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Appl Environ Microbiol. 2009 October; 75(20): 6626–6629.
Published online 2009 August 21. doi:  10.1128/AEM.01387-09
PMCID: PMC2765133

An Mrp-Like Cluster in the Halotolerant Cyanobacterium Aphanothece halophytica Functions as a Na+/H+ Antiporter[down-pointing small open triangle]

Abstract

The mrp homolog gene cluster mrpCD1D2EFGAB (Ap-mrp) was found in a halotolerant cyanobacterium, Aphanothece halophytica, amplified, and expressed in Escherichia coli mutant TO114. Ap-mrp complemented the salt-sensitive phenotype of TO114 and exhibited Na+/H+ and Li+/H+ exchange activities, indicating that Ap-Mrp functions as a Na+/H+ antiporter.

Bacterial mrp (multiple resistance and pH adaptation) loci encode highly unusual multisubunit cation/proton antiporters (CPAs) (13). Mrp systems are widespread among bacteria and archaea and, because of their unique complexity, are classified as the separate CPA3 family (12). They are known as group 1, group 2, and others (mrp-like gene clusters) (13). Group 1 and group 2 Mrp antiporters have been shown to have roles in alkaline pH homeostasis and Na+ resistance (5, 8, 9), sporulation (9), symbiotic nitrogen fixation (11), pathogenesis (10), arsenite resistance (8), and bile salt resistance (7). Cyanobacterial Mrp-like clusters have been reported to be involved in salt stress tolerance (1, 2) and CO2 deficiency-induced expression (1, 17, 18). However, the molecular properties of cyanobacterial Mrp systems have not yet been reported.

Aphanothece halophytica is a halotolerant cyanobacterium, isolated from the Dead Sea, which accumulates betaine and can grow under a wide range of salinity conditions (0.25 to 3.0 M NaCl) as well as at alkaline pH (6, 16). Previous studies have shown that A. halophytica has an NhaP-type Na+/H+ antiporter (CPA1), Ap-NhaP1, with novel ion specificity (15), and two NapA1-type Na+/H+ antiporters (CPA2) of similar size, Ap-NapA1-1 and Ap-NapA1-2 (19). Although the Na+/H+ exchange activity of Ap-NhaP1 was high over a wide pH range, Ap-NapA1-1 and Ap-NapA1-2 exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities, with higher activities at alkaline pH.

From shotgun sequencing of the whole genome, we found in this study that A. halophytica contains an mrp-like gene cluster. It consists of eight open reading frames (ORFs) which seem to be one large transcriptional unit. Each of the ORFs starts with ATG. The entire cluster consists of 5,668 bp (accession number AB507743). A homology search revealed that eight ORFs encode the proteins Ap-MrpC, Ap-MrpD1, Ap-MrpD2, Ap-MrpE, Ap-MrpF, Ap-MrpG, Ap-MrpA, and Ap-MrpB, as shown in Fig. Fig.1.1. The organization of the A. halophytica mrp-like gene cluster is similar to that of the marine costal cyanobacterium Synechococcus sp. strain PCC 7002 but different from other cyanobacterium mrp clusters. One unique feature is an apparent mrpD duplication. The mrpD repeat also occurs in Synechocystis sp. strain PCC 6803, but in that strain, the repeated unit is mrpC mrpD. Each protein in the Ap-Mrp cluster shows significant similarity (>60%) with the corresponding protein of cyanobacteria but low homology to the proteins of group 1 and group 2 clusters (data not shown). Moreover, Bacillus pseudofirmus OF4 MrpA (Bp-MrpA) possesses an oxidoreductase domain and a C-terminal MrpB homolog sequence. Cyanobacterial MrpA does not contain an oxidoreductase domain and only contains a sequence homologous to MrpB. Therefore, MrpB′ might be a more appropriate name for cyanobacterial MrpA, although we have used “MrpA,” following the naming convention of Blanco-Rivero et al. (1). The fact that homology between Ap-MrpD1 and Ap-MrpD2 is higher than any other MrpD suggests that a duplication of Ap-MrpD occurred after the evolution of the Ap-Mrp system. Another interesting aspect of the Ap-mrp system is the clustering together with Ap-bicA and Ap-napA1-2 (Fig. (Fig.1).1). BicA is a medium-affinity, high-flux Na+-dependent inducible HCO3 transporter (18). Among the organisms shown in Fig. Fig.1,1, Synechococcus sp. strain PCC 7002 and Cyanothece sp. strain ATCC 51142 also have bicA and napA genes, whereas Synechococcus sp. strain PCC 7942 has only the sbtA gene, which encodes a high-affinity, low-flux Na+-dependent HCO3 transporter. The implications of the tripartite clustering of transporter genes bicA, napA1-2, and mrp are discussed below.

FIG. 1.
Gene arrangements of mrp-like gene clusters. Gene arrangements of the mrp region of A. halophytica and several cyanobacteria are shown in the upper portion of the figure. Genetic information for Anabaena sp. strain PCC 7120, Cyanothece sp. strain ATCC ...

Until now, no functional properties of cyanobacterial Mrp systems have been demonstrated. To characterize the molecular properties of Ap-Mrp, the Ap-mrp cluster gene was amplified by PCR using specific forward (5′-AAGGATCCAGTTTGTCATTAGTCATT-3′) and reverse (5′-TGGGATCCTTAAAGTAAACCACGATAG-3′) primers. The amplified DNA fragment was ligated into the BamHI restriction site of the pBSK+ plasmid (Stratagene, CA) and sequenced. The DNA sequence was confirmed to be the same as that obtained using shotgun sequencing. The resulting plasmid, pApMrp, was expressed in Escherichia coli host cells (TO114), which lack Na+/H+ antiporter genes (nhaA, nhaB, and chaA) (4). Figure Figure2A2A shows that the E. coli TO114 cells transformed with pApMrp could grow at a rate similar to that of TO114 cells transformed with an empty vector in LBK (Luria broth with KCl instead of NaCl) medium at pH 7.2 and 37°C. However, TO114 cells transformed with vector alone could not grow in the presence of 0.2 M NaCl (Fig. (Fig.2A).2A). By contrast, cells transformed with pApMrp were able to grow under these conditions (Fig. (Fig.2A).2A). Figure Figure2B2B shows that the growth of cells transformed with vector alone was severely inhibited in the presence of 4 mM LiCl, whereas the cells transformed with pApMrp were able to grow under the same conditions.

FIG. 2.
Effects of NaCl and LiCl on the growth of E. coli cells expressing Ap-Mrp in LB medium containing 30 mM KCl and the indicated concentrations of NaCl (A) or LB medium containing 30 mM KCl and 4 mM LiCl (B). Control TO114 cells and transformant TO114 cells ...

To examine the antiporter activity of Ap-Mrp directly, everted membrane vesicles were prepared and antiporter activities were monitored by measuring lactate-induced fluorescence quenching (Q) and salt-induced fluorescence dequenching ([open triangle]Q) as described previously (4, 15). As shown in Fig. Fig.3A,3A, dequenching ([open triangle]Q × 100/Q) was observed upon addition of NaCl in Ap-Mrp-expressing cells but not in control cells, indicating that Ap-Mrp has Na+/H+ antiporter activity. Dequenching was also observed upon addition of LiCl, but not upon addition of KCl (Fig. (Fig.3B)3B) or divalent cations such as Ca2+ and Mg2+ (data not shown). Dequenching by NaCl and LiCl increased with an increase in pH (Fig. (Fig.3C).3C). As shown in Fig. Fig.3D,3D, from the dependence of NaCl and LiCl concentrations on the dequenching, the apparent Km values of Ap-Mrp for Na+ and Li+ were determined to be 2.0 and 2.5 mM. These values are slightly higher than those for the Vibrio cholerae group 2 Mrp system (3) and much higher than the apparent Km for Na+ from group 1 Mrp systems (14). The apparent Km value of Ap-Mrp for Na+ (2.0 mM) is slightly higher than that of Ap-NapA1-1 (0.8 mM) but similar to that of Ap-NapA1-2 (1.8 mM) and would be low enough compared with the internal Na+ concentration, at high salinity as demonstrated previously (15, 19). The apparent Km value for Li+ (2.5 mM) is significantly higher than those for Ap-NapA1-1 (0.05 mM) and Ap-NapA1-2 (0.3 mM). Anyway, these results clearly demonstrate that a cyanobacterial Mrp, Ap-Mrp, functions as a Na+/H+ and Li+/H+ antiporter. To our knowledge, this is the first report demonstrating that the Mrp-like cluster classified as “others” also functions as a Na+/H+ antiporter.

FIG. 3.
Cation-proton exchange activities measured by the acridine orange fluorescence quenching method. Control TO114 cells and transformed TO114 cells expressing Ap-Mrp were grown in LBK medium at pH 7.5, from which everted membrane vesicles were prepared. ...

The knockout of the mrp gene is a useful method to study the physiological function of Mrp. Since transformation of A. halophytica has not been reported, we used a freshwater cyanobacterium, Synechococcus sp. strain PCC 7942, for which the Mrp system has not been studied. The mrpA gene of Synechococcus sp. strain PCC 7942 (Syn7942-mrpA) was disrupted by insertion of the spectinomycin resistance gene (spc). Complete segregation of Syn7942-mrpA was confirmed by PCR amplification (data not shown). The growth rates of wild-type and Syn7942-ΔmrpA cells were almost the same when they were grown in BG11 medium at pH 7.5, but more severe inhibition was observed for the mutant cells when the cells were grown at alkaline pH (pH 9.0) or at high salinity (Fig. (Fig.4A).4A). Here, we provide a functional explanation of how a protective function of a Na+/H+ antiporter (Ap-Mrp) can be achieved at “0 mM Na+.” Under these conditions, no driving force for the proposed uptake of H+ against the pH gradient (with the inside more acidic) would be present. It seems much more likely that the physiological function of the Mrp transporters is restricted to the export of Na+ ions driven by the electrochemical membrane potential. At high pH, such a function is impaired, and therefore cells lacking an Mrp system are much more sensitive toward salt stress.

FIG. 4.
Effects of disruption of the MrpA subunit of Synechococcus sp. strain PCC 7942 on growth and photosynthetic activity. Wild-type and mrpA-disrupted mutant Synechococcus sp. strain PCC 7942 cells were grown in BG11 medium containing 0 or 0.3 M NaCl at pH ...

Figure Figure4B4B shows that the Syn7942-ΔmrpA cells exhibited a rapid decrease (within 10 s) of photosynthetic electron transport activity ([open triangle]F) after salt shock. Moreover, Fig. Fig.4C4C shows that the levels of F in Syn7942-ΔmrpA cells significantly increased upon the salt shock compared with the wild-type cells, suggesting the accumulation of reductants due to the inhibition of electron transport. These data suggest a direct or close link between photosynthetic activity and Ap-Mrp at alkaline pH, namely, Ap-Mrp might utilize the reductants directly or via the electrochemical membrane potential produced by photosynthesis. Obviously, further studies are required to understand the molecular mechanisms of Ap-Mrp.

What is the physiological role of tripartite clustering of transporter genes bicA, napA1-2, and mrp? Of course further studies are required to answer this. But one plausible explanation is the coupling of HCO3 uptake and Na+ circulation. At alkaline pH, HCO3 is the major component of inorganic carbon. Since it is presumed that BicA and SbtA are Na+-induced Na+/HCO3 symporters (18), the role of BicA or SbtA would become important for CO2 uptake at alkaline pH. In addition, at alkaline pH, protection of intracellular pH increase is important and might be carried out by Ap-Mrp and Ap-NapA1-2. Upon the transport of H+ into the cell, Na+ is extruded to the cell exterior. Extracellular Na+ could promote the uptake of HCO3 into the cell, which could then be used for photosynthesis. Thus, cooperation between Ap-BicA, Ap-NapA1-2, and Ap-Mrp would be beneficial for growth under conditions of alkaline pH, high salinity, and low CO2.

Until now, four Na+/H+ antiporter genes have been functionally characterized in A. halophytica. Ap-NhaP1 exhibited very high exchange activity over a wide range of pH values (15). By contrast, three Na+/H+ antiporters, including Ap-NapA1-1, Ap-NapA1-2 (19), and Ap-Mrp, have high exchange activities at alkaline pH. These facts suggests that Na+/H+ antiporters are important for alkaline pH tolerance as well as for high-salinity stress tolerance.

Nucleotide sequence accession number.

Data for the Ap-mrp region are available in the DDBJ databases under accession number AB507743.

Acknowledgments

This work was supported in part by the Salt Science Research Foundation, grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, and the High-Tech Research Center of Meijo University.

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 August 2009.

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