Rungaroon Waditee

Overexpression of a Na+/H+ antiporter confers salt tolerance on a freshwater cyanobacterium, making it capable of growth in sea water

The salt tolerance of a freshwater cyanobacterium, Synechococcus sp. PCC 7942, transformed with genes involved in the synthesis of a Na+/H+ antiporter, betaine, catalase, and a chaperone was examined. Compared with the expression of betaine, catalase, and the chaperone, the expression of the Na+/H+ antiporter gene from a halotolerant cyanobacterium (ApNhaP) drastically improved the salt tolerance of the freshwater cyanobacterium. The Synechococcus cells expressing ApNhaP could grow in BG11 medium containing 0.5 M NaCl as well as in sea water, whereas those expressing betaine, catalase, and the chaperone could not grow under those conditions. The coexpression of ApNhaP with catalase or ApNhaP with catalase and betaine did not further enhance the salt tolerance of Synechococcus cells expressing ApNhaP alone when grown in BG11 medium containing 0.5 M NaCl. Interestingly, the coexpression of ApNhaP with catalase resulted in enhanced salt tolerance of cells grown in sea water. These results demonstrate a key role of sodium ion exclusion by the Na+/H+ antiporter for the salt tolerance of photosynhetic organisms.

Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica.

Glycine betaine (N,N,N-trimethylglycine) is an important osmoprotectant and is synthesized in response to abiotic stresses. Although almost all known biosynthetic pathways of betaine are two-step oxidation of choline, here we isolated twoN-methyltransferase genes from a halotolerant cyanobacterium Aphanothece halophytica. One of gene products (ORF1) catalyzed the methylation reactions of glycine and sarcosine with S-adenosylmethionine acting as the methyl donor. The other one (ORF2) specifically catalyzed the methylation of dimethylglycine to betaine. Both enzymes are active as monomers. Betaine, a final product, did not show the feed back inhibition for the methyltransferases even in the presence of 2 m. A reaction product, S-adenosyl homocysteine, inhibited the methylation reactions with relatively low affinities. The co-expressing of two enzymes in Escherichia coli increased the betaine level and enhanced the growth rates. Immunoblot analysis revealed that the accumulation levels of both enzymes in A. halophytica cells increased with increasing the salinity. These results indicate thatA. halophytica cells synthesize betaine from glycine by a three-step methylation. The changes of amino acids Arg-169 to Lys or Glu in ORF1 and Pro-171 to Gln and/or Met-172 to Arg in ORF2 significantly decreased V max and increasedK m for methyl acceptors (glycine, sarcosine, and dimethylglycine) but modestly affected K m forS-adenosylmethionine, indicating the importance of these amino acids for the binding of methyl acceptors. Physiological and functional properties of methyltransferases were discussed.

Functional Characterization of Choline Monooxygenase, an Enzyme for Betaine Synthesis in Plants

In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO). Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine non-accumulating plant. Here, we examined the functional properties of CMO expressed in Escherichia coli, cyanobacterium, andArabidopsis thaliana. We found that E. colicells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDH-deleted E. coli mutant. Changes of Cys-181 in spinach CMO to Ser, Thr, and Ala and His-287 to Gly, Val, and Ala abolished the accumulation of betaine. The ArabidopsisCMO-like gene was transcribed in Arabidopsis, but its protein was not detected. When the Arabidopsis CMO-like gene was expressed in E. coli, the protein was detected but was found not to promote betaine sysnthesis. Overexpression of spinach CMO in E. coli, Synechococcus sp. PCC7942, andArabidopsis conferred resistance to abiotic stress. These facts clearly indicate that CMO, but not the CMO-like protein, could oxidize choline and that Cys-181 and His-287 are involved in the binding of Fe-S cluster and Fe, respectively.

Potassium/Proton Antiport System of Escherichia coli

The intracellular level of potassium (K+) in Escherichia coli is regulated through multiple K+ transport systems. Recent data indicate that not all K+ extrusion system(s) have been identified (15). Here we report that the E. coli Na+ (Ca2+)/H+ antiporter ChaA functions as a K+ extrusion system. Cells expressing ChaA mediated K+ efflux against a K+ concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K+ under conditions in which wild-type cells extruded K+. The K+/H+ antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K+, which is toxic for these cells. These results suggest that ChaA K+/H+ antiporter activity enables E. coli to adapt to K+ salinity stress and to maintain K+ homeostasis.

Metabolic engineering for betaine accumulation in microbes and plants.

Plants accumulate a variety of osmoprotectants that improve their ability to combat abiotic stresses. Among them, betaine appears to play an important role in conferring resistance to stresses. Betaine is synthesized via either choline oxidation or glycine methylation. An increased betaine level in transgenic plants is one of the potential strategies to generate stress-tolerant crop plants. Here, we showed that an exogenous supply of serine or glycine to a halotolerant cyanobacterium Aphanothece halophytica, which synthesizes betaine from glycine by a three-step methylation, elevated intracellular accumulation of betaine under salt stress. The gene encoding 3-phosphoglycerate dehydrogenase (PGDH), which catalyzes the first step of the phosphorylated pathway of serine biosynthesis, was isolated from A. halophytica. Expression of the Aphanothece PGDH gene in Escherichia coli caused an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis plants, in which the betaine synthetic pathway was introduced via glycine methylation, further increased betaine levels and improved the stress tolerance. These results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways.

Halotolerant cyanobacterium Aphanothece halophytica contains a betaine transporter active at alkaline pH and high salinity.

ABSTRACT 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, γ-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.

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na+ and K+ concentrations is important for bacterial cells, which, in the absence of Na+ extrusion systems, cannot grow in the presence of high external Na+. Likewise, bacteria require K+ uptake systems when the external K+ concentration becomes too low to support growth. At present, we have little knowledge of K+ toxicity and bacterial outward‐directed K+ transport systems. We report here that high external concentrations of K+ at alkaline pH are toxic and that bacteria require K+ efflux and/or extrusion systems to avoid excessive K+ accumulation. We have identified the first example of a bacterial K+(specific)/H+ antiporter, Vp‐NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter‐1 (CPA1) family, was able to mediate K+ extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na+/H+ antiporters, Vp‐NhaA and Vp‐NhaB, which also exhibit a novel ion specificity toward K+, implying that they work as Na+(K+)/H+ exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K+ extrusion against a K+ chemical gradient, indicating that E. coli also possesses an unidentified K+ extrusion system(s).

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na+/H+ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

ABSTRACT Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.

Degradation of Glycinebetaine by Betaine-Homocysteine Methyltransferase in Aphanothece halophytica: Effect of Salt Downshock and Starvation

We have investigated conditions leading to the degradation of glycinebetaine in Aphanothece halophytica and have shown the activity of betaine-homocysteine methyltransferase (BHMT). The intracellular glycinebetaine level was decreased approximately 50% after 36 h salt downshock from 2.0 m NaCl medium to 0.5 m NaCl medium. A slight additional decrease of glycinebetaine occurred when salt downshock was combined with dark treatment. The omission of carbon and nitrogen sources in the growth medium further decreased intracellular glycinebetaine. The activity of BHMT increased from 0 to 460 nmol h−1mg−1 after 3 h salt downshock. Higher strength of salt downshock resulted in higher activity of the enzyme. Small increase of the enzyme activity was also observed when A. halophytica was deprived of carbon and nitrogen sources in the growth medium.

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet ☆

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.