Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Laloknam, S. Articles by Takabe, T. Search for Related Content PubMed PubMed Citation Articles by Laloknam, S. Articles by Takabe, T. Agricola Articles by Laloknam, S. Articles by Takabe, T.

 Previous Article  |  Next Article 

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 Takabe^2,3*

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

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 (BetT_{A. 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 BetT_{A. halophytica} is acidic, 4.58. BetT_{A. halophytica} specifically catalyzed the transport of betaine. Choline, -aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT_{A. halophytica}. Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT_{A. halophytica} is a Na⁺-betaine symporter. Betaine uptake activities of BetT_{A. halophytica} were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT_{A. 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 BetT_{A. 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. BetT_{A. halophytica} is the first identified transporter for compatible solutes in cyanobacteria.

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 BetT_{A. 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.

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 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 betT_{A. 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 BetT_{A. halophytica}, was fused in frame to six histidines at the C terminal and transferred first to E. coli DH5 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 K_m and V_max 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 BetT_{A. halophytica} in a freshwater cyanobacterium.
The expression plasmid for betT_{A. halophytica}, which contains its own promoter, was constructed as previously described (23). The promoter region of betT_{A. 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 betT_{A. 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 BetT_{A. halophytica} was predicted by use of the computer program TopPredII (10).

Nucleotide sequence accession number.
Nucleotide sequence data for BetT_{A. halophytica} are available in the DDBJ database under the accession number AB250783.

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 (BetT_{A. halophytica}) consists of 565 amino acids with a calculated molecular mass of 64,655 Da. A ClustalW analysis showed that BetT_{A. 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. BetT_{A. 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, BetT_{A. 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 BetT_{A. halophytica} in E. coli and its complementation of Na⁺-sensitive phenotype.
To examine the functional properties of BetT_{A. 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, BetT_{A. halophytica} could be expressed in MKH13 cells. The expression level of BetT_{A. halophytica} was almost independent of the concentration of NaCl in the growth medium. The addition of IPTG slightly increased the expression level of BetT_{A. halophytica}.

View larger version (19K): [in this window] [in a new window]

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 betT_{A. 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 BetT_{A. halophytica} could allow the MKH13 cells to grow under high salinity in the presence of proline. The MKH13 cells transformed with betT_{A. 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 BetT_{A. 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 BetT_{A. 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 BetT_{A. 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 BetT_{A. halophytica} is involved in the uptake of betaine by the MKH13 cells, thus allowing their growth under high salinity.

View larger version (38K): [in this window] [in a new window]

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 BetT_{A. halophytica}-expressing cells supplemented with 1 mM betaine was higher than that of other cells. Under an extreme alkaline condition, pH 10, only BetT_{A. halophytica}-expressing cells supplemented with 1 mM betaine could grow (Fig. 2B). These data suggest that BetT_{A. 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 BetT_{A. halophytica} in MKH13 cells.
To examine directly the transporter activity of BetT_{A. 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 betT_{A. 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). V_max 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 K_m 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 betT_{A. halophytica} under any condition (data not shown).

View larger version (21K): [in this window] [in a new window]

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 BetT_{A. halophytica} in MKH13 cells.
We examined the pH dependence of betaine uptake by BetT_{A. halophytica} in MKH13 cells. As shown in Fig. 4A, MKH13 cells transformed with betT_{A. halophytica} preferentially took up [1-¹⁴C]betaine at alkaline pHs. The V_max value at pH 8.5 was about sevenfold higher than that at pH 5.5, and the K_m 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 betT_{A. halophytica} at any pH examined (data not shown).

View larger version (20K): [in this window] [in a new window]

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.

View this table: [in this window] [in a new window]

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

These properties of BetT_{A. 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 BetT_{A. 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 BetT_{A. halophytica} was about twofold higher than that by AmT1 and AmT2. Taken together, these results indicate that BetT_{A. halophytica} is a betaine-specific transporter active at alkaline pH.

View larger version (12K): [in this window] [in a new window]

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 BetT_{A. 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 BetT_{A. 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 -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 BetT_{A. halophytica} (data not shown). These results strongly suggest that the BetT_{A. halophytica} is a transporter specific for betaine.

Sodium is required for betaine uptake by BetT_{A. 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 BetT_{A. halophytica}-expressing cells supplemented with betaine accumulated Na⁺ at a higher rate than other cells. This suggests that BetT_{A. halophytica} plays a role in the rapid accumulation of Na⁺. Moreover, the lower panel of Fig. 6A shows that only those cells expressing BetT_{A. 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 BetT_{A. halophytica} is a Na⁺-betaine symporter.

View larger version (17K): [in this window] [in a new window]

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 V_max 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 K_m values for betaine uptake were slightly affected by the increase of salinity.

View larger version (12K): [in this window] [in a new window]

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.

View this table: [in this window] [in a new window]

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 K_m values were decreased, while the V_max 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 BetT_{A. halophytica} is a major betaine transporter in A. halophytica cells.

View this table: [in this window] [in a new window]

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

Transformation of Synechococcus sp. strain PCC 7942 with betT_{A. halophytica}.
To examine the functional properties of BetT_{A. halophytica}, a freshwater cyanobacterium, Synechococcus sp. strain PCC 7942, was transformed with the betT_{A. 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 betT_{A. 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 K_m 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 V_max 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 betT_{A. halophytica}.

View larger version (12K): [in this window] [in a new window]

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 BetT_{A. 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 betT_{A. 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 BetT_{A. 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 BetT_{A. halophytica} in salt tolerance in A. halophytica.

View larger version (18K): [in this window] [in a new window]

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.

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 betT_{A. halophytica} (Fig. 2) and that the transformed cells exhibited uptake of betaine (Fig. 3 to 6), BetT_{A. 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 BetT_{A. halophytica} (Fig. 3A) suggests that BetT_{A. 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 BetT_{A. 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 BetT_{A. 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 BetT_{A. 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 BetT_{A. halophytica} protein increased with an increase in salinity. This increased level of BetT_{A. 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 BetT_{A. 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 BetT_{A. 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 BetT_{A. halophytica}. The overexpression of the NhaP_{A. 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 BetT_{A. halophytica}, NhaP_{A. 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.

 
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.

 
* 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Apse, M. P., and E. Blumwald. 2002. Engineering salt tolerance in plants. Curr. Opin. Biotechnol. 13:146-150.[CrossRef][Medline]
2
2. Baliarda, A., H. Robert, M. Jebbar, C. Blanco, and C. Le Marrec. 2003. Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila. Curr. Microbiol. 47:347-351.[CrossRef][Medline]
3
3. Boscari, A., K. Mandon, L. Dupont, M. C. Poggi, and D. Le Rudulier. 2002. BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti. J. Bacteriol. 184:2654-2663.[Abstract/Free Full Text]
4
4. Boscari, A., K. Mandon, M. C. Poggi, and D. Le Rudulier. 2004. Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110. Appl. Environ. Microbiol. 70:5916-5922.[Abstract/Free Full Text]
5
5. Bremer, E., and R. Krämer. 2000. Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes in bacteria, p. 79-97. In G. Storz and R. Hengge-Aronis (ed.), Bacterial stress responses. ASM Press, Washington, D.C.
6
6. Culham, D. E., B. Lasby, A. G. Marangoni, J. L. Milner, B. A. Steer, R. W. van Nues, and J. M. Wood. 1993. Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP. J. Mol. Biol. 229:268-276.[CrossRef][Medline]
7
7. Haardt, M., B. Kempf, E. Faat, and E. Bremer. 1995. The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12. Mol. Gen. Genet. 246:783-786.[CrossRef][Medline]
8
8. Hamada, A., T. Hibino, T. Nakamura, and T. Takabe. 2001. Na⁺/H⁺ antiporter from Synechocystis sp. PCC 6803, homologous to SOS1, contains an aspartic residue and long C-terminal tail important for the carrier activity. Plant Physiol. 125:437-446.[Abstract/Free Full Text]
9
9. Hibino, T., N. Kaku, H. Yoshikawa, T. Takabe, and T. Takabe. 1999. Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions. Plant Mol. Biol. 40:409-418.[CrossRef][Medline]
10
10. Hofmann, K., and W. Stoffel. 1992. PROFILEGRAPH: an interactive graphical tool for protein sequence analysis. Comput. Appl. Biosci. 8:331-337.[Abstract/Free Full Text]
11
11. Kappers, R. M., B. Kempf, and E. Bremer. 1996. Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD. J. Bacteriol. 178:5071-5079.[Abstract/Free Full Text]
12
12. Kempf, B., and E. Bremer. 1998. Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol. 170:319-330.[CrossRef][Medline]
13
13. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132.[CrossRef][Medline]
14
14. Nomura, M., M. Ishitani, T. Takabe, A. K. Rai, and T. Takabe. 1995. Synechococcus sp. PCC 7942 transformed with Escherichia coli bet genes produces glycine betaine from choline and acquires resistance to salt stress. Plant Physiol. 107:703-708.[Abstract]
15
15. Padan, E., E. Bibi, M. Ito, and T. A. Krulwich. 2005. Alkaline pH homeostasis in bacteria: new insights. Biochim. Biophys. Acta 1717:67-88.[Medline]
16
16. Peter, H., A. Burkovski, and R. Kramer. 1996. Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine. J. Bacteriol. 178:5229-5234.[Abstract/Free Full Text]
17
17. Peter, H., B. Weil, A. Burkovski, R. Kramer, and S. Morbach. 1998. Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP. J. Bacteriol. 180:6005-6012.[Abstract/Free Full Text]
18
18. Poolman, B., J. J. Spitzer, and J. M. Wood. 2004. Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions. Biochim. Biophys. Acta 1666:88-104.[Medline]
19
19. Rentsch, M., B. Hirner, E. Schmelzer, and W. B. Frommer. 1996. Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. Plant Cell 8:1437-1446.[Abstract]
20
20. Rontein, D., G. Basset, and A. D. Hanson. 2002. Metabolic engineering of osmoprotectant accumulation in plants. Metab. Eng. 4:49-56.[CrossRef][Medline]
21
21. Rubenhagen, R., H. Roensch, H. Jung, R. Kramer, and S. Morbach. 2000. Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes. J. Biol. Chem. 275:735-741.[Abstract/Free Full Text]
22
22. Sleator, R. D., C. G. M. Gahan, T. Abee, and C. Hill. 1999. Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28. Appl. Environ. Microbiol. 65:2078-2083.[Abstract/Free Full Text]
23
23. Waditee, R., T. Hibino, T. Nakamura, A. Incharoensakdi, and T. Takabe. 2002. Overexpression of a Na⁺/H⁺ antiporter confers salt tolerance on a freshwater cyanobacterium, making it capable of growth in sea water. Proc. Natl. Acad. Sci. USA 99:4109-4114.[Abstract/Free Full Text]
24
24. Waditee, R., T. Hibino, Y. Tanaka, T. Nakamura, A. Incharoensakdi, S. Hayakawa, Y. Futsuhara, Y. Kawamitsu, T. Takabe, and T. Takabe. 2002. Functional characterization of betaine/proline transporters in betaine-accumulating mangrove. J. Biol. Chem. 277:18373-18382.[Abstract/Free Full Text]
25
25. Waditee, R., T. Hibino, Y. Tanaka, T. Nakamura, A. Incharoensakdi, and T. Takabe. 2001. Halotolerant cyanobacterium Aphanothece halophytica contains an Na⁺/H⁺ antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail. J. Biol. Chem. 276:36931-36938.[Abstract/Free Full Text]
26
26. Waditee, R., Y. Tanaka, K. Aoki, T. Hibino, H. Jikuya, J. Takano, T. Takabe, and T. Takabe. 2003. Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica. J. Biol. Chem. 278:4932-4942.[Abstract/Free Full Text]
27
27. Waditee, R., M. N. Bhuiyan, V. Rai, K. Aoki, Y. Tanaka, T. Hibino, S. Suzuki, J. Takano, A. T. Jagendorf, T. Takabe, and T. Takabe. 2005. Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc. Natl. Acad. Sci. USA 102:1318-1323.[Abstract/Free Full Text]
28
28. Wood, J. M., D. E. Culham, A. Hillar, Y. I. Vernikovska, F. Liu, J. M. Boggs, and R. A. Keates. 2005. A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli. Biochemistry 44:5634-5646.[CrossRef][Medline]
29
29. Wutipraditkul, N., R. Waditee, A. Incharoensakdi, T. Hibino, Y. Tanaka, T. Nakamura, M. Shikata, T. Takabe, and T. Takabe. 2005. Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na⁺/H⁺ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH. Appl. Environ. Microbiol. 71:4176-4184.[Abstract/Free Full Text]
30
30. Yamaguchi, T., and E. Blumwald. 2005. Developing salt-tolerant crop plants: challenges and opportunities. Trends Plant Sci. 10:615-620.[CrossRef][Medline]
31
31. Zhu, J. K. 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Plant Biol. 6:441-445.[CrossRef][Medline]

---

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.

This article has been cited by other articles:

• Scanlan, D. J., Ostrowski, M., Mazard, S., Dufresne, A., Garczarek, L., Hess, W. R., Post, A. F., Hagemann, M., Paulsen, I., Partensky, F. (2009). Ecological Genomics of Marine Picocyanobacteria. Microbiol. Mol. Biol. Rev. 73: 249-299 [Abstract] [Full Text]  
• Juste, A., Lievens, B., Frans, I., Marsh, T. L., Klingeberg, M., Michiels, C. W., Willems, K. A. (2008). Genetic and physiological diversity of Tetragenococcus halophilus strains isolated from sugar- and salt-rich environments. Microbiology 154: 2600-2610 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Laloknam, S. Articles by Takabe, T. Search for Related Content PubMed PubMed Citation Articles by Laloknam, S. Articles by Takabe, T. Agricola Articles by Laloknam, S. Articles by Takabe, T.