI-R was 5'-GTGGATCCTCAATGATGATGATGATG-3'. The resulting plasmid was designated as pUC303-ApBetT and used to transform Synechococcus sp. strain PCC 7942 cells (23). For the salt stress experiments, Synechococcus cells were subcultured in BG11 medium as described above and supplemented with 10 µg ml–1 streptomycin. Cells at the late logarithmic phase were transferred into fresh medium containing various concentrations of NaCl (0 to 0.5 M) or seawater.

Other methods.
The nucleotide sequences were determined using an ABI310 genetic analyzer (Applied Biosystems, Foster City, CA). Cellular ions were determined with a Shimadzu PIA-1000 personal ion analyzer. Protein was determined by Lowry's method as described previously (9, 25). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were carried out as described previously (8, 25). An antibody raised against the six-His tag was obtained from R&D Systems (Minneapolis). The hydropathy profile of proteins was predicted by a computer-assisted procedure according to the method of Kyte and Doolittle (13). The possible transmembrane (TM) structure of BetTA. halophytica was predicted by use of the computer program TopPredII (10).

Nucleotide sequence accession number.
Nucleotide sequence data for BetTA. halophytica are available in the DDBJ database under the accession number AB250783.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of betaine transporter gene from A. halophytica.
Hitherto, betaine transporters had not been reported for any cyanobacterium. Here, one open reading frame similar to the gene encoding the betaine transporter OpuD from Bacillus subtilis was found from the shotgun clones of A. halophytica. The gene was isolated by PCR amplification, and its sequence was determined. The predicted gene product (BetTA. halophytica) consists of 565 amino acids with a calculated molecular mass of 64,655 Da. A ClustalW analysis showed that BetTA. halophytica has 12 TM segments and could be classified as a member of the betaine-choline-carnitine transporter (BCCT) family, of which one common functional feature is that its members transport molecules with a quaternary ammonium group. BetTA. halophytica exhibited the highest level of similarity (53%) to ButA from a moderate halophilic lactic acid bacterium, Tetragenococcus halophila (2), lower levels of similarity to BetP from Corynebacterium glutamicum (40%) (16) and to OpuD from Bacillus subtilis (40%) (11), and very low levels of similarity to betaine/proline transporters ProP (29%) from E. coli (6) and AmT1 (28%) from mangrove (24). Alignment of eight transporters, BetTA. halophytica from A. halophytica, ProP from E coli (6), BetP (16) and EctP (17) from C. glutamicum, ButA from T. halophila (2), OpuD from B. subtilis (11), BetS from Sinorhizobium meliloti (3), and BetL from Listeria monocytogenes (22), showed a highly conserved region in TM8, (347 WTVFYWGWWISWSPFVGMFIA 367). Thirteen amino acid residues in this region are conserved among the eight proteins. Interestingly, a loop region connecting TM8 and TM9 is also highly conserved.

Expression of BetTA. halophytica in E. coli and its complementation of Na+-sensitive phenotype.
To examine the functional properties of BetTA. halophytica, its gene was expressed in E. coli mutant MKH13 cells in which betT, putPA, proP, and proU genes had been deleted. As shown in Fig. 1, BetTA. halophytica could be expressed in MKH13 cells. The expression level of BetTA. halophytica was almost independent of the concentration of NaCl in the growth medium. The addition of IPTG slightly increased the expression level of BetTA. halophytica.


Figure 1
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  		FIG. 1. Western blot analysis of BetTA. halophytica in salt-sensitive E. coli MKH13 cells. BetTA. halophytica-expressing MKH13 cells were grown at 37°C in MMA (pH 7.0) containing 0.2% glucose, ampicillin (50 µg/ml), and indicated concentrations of NaCl until the optical density at 620 nm reached 0.3. Then, the indicated concentrations of IPTG were added. After 5 h of incubation, the cells were harvested and sonicated, and membrane fractions were used for Western blotting.

 
MKH13 cells transformed with the control plasmid could not grow on an agar plate containing NaCl at 0.75 M or higher at pH 7.0 regardless of the presence or absence of 1 mM betaine (data not shown). The cells transformed with betTA. halophytica could grow in the medium containing 0.75 M NaCl with added betaine. Similar results were obtained when experiments were performed at pH 9.0 but not at pH 5.0.

Since the MKH13 cells do not contain the proline transporters ProP and ProU (2, 12, 17), we therefore tested whether BetTA. halophytica could allow the MKH13 cells to grow under high salinity in the presence of proline. The MKH13 cells transformed with betTA. halophytica could not grow in MMA containing 0.75 M NaCl and 1 mM proline with or without 1 mM IPTG (data not shown). These results suggest that BetTA. halophytica could take up betaine but not proline at neutral or alkaline pHs.

Growth of MKH13 cells in the minimal medium at various salinities and pHs.
The E. coli MKH13 cells are unable to grow in high-osmolarity medium containing betaine due to the lack of a betaine transport system as well as betaine synthesis genes (5, 7, 12). Figure 2A shows that in the absence of NaCl, both control MKH13 cells and BetTA. halophytica-expressing cells exhibited similar growth patterns regardless of the presence or absence of choline and betaine. The increase of NaCl concentrations resulted in slow growth of all the cells. However, the growth rate of BetTA. halophytica-expressing cells supplemented with 1 mM betaine was higher than that of the other cells. No enhancement of growth was observed when betaine was replaced by choline or proline. The addition of betaine to the control cells failed to promote growth under high-salinity conditions. All these data indicate that BetTA. halophytica is involved in the uptake of betaine by the MKH13 cells, thus allowing their growth under high salinity.


Figure 2
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  		FIG. 2. Effects of NaCl and pH on the growth of E. coli MKH13 cells expressing BetTA. halophytica. The MKH13 cells transformed with pApbetT or pTrcHis2C were grown in MMA containing the indicated concentrations of NaCl at pH 7.0 (A) or in the medium at indicated pHs (B). Betaine or choline (1 mM) was added when indicated. Growth was monitored by the absorbance at 620 nm. The pH values were adjusted by KOH or 2-(N-morpholino)ethanesulfonic acid. Open symbols represent the vector-expressing cells, whereas closed symbols represent BetTA. halophytica-expressing cells. Shapes: circles, without supplementation; squares, supplemented with betaine (1 mM); triangles, supplemented with choline (1 mM). Each value shows the average of three independent measurements.

 
Since the complementation of the salt-sensitive phenotype of MKH13 was observed at pHs 7.0 and 9.0 but not at pH 5.0, we examined the growth of MKH13 cells in MMA containing 0.5 M NaCl at various pHs. The growth rate of control MKH13 cells was very slow at pH 5.0 (Fig. 2B), increased upon the increase of pH up to pH 7.0, and decreased at pH 8.0 up to pH 10.0. All cells showed similar pH-dependent growth patterns. The growth rate of BetTA. halophytica-expressing cells supplemented with 1 mM betaine was higher than that of other cells. Under an extreme alkaline condition, pH 10, only BetTA. halophytica-expressing cells supplemented with 1 mM betaine could grow (Fig. 2B). These data suggest that BetTA. halophytica is active even at alkaline pH for the uptake of betaine by the MKH13 cells, thus allowing their growth at alkaline pH.

Kinetic properties of BetTA. halophytica in MKH13 cells.
To examine directly the transporter activity of BetTA. halophytica, we determined the kinetics of this transporter. No measurable uptake of [1-14C]betaine was observed for the MKH13 cells transformed with pTrcHis2C (Fig. 3A). The MKH13 cells transformed with betTA. halophytica could not take up betaine in the absence of NaCl (Fig. 3A). The addition of NaCl (0.25 M and 0.5 M) significantly increased the uptake of betaine (Fig. 3A). Vmax values for uptake in the presence of 0.25 M and 0.5 M NaCl were 10.8 and 18.2 nmol · betaine min–1 · mg protein–1, respectively, while their Km values were 115 µm and 128 µM, respectively (Fig. 3B). No measurable uptake of [1-14C]proline was observed for the MKH13 cells transformed with betTA. halophytica under any condition (data not shown).


Figure 3
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  		FIG. 3. Kinetics of betaine uptake by BetTA. halophytica in MKH13 cells. (A) Time course of betaine uptake at pH 7.0. Concentrations of NaCl in the assay media were as follows (symbols are indicated in parentheses): 0.0 M (open circles), 0.25 M (open squares), and 0.5 M (closed circles). ApBetT, BetTA. halophytica. (B) Double reciprocal plots of betaine transport kinetics by BetTA. halophytica-expressing MKH13 cells assayed in the presence of 0.25 M NaCl (upper panel) or 0.5 M NaCl (lower panel). Each value shows the average of three independent measurements. V and [S] in panel B represent the betaine uptake rate and the concentration of betaine, respectively.

 
pH dependence of betaine uptake by BetTA. halophytica in MKH13 cells.
We examined the pH dependence of betaine uptake by BetTA. halophytica in MKH13 cells. As shown in Fig. 4A, MKH13 cells transformed with betTA. halophytica preferentially took up [1-14C]betaine at alkaline pHs. The Vmax value at pH 8.5 was about sevenfold higher than that at pH 5.5, and the Km value at pH 8.5 was about threefold lower than that at pH 5.5 (Table 1). The optimum pH for betaine uptake was 9.0 (Fig. 4B). For proline uptake, no measurable level was observed for the MKH13 cells transformed with betTA. halophytica at any pH examined (data not shown).


Figure 4
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  		FIG. 4. pH dependence of betaine uptake by BetTA. halophytica in MKH13 cells. (A) Kinetics of betaine uptake by BetTA. halophytica-expressing MKH13 cells. pH values in assay media are 5.5 (squares), 7.0 (circles), or 8.5 (triangles). The uptake medium contained 0.5 M NaCl. The values of pH were adjusted by KOH or 2-(N-morpholino)ethanesulfonic acid. (B) Effect of pH on betaine uptake by BetTA. halophytica in MKH13 cells. Each value shows the average of three independent measurements.

 

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  		TABLE 1. Kinetic parameters for betaine uptake by BetTA. halophytica-expressing MKH13 cells at three different pHs

 
These properties of BetTA. halophytica are quite different from those of the mangrove betaine transporters AmT1 and AmT2 (24). Mangrove AmT1 and AmT2 could transport both betaine and proline, whereas BetTA. halophytica could not transport proline (Fig. 5A and B). Betaine uptake by AmT1 and AmT2 increased with the decrease of pH, with the optimum pH being around 6.0 (Fig. 5C). At pH 7.0, the betaine uptake rate by BetTA. halophytica was about twofold higher than that by AmT1 and AmT2. Taken together, these results indicate that BetTA. halophytica is a betaine-specific transporter active at alkaline pH.


Figure 5
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  		FIG. 5. Comparison of betaine and proline uptake by BetTA. halophytica, AmT1, and AmT2 in MKH13 cells. (A and B) Time course of betaine (A) or proline (B) uptake by MKH13 cells. MKH13 cells were transformed with vector (open circles), AmT1 (closed squares), AmT2 (open squares), or BetTA. halophytica (closed circles). (C) pH dependence of betaine uptake by BetTA. halophytica, AmT1, and AmT2 in MKH13 cells at pH 7.0 and 0.5 M NaCl. Black bars, BetTA. halophytica; gray bars, AmT1; white bars, AmT2. Each value shows the average of three independent measurements.

 
Competitions for betaine uptake mediated by BetTA. halophytica.
To obtain information on substrate specificity, we performed competition experiments. Consistent with the betaine uptake experiments (Fig. 4A), the [1-14C]betaine uptake by BetTA. halophytica in MKH13 cells was inhibited by about 80% or 90% when 100-fold "cold" betaine was included in uptake medium that contained 0.25 M NaCl or 0.5 M NaCl, respectively (data not shown). However, choline slightly inhibited betaine uptake by about 20% or 10% when the uptake medium contained 0.25 M NaCl or 0.5 M NaCl, respectively. Betaine aldehyde also exhibited similarly weak inhibition. By contrast, no other compound tested, including {gamma}-aminobutyric acid, proline, glutamate, aspartate, glutamine, asparagine, glycine, sarcosine, dimethylglycine, lysine, histidine, alanine, leucine, isoleucine, serine, cysteine, threonine, valine, phenylalanine, tryptophan, and methionine, inhibited the betaine uptake by BetTA. halophytica (data not shown). These results strongly suggest that the BetTA. halophytica is a transporter specific for betaine.

Sodium is required for betaine uptake by BetTA. halophytica.
To obtain information on the cosubstrate, we examined the uptake of Na+, K+, and betaine by MKH13 cells. Figure 6A shows that upon the addition of NaCl, uptake of Na+ occurred in all cells. However, the BetTA. halophytica-expressing cells supplemented with betaine accumulated Na+ at a higher rate than other cells. This suggests that BetTA. halophytica plays a role in the rapid accumulation of Na+. Moreover, the lower panel of Fig. 6A shows that only those cells expressing BetTA. halophytica and supplemented with betaine could accumulate betaine. These results suggest that Na+ was taken up together with betaine. The molar ratio of Na+ and betaine uptake was approximately equal to 1 (Fig. 6A). Figure 6B shows that upon the addition of KCl, the intracellular K+ level increased with time. However, the accumulation levels of K+ were quite similar in all cells. By contrast, no betaine uptake activity could be detected for any of the cells upon the addition of KCl (Fig. 6B, lower panel). Figure 6C shows that the uptake of both Na+ and betaine increased in a concentration-dependent manner with respect to NaCl and betaine. We also examined the effects of different cations and anions. Li+, Cs+, Rb+, NH4+, Ca2+, and Mg2+ could not replace Na+ (data not shown). By contrast, NO3– and H2PO4– could replace Cl– (data not shown). Sucrose and sorbitol were also ineffective for betaine uptake (data not shown). These results strongly suggest that the BetTA. halophytica is a Na+-betaine symporter.


Figure 6
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  		FIG. 6. Uptake of Na+, K+, and betaine by BetTA. halophytica in MKH13 cells. (A) Time course of Na+ and betaine uptake upon the addition of 0.5 M NaCl to the assay medium. (B) Time course of K+ and betaine uptake upon the addition of 0.5 M KCl to the assay medium. MKH13 cells were transformed with vector (circles) or BetTA. halophytica (squares). Open symbols, without supplementation; closed symbols, supplemented with 1 mM betaine. (C) Uptake of Na+ and betaine by BetTA. halophytica in MKH13 cells under various concentrations of NaCl and betaine. Concentrations of NaCl were 0.1 (white bars), 0.3 (gray bars), and 0.5 (black bars) M. Each value shows the average of three independent measurements.

 
Betaine uptake in A. halophytica cells.
Next, we examined betaine uptake by A. halophytica cells. A. halophytica cells were grown in medium containing 0.5 M NaCl and harvested. After the cells were washed, betaine uptake was measured at various salinity conditions. As shown in Fig. 7A, A. halophytica cells could actively take up betaine in assay medium containing 0.5 M or 2.0 M NaCl. The Vmax value for betaine uptake for cells supplemented with 2.0 M NaCl was almost twofold higher than that for cells supplemented with 0.5 M NaCl (Table 2). However, the Km values for betaine uptake were slightly affected by the increase of salinity.


Figure 7
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  		FIG. 7. Betaine uptake by A. halophytica cells. (A) Time course of betaine uptake. The concentrations of NaCl in the assay media are 0.5 M and 2.0 M. (B) Kinetics of betaine uptake at three different pH values. The pHs in the assay media were 5.5 (squares), 7.0 (circles), and 8.5 (triangles). (C) pH dependence of betaine uptake. The assay medium contained 0.5 M NaCl at the indicated pH. Each value shows the average of three independent measurements.

 

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  		TABLE 2. Kinetic parameters for betaine uptake by A. halophytica cells at two different concentrations of NaCl

 
The pH dependence of betaine uptake by A. halophytica was also similar to that for MKH13 cells. With increasing pH, the Km values were decreased, while the Vmax values were increased (Fig. 7B and Table 3). The optimum pH for betaine uptake was 9.0 (Fig. 7C). Although we cannot rule out the existence of other betaine transporters with similar pH and salt dependence, these results suggest that BetTA. halophytica is a major betaine transporter in A. halophytica cells.


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  		TABLE 3. Kinetic parameters for betaine uptake by A. halophytica cells at three different pHs

 
Transformation of Synechococcus sp. strain PCC 7942 with betTA. halophytica.
To examine the functional properties of BetTA. halophytica, a freshwater cyanobacterium, Synechococcus sp. strain PCC 7942, was transformed with the betTA. halophytica gene. The cells transformed with vector pUC303 failed to take up betaine regardless of the presence or absence of NaCl (Fig. 8A, upper panel). This result suggests that Synechococcus cells lack a betaine transporter. It should be mentioned that we previously observed choline uptake in wild-type Synechococcus cells (14). No measurable uptake of [1-14C]betaine was observed for the Synechococcus cell transformed with betTA. halophytica when the growth medium lacked NaCl (Fig. 8A, lower panel). The betaine uptake increased with increasing concentrations of NaCl from 0.1 M to 0.3 M. The Km values for betaine uptake were not affected by the salinity (284 µM at 0.1 M NaCl compared to 253 µM at 0.3 M NaCl). The Vmax values at 0.1 and 0.3 M NaCl were 1.2 and 1.6 nmol · min–1 · mg protein–1, respectively. The optimum pH for betaine uptake was 9.0 (Fig. 8B). These properties are similar to those for MKH13 cells transformed with betTA. halophytica.


Figure 8
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  		FIG. 8. Kinetics (A) and pH dependence (B) of betaine uptake by BetTA. halophytica in Synechococcus sp. strain PCC 7942 cells. (A) Time course of betaine uptake in Synechococcus sp. strain PCC 7942 cells transformed with pUC303 or pUC303-Ap-BetT assayed in the absence (open circles) or the presence of 0.1 M NaCl (open squares) or 0.3 M NaCl (closed circles) at pH 7.0. (B) pH dependence of betaine uptake rate. The uptake medium contained 0.1 M NaCl. Each value shows the average of three independent measurements.

 
Overexpression of BetTA. halophytica conferred salt tolerance on a freshwater cyanobacterium, Synechococcus sp. strain PCC 7942.
We examined the salt tolerance of Synechococcus sp. strain PCC 7942 cells at pH 7.0. As shown in Fig. 9A, the growth rates of pUC303-transformed and betTA. halophytica-transformed cells were almost the same when the cells were grown in BG11 medium lacking NaCl. The presence of 0.3 M NaCl in BG11 medium significantly reduced the growth rate of the control cells but only slightly inhibited the growth of the transformant cells supplemented with betaine (Fig. 9B). Only the transformant cells supplemented with betaine could grow at a higher concentration of NaCl or even in seawater (Fig. 9C and D). Interestingly, the Western blot analysis shows that BetTA. halophytica protein was detected in the plasma membrane fractions and that its level increased with increased levels of salinity (Fig. 9E). These results suggest an important role for BetTA. halophytica in salt tolerance in A. halophytica.


Figure 9
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  		FIG. 9. Salt tolerance of cells of Synechococcus sp. strain PCC 7942 expressing BetTA. halophytica. Cells of freshwater Synechococcus sp. strain PCC 7942 transformed with vector (pUC303) or BetTA. halophytica were grown in BG11 medium containing 0.0 M NaCl (A), 0.3 M NaCl (B), or 0.5 M NaCl (C) or in seawater (D) with the supplementation of 1 mM choline or betaine. (E) Immunoblot analysis of BetTA. halophytica-expressing cells at indicated concentrations of NaCl. Thylakoid membrane (TM) and plasma membrane (PM) fractions were prepared from wild-type and transformant cells. In each lane, 50-µg portions of membrane proteins were loaded. Transporter proteins were detected using an antibody raised against the His tag. Each value shows the average of three independent measurements. ApBetT, BetTA. halophytica.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we show that a halotolerant cyanobacterium, A. halophytica, has at least one BCCT-type betaine transporter. Based on the data showing that the choline, betaine, and proline transport-deficient E. coli MKH13 mutant cells became Na+ tolerant by transformation with betTA. halophytica (Fig. 2) and that the transformed cells exhibited uptake of betaine (Fig. 3 to 6), BetTA. halophytica could be assigned as a transporter specific for betaine. The requirement for Na+ but not for other cations, such as K+, Li+, Rb+, Ca2+, and Mg2+, for the uptake of betaine by BetTA. halophytica (Fig. 3A) suggests that BetTA. halophytica is a Na+-betaine symporter. This suggestion is also supported by the results showing that the addition of NaCl increased Na+ uptake concomitantly with the increase of betaine uptake with a similar mole ratio (Fig. 6A).

The interesting finding in this study is the high betaine transport activity, particularly at alkaline pHs. To our knowledge, an alkaline optimal pH, such as 9.0, has not previously been reported. For alkaliphiles, active uptake of betaine at alkaline pHs would be beneficial for the survival of cells under these severe conditions. H+ uptake by the Na+/H+ antiporter is important to keep the cytoplasmic pH neutral, and the Na+/H+ antiporter could extrude Na+ out of the cell. To maintain H+ homeostasis at alkaline pH, a reentry route for Na+ to be a substrate for the Na+/H+ antiporter is required (15). The Na+-betaine symporter could be a reentry route. Cooperation of the Na+-betaine symporter and the Na+/H+ antiporter would lead to the adjustment of pH homeostasis as well as the accumulation of betaine essential for osmotic balance. This strategy is particularly important for A. halophytica to survive under salt stress and alkaline pHs.

The pH dependence of the betaine transporter from A. halophytica is quite different from that of the mangrove betaine transporters AmT1 and AmT2 (24). Betaine uptake by AmT1 and AmT2 decreased, with an increase of pH in the range from 6 to 9 and with an optimum pH of around 6.0 (Fig. 5C). By contrast, in the same pH range of 6 to 9, an increase of pH resulted in an enhanced betaine uptake by BetTA. halophytica-expressing cells. Although the importance of a charged C-terminal domains of BetP (21) and ProP (28) for osmosensing have been demonstrated previously, no structural information on the pH sensing of the betaine transporters is available. Therefore, it would be interesting to identify the amino acid residues involved in the pH sensing of BetTA. halophytica and AmT.

The substrate specificity of the betaine transporter in A. halophytica also differs from the substrate specificity of that in mangrove. The former transporter is highly specific for betaine, whereas the latter can transport both betaine and proline (24). For the cation requirement, both NaCl and KCl could increase betaine uptake mediated by mangrove AmT1, whereas only NaCl could increase betaine uptake mediated by BetTA. halophytica.

Figure 8A shows that Synechococcus cells transformed with vector pUC303 failed to take up betaine even in the presence of NaCl. Since the transformation of Synechococcus sp. strain PCC 7942 cells is relatively simple, Synechococcus cells might be used as a model system to study the effects of betaine synthesis and betaine transport on the stress tolerance of cyanobacteria.

It is well demonstrated that the betaine transporter is essentially regulated by the activation of the transporter (18). However, Fig. 9E shows that the level of BetTA. halophytica protein increased with an increase in salinity. This increased level of BetTA. halophytica in the halotolerant cyanobacterium A. halophytica might reflect the importance of the betaine transporter in maintaining osmotic balance under salt stress. Molecular mechanisms of this osmoregulation remain to be clarified.

Figure 9C and D show that overexpression of BetTA. halophytica could confer salt tolerance to Synechococcus cells in such a way that they are capable of growth in medium containing 0.5 M NaCl and even in seawater supplemented with betaine. The import of betaine present in the medium by BetTA. halophytica plays a key role in sustaining the growth of Synechococcus cells under high-salinity conditions. Thus far, attempts to improve salt tolerance by the genetic transformation of the betaine transporter have not been reported, except for one recent paper (4). The Bradyrhizobium japonicum strain USDA110, of the family Rhizobiaceae, was transformed with the betS gene of Sinorhizobium meliloti, which encodes a major BCCT-type betaine transporter. Whereas betaine transport was absent in the USDA110 strain, salt-treated transformed cells accumulated large amounts of betaine (4). The transformant could grow in medium containing 80 mM NaCl but not in medium containing 100 mM NaCl, whereas the wild-type USDA110 strain could not grow at 80 mM NaCl. Since the wild-type Synechococcus strain PCC 7942 cells could not grow in medium containing 380 mM NaCl (14, 23), the present study shows a significant improvement in salt tolerance, mediated by BetTA. halophytica. The overexpression of the NhaPA. halophytica antiporter from A. halophytica (23) and the overexpression of glycine methylation genes from A. halophytica (27) significantly improved the salt tolerance of the freshwater cyanobacterium Synechococcus, making it capable of growth in seawater. Therefore, overexpression of BetTA. halophytica, NhaPA. halophytica, and methylation genes together in cyanobacteria is a model that is both interesting and suitable for understanding the limiting factors hindering the improvement of salt tolerance in any organism. Recently, we showed that the overexpression of glycine methylation genes significantly improves the stress tolerance of a model plant, Arabidopsis (27). Therefore, the introduction of a set of genes with multiple functions into important crop plants, such as rice, is also an interesting challenge for the development of salt-tolerant crop plants.


   ACKNOWLEDGMENTS
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, the High-Tech Research Center of Meijo University. S.L. and A.I. were supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program. A.I. was supported by the Thai government through the Thailand-Japan Technology Transfer Project. T.B. was supported by the Thailand Research Fund (TRF) New Researchers Grant (TRG4580087) and by the Thai government through the Thailand-Japan Technology Transfer Project.

We thank Eiko Tsunekawa for her expert technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Research Institute of Meijo University, Tenpaku-ku, Nagoya 468-8502, Japan. Phone: 81-52-838-2277. Fax: 81-52-832-1545. E-mail: takabe{at}ccmfs.meijo-u.ac.jp. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, September 2006, p. 6018-6026, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00733-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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