Received 4 May 2005/Accepted 26 August 2005

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 2005, p Vol. 187, No /05/$ doi: /jb Copyright 2005, American Society for Microbiology. All Rights Reserved. The Activity Profile of the NhaD-Type Na (Li )/H Antiporter from the Soda Lake Haloalkaliphile Alkalimonas amylolytica Is Adaptive for the Extreme Environment Jun Liu, 1,2 Yanfen Xue, 1 Quanhui Wang, 1,2 Yi Wei, 3 Talia H. Swartz, 3 David B. Hicks, 3 Masahiro Ito, 4 Yanhe Ma, 1 * and Terry A. Krulwich 3 Institute of Microbiology, Chinese Academy of Sciences, Beijing , People s Republic of China 1 ; Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York ; Graduate School of Life Sciences, Toyo University, Oura-gun, Gunma , Japan 4 ; and Graduate School of Chinese Academy of Sciences, Beijing , People s Republic of China 2 Received 4 May 2005/Accepted 26 August 2005 In extreme alkaliphiles, Na /H antiporters play a central role in the Na cycle that supports ph homeostasis, Na resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high ph with high salinity. Here, studies were conducted on a Na (Li )/H antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different ph values and Na concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 ( nhaa nhab), the ph optimum for NhaD-dependent Na (Li )/H antiport was at least 9.5, the highest ph that could be tested; no activity was observed at ph <8.5. NhaD supported low Na /H antiport activity at ph 9.5 that was detectable over a range of Na concentrations from 10 mm to at least 800 mm, with a 600 mm optimum. Although A. amylolytica nhad was isolated by complementing the Li sensitivity of the triple mutant E. coli strain KNabc ( nhaa nhab chaa), sustained propagation of nhadbearing plasmids in this strain resulted in a glycine (Gly 327 )3serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at Na concentrations up to 200 mm but lower activity at 400 to 600 mm Na, with a ph optimum and minimal ph for activity lower than those of wild-type NhaD. Downloaded from Monovalent cation/h antiporters of bacteria extrude cytoplasmic monovalent cations in exchange for H from the outside medium (for reviews, see references 38 and 40). The exchange is energized by the electrochemical gradient of protons ( p, alkaline and negative inside) that is generated across the cytoplasmic membrane by distinct proton-pumping complexes such as those of the respiratory chain (56). Secondary monovalent cation/h antiporters that are energized in this way are found in many different families and superfamilies within the sequence-based Transporter Classification (TC) (48) where they are assigned TC numbers ( (i) the large major facilitator superfamily (TC 2A.1) contains several drug and multidrug/h antiporters that catalyze Na (K )/H, as well as drug/h antiport (24); (ii) the Ca 2 : cation antiporter (CACA) family (TC 2.A.19) contains at least one Ca 2 /H antiporter, ChaA (TC 2.A ) from E. coli, that also catalyzes Na /H antiport (17, 34, 49); (iii) the cation:proton antiporter-1 family (CPA-1) (TC 2.A.36) encompasses a large number of eukaryotic Na /H antiporters (exchangers), as well as bacterial examples, e.g., NhaG, NhaP, and NhaK (3, 7); (iv) the CPA-2 family (TC 2.A.37) contains diverse bacterial cation transporters, including both eukaryotic and prokaryotic Na /H antiporters; the extensively studied * Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Beijing , People s Republic of China. Phone: Fax: mayanhe@sun.im.ac.cn. NhaA antiporter from E. coli has recently been suggested to belong in the CPA-2 family (3, 41); (v) the CPA-3 family (TC 2.A.63) contains a widely distributed family of monovalent cation/h antiporters that are encoded by conserved six to seven gene operons and are called Mrp, Sha, Pha, or Mnh in different bacteria (53); and (vi) three additional Na /H antiporter families, the NhaB (TC 2.A.34), NhaC (TC 2.A.35), and NhaD (TC 2.A.62) families, have all been grouped within an ion transporter superfamily (no assigned TC number) (44). Most bacteria have multiple monovalent cation/h antiporters, often with several paralogues from one antiporter family, as well as antiporters from other families (36). The precise roles of each antiporter within any single organism are not yet fully defined, but physiological roles have been clearly established for some monovalent cation/h antiporters. These antiporters play a major role in alkali-tolerance of respiring bacterial cells, protect bacterial cells from cytotoxic effects of Na and Li and also establish an inwardly directed Na gradient that energizes transport of some solutes and motility systems (16, 36, 39). The role of monovalent cation/h antiporters in alkaline ph regulation was first demonstrated in extremely alkaliphilic bacteria, in which Na /H antiporters are specifically required for maintenance of a cytoplasmic ph substantially below the external ph (11, 19 22, 30). In contrast, the ph homeostasis needs of nonalkaliphilic bacteria are met by both Na /H and K /H antiporters (4, 26, 32, 43). Although the key role of Na /H antiporters in the physiology of alkaliphiles is well on October 15, 2018 by guest 7589

2 7590 LIU ET AL. J. BACTERIOL. recognized (21), identification and characterization of specific alkaliphile antiporter proteins has been limited to alkaliphilic Bacillus species from soil. These studies have identified the Mrp antiporter as having a major role in alkaline ph homeostasis of alkaliphilic Bacillus and suggested ancillary roles for NhaC and one or more other antiporters yet to be characterized (23, 36); an NhaP-like Na /H antiporter and MleN, an Na -lactate/malic acid antiporter (an NhaC paralogue) are also annotated in the Bacillus halodurans C-125 genome ( and could contribute to ph homeostasis and/or Na -resistance (36). To date, no attempts have been made to identify and characterize the Na /H antiporters of haloalkaliphiles from alkaline soda lakes, although these lakes are rich sources of alkaliphiles and are widely distributed around the earth. Alkaline soda lakes are the most stable high ph environment among the natural habitats of extreme alkaliphiles, with both high salinity and alkalinity that is often greater than ph 11.5 (10, 18, 58). A very diverse group of soda lake haloalkaliphiles has been extensively characterized, resulting in recognition of numerous new bacterial genera (18, 29, 52, 59). In the present study, we present the first characterization of a Na (Li )/H antiporter from a soda lake bacterium. The bacterium, Alkalimonas amylolytica, is a gramnegative proteobacterium of the 3 class that was recently isolated from Lake Chahannor in China; it has a respirationbased metabolism and grows at NaCl concentrations up to 7% in a range of ph from 7.5 to 11, with an optimum at ph 10 to 10.5 (28). Identification of A. amylolytica nhad was carried out by a standard approach for identifying Na /H antiporters from bacteria whose genomes have not yet been sequenced. This involves a complementation screen of DNA libraries in antiporter-deficient strains of Escherichia coli that identify candidate Na (Li )/H antiporter-encoding clones by their restoration of Na (Li ) resistance (37, 40). MATERIALS AND METHODS Strain or plasmid TABLE 1. Bacterial strains and plasmids Properties Source or reference Strains A. amylolytica Extreme alkaliphiles, gram 28 negative E. coli DH5 F 80dlacZ M15 (laczyaargf) GIBCO-BRL U169 reca1 enda1 hsdr17 (r K m K ) phoa supe44 thi-1 gyra96 rela1 E. coli KNabc TG1( nhaa nhab chaa) 33 E. coli EP432 melb(lid a ) nhaa1::kan 42 E. coli C43(DE3) nhab1::cam laczy thr-1 BL21 derivative 31 Plasmids puc18 Cloning vector; Amp r Promega pgem3zf( ) Cloning vector; Amp r Promega pl8 puc18 derivative, contains the This study wild-type nhad gene from A. amylolytica pz2 puc18 derivative, contains the This study mutant nhad gene from A. amylolytica pa2 pgem3zf( ) derivative, This study contains the wild-type nhad gene from A. amylolytica pa2n3 pgem3zf( ) derivative; This study contains the mutant nhad gene from A. amylolytica pet21b( ) Expression vector; Amp r Novagene peta2 pet21b( ) derivative, contains This study the wild-type nhad from A. amylolytica peta2n3 pet21b( ) derivative, contains the mutant nhad (G327S) from A. amylolytica This study a LiD, Li dependent. Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in the present study are listed in Table 1. A. amylolytica was grown at 37 C, with shaking, in Horikoshi I medium (13). Antiporter-deficient E. coli strains KNabc and EP432 and transformants thereof were grown in LBK medium (9) at ph 7.5 with cation additions noted under specific experiments. E. coli C43 was grown in LB medium (50). Appropriate antibiotics were added for plasmid transformants of mutant strains, using the following concentrations: ampicillin, 100 g/ml; chloramphenicol, 50 g/ml; and kanamycin, 50 g/ml. DNA library construction and complementation screen in antiporter-deficient E. coli that identified nhad. Standard molecular methods were used for DNA isolation, cloning, and restriction analyses (50). For construction of a DNA library, chromosomal DNA was isolated from A. amylolytica and partially digested with Sau3A1. Fragments in the range of 3 to 8 kb were ligated with BamHI-digested puc18, and the ligation mixture was used to transform the triple antiporter mutant strain E. coli KNabc. The transformants were screened on LBK-ampicillin plates (ph 7.5), containing either 200 mm NaCl or 10 mm LiCl. No growth was observed on the NaCl-containing plates but a clone that supported growth on the LiCl-containing plates was isolated. Initial sequence analysis was carried out on the 3.5-kb insert in this clone, pl8, to assess whether there was a candidate antiporter gene. A putative monocistronic nhad homologue was identified. A subclone, pz2, was prepared in puc18 by digesting pl8 with SphI and religated to remove approximately 1 kb of sequence upstream of nhad, leaving the nhad coding region together with 399 bp of upstream DNA and 611 bp of downstream DNA. Transformants of E. coli KNabc with the ligation mixture were plated on LBK-ampicillin plates (ph 7.5), containing 200 mm NaCl, yielding plasmid pz2. As discussed in Results, the sequence of the nhad gene in pz2 differed by one nucleotide change from the version in pl8 and the corresponding chromosomal sequence. Initial sequence analyses were conducted by the Shanghai BioAsia Biotechnology company and subsequent sequence analyses were conducted in the Mount Sinai School of Medicine DNA Core Facility. Cloning and mutagenesis of chromosomal antiporter gene. The chromosomal copy of the nhad gene and sufficient upstream region to contain the most likely promoter region(s) was amplified by PCR with the following primers: forward primer 5 -ACTGGAGCTCAAATAGCCCAGATTGG-3, which contains a SacI site, and reverse primer 5 -ATCGTCTAGAGGTTTAGTCGTAGATATG-3, which contains an XbaI site. The products of several independent PCRs had identical sequences. One such 1.6-kb fragment was digested with SacI and XbaI and cloned into SacI- and XbaI-digested pgem3zf (Promega). The resulting recombinant plasmid that contained the chromosomal nhad locus was designated pa2. A mutant version of pa2, designated pa2n3 was constructed, in which a unique 849-bp EcoRV-XbaI fragment from pz2 that contained the region with the mutation replaced the corresponding wild-type fragment. Sequence analysis confirmed that the insert in pa2n3 differed from that in pa2 only by the single nucleotide that distinguished the nhad in pz2 from chromosomal and pl8 sequence. The sequence of wild-type A. amylolytica nhad was deposited in GenBank under accession number AY Preparation and assays of everted membrane vesicles. Transformants of E. coli EP432 ( nhaa nhab) that expressed nhad or nhad-g327s were used for assays of antiport by fluorescence-based assays in everted membrane vesicles. The stability of both wild-type and mutant genes in the E. coli EP432 transformants was monitored by sequencing of plasmids after passage and growth in this antiporter-deficient strain for use in the assays; no mutations were detected. Everted membrane vesicles were prepared by breaking cells using a French pressure cell as described by others (2, 47); the buffers used in the preparation were 10 mm Tris-HCl (ph 7.5), containing 140 mm choline chloride, 0.5 mm dithiothreitol, 10% glycerol, a protease inhibitor tablet (Roche), and 1 mm

3 VOL. 187, 2005 ALKALIMONAS AMYLOLYTICA NhaD 7591 FIG. 1. Assays of Li /H antiport activity at ph 9.5 in vesicles of E. coli EP432 transformants. Vesicles from transformants expressing empty vector (pgem), wild-type NhaD (pa2), or mutant NhaD (pa2n3) were assayed in 2 ml containing 50 mm BTP buffer, 140 mm choline chloride, 5 mm MgCl 2,1 M AO, and 60 g of vesicle protein. Respiration was initiated by the addition of Tris-succinate to a final concentration of 2.5 mm at the point indicated by the downward arrow, and LiCl was added, to a final concentration of 10 mm, at the point indicated. The traces shown are representative traces from two to three independent experiments. phenylmethylsulfonyl fluoride. Protein content was measured by the Lowry method using lysozyme as the standard (27). Assays of monovalent cation/h antiport were conducted with acridine orange (AO) as a fluorescent probe of the transmembrane ph gradient ( ph, acid in) as described by Goldberg et al. (9). Except as indicated for specific experiments, the assay mixtures were made up to a total volume of 2 ml containing: 50 mm 1,3-Bis[tris(hydroxymethyl)methylamino] propane (BTP) buffer, 140 mm choline chloride, 5 mm MgCl 2,1 M AO, and 60 g of vesicle protein. Respiration was initiated by the addition of Tris-succinate to a final concentration of 2.5 mm; in some experiments 5 mm KCl was added before the succinate, as described in Results. Addition of the electron donor resulted in AO quenching in response to the development of a ph gradient, acid in, as protons were pumped into the everted vesicles. After the quench reached steady state, test cations, as chloride salts, were added to assess their ability to act as substrates for antiport with H from inside the vesicles. The magnitude of this response was recorded as the increase in AO fluorescence (dequenching) right after cation addition, and the percent dequenching was calculated relative to the initial respiration-dependent quench. The percent dequenching observed over a range of Na or Li concentrations was used to calculate apparent K m values for these substrate cations as described by others (15, 25, 45, 46, 55); this makes the maximal percent dequenching a surrogate value for V max, although this value was recorded at a single time after addition of the substrate. As shown by others, properties based on these values relate well to independent in vitro assessments of the catalytic properties of several antiporters and their mutant forms (e.g., see references 46 and 55). On the other hand, this assay does not always detect antiporter activity under conditions of ph and cation concentration in which the antiporter exerted effects in vivo, probably because of fluctuations of ph during growth and large differences between the assay mixture and the in vivo milieu, e.g., ionic strength and composition, solute composition, and overall protein concentration (8, 35). In some assays, the use of high concentrations of added NaCl, e.g., 600 mm NaCl, resulted in a small change in the assay volume and also imposed a greater change in extravesicular osmolarity than the range of 1 to 25 mm. At 600 mm cation addition resulted in a small increase in the quenching in control vesicles. This was observed whether the salt added contained an antiporter substrate, Na or Li, or a nonsubstrate, K or choline. The small increase of quenching resulted in a new flat baseline for quenching in the control. The assays of the Aa-NhaD and Aa-NhaD-G327S vesicles were conducted identically to the control reactions; the same increase in quenching was observed at ph values at which the antiporter was inactive and the calculated percent dequenching took account of this increase where antiporter activity was observed. All assays were conducted in duplicate or triplicate in two to three independent experiments on different preparations. Preparation of C-terminally hexahistidine-tagged NhaD forms and Western analyses. Both NhaD and NhaD G327S were amplified by PCR using the primers 5 -GGTTCTAGACGCTAAACGCTGTGCTACAA-3 and 5 -TGCCTC GAGGTCGTAGATATGAAACAAGTCTGC-3, the first of which introduced an XbaI site and the second of which introduced an XhoI site. The doubly FIG. 2. Na /H antiport activity of wild-type NhaD, with 10 versus 600 mm added Na, as a function of ph. The assay protocol was as described in the legend to Fig. 1 except that 5 mm KCl was added prior to the energization with Tris-succinate. The data are shown for assays with 10 mm NaCl (open symbols) or 600 mm NaCl addition (solid symbols). The error bars indicate standard deviations from duplicate assays from two to three independent experiments. digested products were cloned into XbaI- and XhoI-digested pet21b( ) vector (Novagen) so that the a hexahistidine tag was introduced at the C terminus of NhaD. The two transformants were grown in LB plus ampicillin to an A 600 of 0.6. Expression of the tagged nhad genes was then induced by addition of 0.7 mm IPTG, and the cells were incubated overnight. The amount of NhaD in everted membrane vesicles was assessed by Western analyses. Samples were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (51), transferred to nitrocellulose membranes, and His-tagged proteins were detected by chemiluminescence (Pierce) with the INDIA anti-his probe (Pierce). GenBank accession number. The new gene sequence data were deposited under GenBank accession number AY RESULTS Cloning of an nhad gene from A. amylolytica in an antiporter-deficient E. coli strain and comparison of this gene with the chromosomal sequence. An nhad homologue was found upon sequencing recombinant vector, pl8, which had been isolated from an A. amylolytica DNA library in puc18. The pl8 transformant of antiporter-deficient E. coli KNabc ( nhaa nhab chaa) grew on LBK plus 10 mm LiCl but not on LBK plus 200 mm NaCl at ph 7.5. The sequence of the nhad gene corresponded precisely to that of the chromosomal nhad that was sequenced directly from PCR products. In contrast, growth of an E. coli KNabc transformant with a subclone of pl8, pz2, produced colonies on LBK containing up to 500 mm NaCl. The nhad gene in pz2 was found by sequence analysis to have a single nucleotide change that altered one amino acid of the predicted chromosomal gene product. We hypothesized that the mutant nhad gene of pz2 arose in cultures derived from single colonies

4 7592 LIU ET AL. J. BACTERIOL. FIG. 3. Li /H and Na /H antiport activity of NhaD-G327S as a function of cation concentration at ph 9.5. Fluorescence-based assays of the Li /H (A) and Na /H (B) antiport activity of NhaD-G327S in E. coli EP432 vesicles were conducted at ph 9.5 over a range of concentrations of added LiCl or NaCl; the Michaelis-Menten plot is shown, and a reciprocal plot is shown as an inset in each panel. The assay protocol was identical to that described in the legend to Fig. 1, with no addition of KCl before energization with Tris-succinate. The activity observed in vesicles from the pgem control transformant was subtracted from the NhaD-G327S activity at each concentration to obtain the values shown. The standard deviations of the values, which are derived from duplicate assays in at least two independent experiments, were less than 10% of the mean values. of the E. coli KNabc/pL8 transformant. This was supported by experiments showing that during propagation E. coli KNabc transformants with wild-type nhad-bearing plasmids similar mutations consistently arose. In contrast, the wild-type nhad could be stably maintained in the less-impaired double mutant E. coli EP432 ( nhaa nhab) (data not shown). The deduced 480-amino-acid product of the complementing haloalkaliphile gene was identified as NhaD from BLAST analyses (1). The closest homologues in the databases included the only two NhaD antiporters that have been experimentally verified to have Na (Li )/H antiport activity, i.e., Vibrio parahaemolyticus NhaD (72% identity and 84% similarity) and Vibrio cholerae NhaD (65% identity and 78% similarity), with a putative NhaD from Idiomarina loihiensis listed as the closest homologue (76% identity and 86% similarity). A. amylolytica NhaD was predicted by the HMMTOP program (54) to be a polytopic membrane protein with 14 transmembrane segments (TMS), whereas V. cholerae NhaD was predicted to have 13 TMS (35). The significant difference between the HMMTOP models of the two NhaD homologues is confined to first 30 residues of the N-terminal region, so both models place the site of the Gly3Ser mutational change in the pz2 version, an alteration of Gly 327, in the large cytoplasmic loop that would be between TMS X and XI in the haloalkaliphile NhaD. Wild-type NhaD has low Na (Li )/H antiport activity that functions over a large range of Na concentrations and with a high ph optimum. Assays were initiated to characterize the antiport properties of chromosomally encoded of A. amylolytica NhaD using a fluorescence assay of E. coli EP432 ( nhaa nhab) membrane vesicles expressing nhad from plasmid pa2; vesicles from a transformant with the empty pgem3zf vector was the control plasmid for the assay. The double antiporter mutant E. coli EP432 was used even though it has a little more residual antiport than triple mutant E. coli KNabc ( nhaa nhab chaa) because nhad was unstable in the latter strain. Almost all of the residual background in control vesicles of E. coli EP432 can be suppressed by addition of 5 mm KCl before addition of the electron donor that energizes the vesicles (17). However, KCl was not pre-added in the first assays. This made it possible to assay K /H antiport in addition to the Na /H and Li /H antiport activity, in case NhaD from the soda lake alkaliphile had this capacity; V. parahaemolyticus NhaD had exhibited Na (Li )/H but not K /H (33). Although not shown, wild-type A. amylolytica NhaD exhibited no activity with any test cation (at 10 mm) at ph values of 9 in these in vitro assays; at ph 9.5, NhaD exhibited no Na /H or K /H antiport above the background in control vesicles but did show modest Li /H antiport above the low Li /H antiport background of the control vesicles (Fig. 1, left two traces). In order to minimize the Na /H antiport background of the control vesicles, the activity of wild-type NhaD was further studied by the fluorescence assay of everted vesicles to which 5 mm KCl was added before energization; under these conditions, there was a small background activity that dropped to zero at ph 9. Wild-type NhaD (pa2)-dependent Na /H antiport activity was evident in the ph range from 9 to 9.5, increasing linearly over that range (Fig. 2); higher ph values could not be tested due to the limitations of the heterologous E. coli system, and there are no comparable multiple antiporter mutants of alkaliphiles available in which to conduct the assays. The low activity of wild-type NhaD increased gradually with increasing Na concentration over a broad range, and the small background activity in the control fell to zero at about 400 mm Na ; the difference in NhaD and control activity at 10 mm and 600 mm added Na is shown in Fig. 2. Although not shown, NhaD activity was highest at 600 mm Na and declined at 800 mm Na. NhaD-G327S has an altered activity profile in both ph and substrate concentration. A single nucleotide mutation was found during the propagation of an nhad-bearing plasmid in

5 VOL. 187, 2005 ALKALIMONAS AMYLOLYTICA NhaD 7593 FIG. 4. Na /H antiport activity of NhaD-G327S, with 10 versus 600 mm added Na, as a function of ph. The assay protocol was as described in the legend to Fig. 2., with 5 mm KCl added prior to the energization with Tris-succinate; open symbols are used for data from assays with 10 mm added NaCl, and closed symbols are used for data from assays with 600 mm added NaCl. the E. coli KNabc strain. The mutation found in pz2 and pa2n3 was a GGT-to-AGT change at position 1166 of the pa2 clone and was predicted to change a glycine to a serine residue in the product at position 327. Assays were conducted on E. coli EP432 vesicles from a transformant expressing the mutant NhaD-G327S encoded in pa2n3, as described above for the wild-type antiporter. Initial assays using 10 mm test ions in the absence of pre-added KCl established that this antiporter had a higher Li /H antiport activity than wild-type at ph 9.5, which was the optimal ph under this assay condition (Fig. 1, right trace). NhaD-G327S exhibited significant Na /H antiport activity under these same conditions (Fig. 3B) but no K /H antiport and no Ca 2 /H antiport (as assayed in assay mixtures with or without MgCl 2 ) (data not shown). The larger percent dequenching with the mutant NhaD made it possible to examine antiport activity over a range of concentrations of both LiCl and NaCl. Michaelis-Menten kinetics were observed, with a higher maximal percent dequenching with LiCl (Fig. 3A) than with NaCl (Fig. 3B) and calculated apparent K m values of approximately 3 mm for LiCl (Fig. 3A, inset) and 0.5 mm for NaCl (Fig. 3B, inset). In order to directly compare the Na /H antiport properties of NhaD-G327S with wild-type NhaD under conditions that allow activity of both antiporters, the mutant was next assayed in the protocol in which 5 mm KCl was added before energization. The ph optimum of NhaD-G327S was ph 9 (Fig. 4), lower than in the absence of KCl and at least half a ph unit below that of wild-type NhaD, for which the activity under the same conditions was at highest at ph 9.5 and could not be probed at higher ph values (Fig. 2). Two other differences between the mutant and wild-type activity profiles were evident FIG. 5. Comparative Na /H antiport activity data for wild-type NhaD and NhaD-G327S from assays with 10 or 600 mm added NaCl at three different ph values. Selected data from assays of antiport activity, with 5 mm KCl added before the Tris-succinate, are presented after subtraction of background antiport activity. The error bars represent the standard deviation of the mean values. (compare Fig. 2 and 4): first, the minimum ph at which activity was observed was also lower in the mutant (ph 8.5) than in wild-type (ph 9.0); and second, the mutant NhaD was inhibited at 600 mm Na (Fig. 4), the optimal concentration for the wild-type enzyme. Selected data directly illustrating the comparative properties of wild-type (pa2) and mutant NhaD (pa2n3) are shown in Fig. 5 and highlight another striking property of wild-type NhaD compared to NhaD-G327S, i.e., the low overall activity observed for wild-type NhaD under all conditions. Although the two antiporter genes were expressed from the same natural promoter in the same plasmid, it was possible that the wild-type integrated less well into the membrane in E. coli. This could not be assessed by Western analyses using the wild-type promoter because the signal was too low. To measure relative membrane incorporation of the wild-type and mutant NhaD when their genes were expressed with hexahistidine tags under a stronger promoter, the two genes were expressed under the same IPTG-inducible promoter as each other in the E. coli C43-pET vector system described in Materials and Methods; under these conditions, Western analyses showed slightly higher levels of hexahistidine tagged NhaD in the membranes than tagged NhaD-G327S. The caveat in weighing these results is that the conditions differed from those of the assay conditions, but they do not support the conclusion that the wild-type form exhibits a general assembly defect relative to the mutant NhaD in E. coli (data not shown). DISCUSSION NhaD is widespread among bacteria, but no specific physiological role has yet been discerned for any NhaD (12). The

6 7594 LIU ET AL. J. BACTERIOL. three NhaD homologues that have thus far been studied NhaD from A. amylolytica (Aa-NhaD), V. parahaemolyticus (Vp-NhaD) (33), and V. cholerae (Vc-NhaD) (5, 35) all exhibited Na /H and Li /H antiport and alkaline ph optima in the same type of membrane vesicle assay used here, but the ph profile of Aa-NhaD is significantly different from the others. Aa-NhaD was only active above ph 8.5 and the optimal ph may well be greater than 9.5, the highest ph value tested. In contrast, Vp-NhaD exhibited alkali-stimulated activity from ph 8 to 9 but reduced activity at ph 9.5 (33), whereas Vc- NhaD was active only in a ph range from ph 7.25 to 8.5 with an optimum at ph 8.0 (5). The different ph responses of the NhaD antiporters probably reflect the dependence of the antiporters activity on cytoplasmic ph in their different hosts, as was found for the major Na /H antiporter of E. coli, 12-TMS (388 amino acids) NhaA, and its homologues in different bacteria (41). E. coli NhaA exhibits profound ph-dependent changes in antiporter activity, resulting in activation by alkaline conditions that depends upon the cytoplasmic ph and critically involves a cytoplasmic loop between TMS VIII to IX (40, 41, 55); a mechanism for the participation of this cytoplasmic loop in NhaA activation at alkaline ph has been suggested by the recently reported high-resolution structure of NhaA (14). The location of the G327S mutation in Aa-NhaD is predicted to be in the analogous position on the cytoplasmic face of the putative 14-TMS transporter; this mutation brought the ph profile of Aa-NhaD closer to that of nonalkaliphilic NhaD (33). Experimental data on the cytoplasmic ph of A. amylolytica are not yet available. However, the minimum ph for wild-type Aa- NhaD activity is close to the cytoplasmic ph of 8.2 of alkaliphilic Bacillus pseudofirmus OF4 at external ph values of 10 to 10.5 (21), the optimal ph range for growth for both B. pseudofirmus OF4 and A. amylolytica (21, 28). At an external ph of 11, B. pseudofirmus OF4 still grew, but the growth rate was lower and the cytoplasmic ph was higher, at 9.5 (21). Thus, in the setting of an extreme alkaliphile, an antiporter with the wild-type Aa-NhaD ph profile could play a role in cytoplasmic ph homeostasis at and above the optimal ph for growth. How can we account for the fact that wild-type Aa-NhaD complemented the Li -sensitivity of E. coli KNabc in LBK medium at ph 7.5 (leading to its recognition), i.e., at a ph well below the ph range exhibited for Aa-NhaD in the vesicle assays? During bacterial growth in unbuffered LB(K) media, the ph of the medium rises significantly (6, 57). Perhaps this increase in external ph caused a large enough increase in cytoplasmic ph to activate Aa-NhaD or the activation ph is slightly lower under the conditions of ion composition and possible activators of the cytoplasm. Wild-type Aa-NhaD exhibited two notable properties apart from its ph profile: first, a capacity for Na /H antiport over an extremely broad range of Na concentrations, with a high optimal [Na ], and second, a low activity relative to NhaD antiporters from other bacteria (5, 33). Both of these properties, like the ph profile, were tempered by the single G327S mutation. Presumably, these changes make Aa-NhaD-G327S more adaptive for the E. coli strain in which the mutation arose, whereas the wild-type Aa-NhaD profile is more adaptive for the A. amylolytica niche. The ability of wild-type Aa-NhaD to function optimally at 600 mm, a capacity lost in the mutant (Fig. 5), is consonant with a role under the extremely saline conditions of the soda lake environment where the organism is likely to be subjected to periodic challenge by adversely high cytoplasmic Na and needs antiporters that can function under such circumstances. With regard to the lower activity noted for wild-type Aa-NhaD compared to mutant Aa-NhaD-G327S and other NhaD antiporters, we cannot rule out the possibility that the antiporter is much more active at its true ph optimum in its natural cytoplasmic setting. Nor can we rule out the possibility that Aa-NhaD has some distinct catalytic activity that is more physiologically important than its monovalent cation/ proton antiport activity and more active. However, the low Na /H antiport activity of Aa-NhaD may be an adaptive property of a physiologically important alkaliphile antiporter. At very high ph, the activity level of Na /H antiporters must be carefully poised to meet the demands of Na and alkali resistance without causing adverse energy depletion; this was discussed in connection with the failure of E. coli to grow at alkaline ph in the presence of Na when NhaA, whose activity declines at ph 8.0, was replaced by a mutant form that is fully active up to ph 9.0 (40, 41, 46). Aa-NhaD may be designed to play important antiport roles without putting the overall cell energetics at risk. The current findings indicate that the activity profile of A. amylolytica NhaD is consonant with its natural setting and that these properties are modified by a single mutation in the nhad gene. Once the genetic tools are available, disruption of the gene in A. amylolytica and studies of mutant forms of the antiporter in its natural host will be needed to further clarify the role(s) of Aa-NhaD and the utility of its unusual features. 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