Presence of a na+-stimulated p-type atpase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium aphanothece halophytica

Presence of a Na1-stimulated P-type ATPase in the plasmamembrane of the alkaliphilic halotolerant cyanobacteriumAphanothece halophytica Kanjana Wiangnon1, Wuttinun Raksajit1 & Aran Incharoensakdi1,2 1Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; and 2Center for Environmental Stress Tolerance inPlants, Faculty of Science, Chulalongkorn University, Bangkok, Thailand Department of Biochemistry and Center for Aphanothece cells could take up Na1 and this uptake was strongly inhibited by Environmental Stress Tolerance in Plants,Faculty of Science, Chulalongkorn University, the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na1 exhibited Na1 extrusion ability upon energizing with glucose.
Na1 was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na1-ionophore. Orthovana-date and CCCP strongly inhibited Na1 uptake, whereas N, N0-dicyclohexylcarbo- Received 13 October 2006; revised 11 January diimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na1 but not with K1, Ca21 and Li1. The K First published online 15 February 2007.
and Na1 were 1.66 Æ 0.12 and 25.0 Æ 1.8 mM, respectively, whereas the Vmax valuewas 0.66 Æ 0.05 mmol minÀ1 mgÀ1. Mg21 was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadateand DCCD. Amiloride, a Na1/H1 antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na1-stimulated ATPase in Aphanothece halophy- cyanobacterium; salt stress; Na1 extrusion.
tica. Plasma membranes from cells grown under salt stress condition showedhigher ATPase activity than those from cells grown under nonstress condition.
enhanced salt tolerance (Waditee et al., 2001; Wutipraditkulet al., 2005). Moreover, the overexpression of the NhaP antiporter from A. halophytica could allow the freshwater Salinity is one of the most important problems encountered cyanobacterium Synechococcus to grow in sea water (Wadi- by soil microorganisms and plants leading to the reduction tee et al., 2002). The efflux of Na1 mediated by Na1/H1 is in crop productivity worldwide. Specific mechanisms are categorized as a secondary Na1 transport because it utilizes needed to adjust the internal osmotic status for organisms proton motive force provided by a primary proton pump, thriving in hypersaline environments. One such mechanism involves the ability of the cells to accumulate compatible In most plant cells, the extreme of Na1 was mediated low molecular weight organic solutes such as glycine primarily through the Na1/H1 antiport system. However, betaine (Incharoensakdi & Wutipraditkul, 1999; Sakamoto there have been recent reports on the involvement of a & Murata, 2002). Another mechanism for adaptation to primary Na1 pump, Na1-ATPase, in the efflux of Na1 in high salinity is the removal of Na1 ions from the cytoplasm bacteria and higher plants. V-type Na1-ATPases were found out of the cells via plasma membrane or into the vacuoles in Enterococcus hirae and M-12, Amphibacillus sp. (Kaieda via tonoplast membrane (Apse & Blumwald, 2002). Apha- et al., 1998; Murata et al., 2001) whereas F-type ATPases were nothece halophytica is a halotolerant cyanobacterium, which found in Ilyobacter tartaricus and Acetobacterium woodii is able to grow in a wide range of salinity from 0.25 to 3.0 M (Neumann et al., 1998; Muller et al., 2001). An alkaliphilic NaCl and in external alkaline conditions up to an external Exiguobacterium aurantiacum contains a P-type Na1-ATPase pH of 11.0 (Takabe et al., 1988; Waditee et al., 2003).
(Ueno et al., 2000). P-type Na1-ATPases were also found in Previous studies showed that Na1/H1 antiporters of alkali- the plasma membranes of marine algae, Heterosigma akashi- philic A. halophytica play a crucial role in Na1 efflux with wo and Tetraselmis viridis (Shono et al., 1996; Popova et al.,  2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 1998). Studies on ATPase in cyanobacteria were scarce. P-type 22NaCl (0.45 mCi mmolÀ1) with an illumination of 30 mmol ATPase gene was cloned from Synechococcus 7942 whereas its photon mÀ2 sÀ1 provided by cool-white fluorescent lamps.
function was found to be involved in Cu21 transport (Phung After equilibration for 30 min, the mixture was added with et al., 1994). P-type ATPases were also reported in more 4 mM ATP to initiate the uptake of 22Na1. Na1-ionophore freshwater cyanobacteria such as Synechococcus PCC 6301, (ETH 2120, N,N,N0,N0,-tetracyclohexyl-1,2-phenylenediox- Synechocystis PCC 6803 and Anabaena PCC 7120 (Neisser ydiacetamide) and other ionophores including inhibitors et al., 1994). A P-type ATPase from Synechococcus PCC 7942 were added at 10 min before the addition of ATP. At with a proposed role in K1 influx under hyperosmotic intervals, 50 mL of the reaction mixture was filtered through condition has been reported (Kanamura et al., 1993; Neisser a 0.2 mm nitrocellulose membrane. The radioactivity et al., 1994). ATPase with enhanced activity under hypersaline trapped on the membrane filter was measured with a condition was reported in a marine cyanobacterium Spirulina subsalsa (Gabbay-Azaria et al., 1994). Hitherto, ATPaseinvolved in Na1 transport has not been reported for cyano- bacteria. This study demonstrates the presence of a P-typeATPase, specifically stimulated by Na1. The transport of Na1 The activity of ATPase was assayed by measuring the release by this cyanobacterium appeared to utilize the proton motive of inorganic phosphate resulting from the hydrolysis of ATP (Koyama et al., 1980). The reaction mixture (1 mL) con-tained 20 mM Tris-HCl pH 7.6, 5 mM MgCl2, 4 mM ATP,100 mM NaCl and enzyme. The reaction was started by the Axenic cells of A. halophytica were grown photoautotrophi- To determine ATP content in A. halophytica, cell suspensions cally in BG-11 medium supplemented with 18 mM NaNO3 were centrifuged at room temperature (2500 g, 10 min) and and Turk Island salt solution described previously (Inchar- the pellet was washed twice before suspending with 0.22 M oensakdi & Waditee, 2000). Cells were grown in cotton- phosphate buffer pH 6.8. This was followed by sonication to plugged 250 mL conical flasks containing 100 mL medium completely break the cells. Debris was removed and super- on a rotary shaker at 30 1C under continuous illumination natant was determined for ATP by HPLC. The iso- by cool white fluorescence tubes of 60 mmol photon mÀ2 sÀ1 cratic reverse phase HPLC with a water Resolve C18 (5-mM, spherical) column (Millipore, Milford, MA) wasused and mobile phase was 0.22 M phosphate buffer pH 6.8.
The eluate was monitored by absorption at 259 nm with a Cells at exponential growth phase were harvested and flow rate of 1 mL minÀ1. ATP content was determined based suspended in 20 mM Tris-HCl pH 7.6 containing 1.0 M on the standard curve constructed with the use of known sucrose. The crude membrane vesicles were prepared by concentrations of ATP injected vs. peak areas (Patil et al., treatment of the cell suspension with 0.2% lysozyme and the 1997). Protein concentration was determined by the method resulting spheroplasts were subject to a French pressure cell of Bradford (1976) using bovine serum albumin as standard.
at 900 psi. This crude membrane preparation was centri- In general, three independent experiments were per- fuged twice (4000 g, 20 min) to remove unbroken cells and formed and the mean values Æ SEM are given in the figures.
cell debris. The resulting dark blue green supernatant isreferred to as a mixture of crude vesicles. The plasma membrane vesicles were isolated by aqueous polymer two-phase partitioning using 5.6% (w/w) Dextran T-500 and 5.6% (w/w) polyethylene glycol as described by Norling et al.
(1994). The final plasma membrane vesicles were suspendedin 20 mM Tris-HCl pH 7.6 buffer containing 4 mM benza- To test whether A. halophytica could take up Na1, the effect of NaCl at different concentrations on the uptake of Na1 was investigated. Aphanothece cells showed an increaseduptake of 22Na1 with increasing concentration of 22NaCl (Fig. 1a). Initial uptake rates at 2, 5, 10 and 20 mM NaCl were estimated to be 13.4 Æ 0.5, 38.6 Æ 1.6, 75.0 Æ 3.1 and The membrane vesicles were suspended in 1 mL of 20 mM 130.3 Æ 4.8 nmol minÀ1 mgÀ1, respectively. The uptake of Tris-HCl pH 7.6 (1 mg protein mLÀ1) containing 20 mM 22Na1 was inhibited by the protonophore carbonyl cyanide  2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Na1-stimulated ATPase in Aphanothece halophytica m-chlorophenylhydrazone (CCCP) (Fig. 1a, inset). Slight increased intracellular content of 22Na1 with increasing time uptake of 22Na1 in the CCCP-treated Aphanothece cells was of incubation with 20 mM 22NaCl. Addition of glucose to observed at 10 and 20 mM NaCl, which might reflect the cell suspension at 10 min resulted in a slight decrease of diffusion of Na1 by sodium symport systems.
intracellular 22Na1 at 15 min. An almost complete loss of Aphanothece cells also possessed Na1 extrusion capacity intracellular 22Na1 was evident at 20 min, suggesting 22Na1 upon energizing with glucose (Fig. 1b). Cells showed an extrusion capacity of Aphanothece cells. To determine theinvolvement of ATP in the uptake and extrusion of Na1, thelevels of ATP in Aphanothece cells with and without glucose during the transport process were measured. In deenergized cells, ATP levels were decreased (Fig. 1b, inset) concomitant with the increase of Na1 uptake (Fig. 1b). Similarly, theenergized cells also showed the decreased ATP levels (Fig. 1b,inset) accompanying the increase of Na1 extrusion capacity (Fig. 1b). 22Na1 uptake was also demonstrated using plasmamembrane vesicles upon addition of 4 mM ATP (Fig. 1c).
An increase of 22Na1 uptake into the vesicles was observedwith saturation at around 6 min of incubation. The uptake of 22Na1 was dependent on ATP. Na1-ionophore as well as gramicidin D and monensin abolished the uptake of 22Na1.
The protonophore, CCCP and orthovanadate strongly in-hibited 22Na1 uptake. In contrast, N, N0-dicyclohexylcarbo- diimide (DCCD) caused no inhibition of 22Na1uptake in the first 4 min although slight inhibition was observed Before the characterization of ATPase, it was necessary to determine the ratio of the inside-out and right-side-out vesicles in the membrane preparations. This was accom- plished by measuring ATPase activity of the prepared membrane vesicles treated with 3% toluene according to Fig. 1. Na1 uptake (a), Na1 extrusion (b) in Aphanothece halophytica and Na1 uptake in plasma membrane vesicles (c). In (a), cells at the exponential growth phase (2 mg protein mL À 1) were incubated in 20 mM Tris-HCl buffer pH 7.6 containing 2 (~), 5 (’), 10 (m) and20 mM () 22NaCl (0.45 mCi mmol À 1). At indicated times, the radio- activity trapped in the cells after filtering through a 0.45 mm nitrocellu- lose membrane was counted in a gamma counter. Inset shows 22Na1 uptake rates at different concentrations of 22NaCl without treatment ()or pretreated 10 min with 1 mM CCCP (n). In (b), the experiments werecarried out as in (a) with 20 mM 22NaCl (). At 10 min, the reaction mixture was divided into two portions with the addition of 10 mMglucose (indicated by arrow) to one portion (m). Inset shows ATP levelsof deenergized (no glucose) cells () and energized (with 10 mM glucose) cells (n) at indicated times of Na1 uptake and Na1 extrusion.
In (c), the membrane vesicles (1 mg protein mL À 1) were incubated in (0.45 mCi mmol À 1) with 4 mM ATP (), without ATP (}), with 4 mM ATP plus 1 mM DCCD (~), with 4 mM ATP plus 1 mM CCCP (m), with 4 mM ATP plus 10 mM orthovanadate (&), with 4 mM ATP plus 100 mM monensin (), with 4 mM ATP plus 25 mM Na1-ionophore (’) and with 4 mM ATP plus 10 mM gramicidin D (n).
 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved (1 mg protein mLÀ1) were incubated in 20 mM Tris-HCl buffer pH 7.6containing 4 mM ATP, 5 mM MgCl2 with NaCl (), KCl (’), CaCl2 (m)and LiCl (~) at indicated concentrations. Inset shows a double reciprocal plot of the activity against the NaCl concentration.
Heise et al. (1992). Toluene destroyed the membrane integrity resulting in increased ATPase activities if the membrane preparations contained right-side-out vesicles.
The stimulation of ATPase activity by toluene treatment(data not shown) could not be observed, indicating that themembrane preparations consisted almost entirely of inside- The ATPase activity was only marginal without the addition of NaCl (Fig. 2). Increasing NaCl concentration led to an increase of ATPase activity with saturation at about 100 mM. No stimulation of the activity was observed by theaddition of monovalent cations such as K1 or Li1 ordivalent cation such as Ca21, suggesting that ATPase was specific for Na1. The Km value for Na1 was estimated fromthe 25.0 Æ 1.8 mM. The ATPase activity increased with the ATP concentration with saturation being reached at about 4 mM (Fig. 3a). The Km value for ATP and Vmax estimated bythe Lineweaver–Burke plot were 1.66 Æ 0.12 mM and0.66 Æ 0.05 mmol minÀ1 mgÀ1 protein, respectively (Fig. 3a,inset). The ATPase activity required the presence of MgCl with optimal activity at 5 mM (Fig. 3b). Concentration of 2 higher than 5 mM led to a decline in ATPase activity.
Optimal pH for the ATPase was around pH 7.5 (Fig. 3c).
Fig. 3. Dependence of ATPase on ATP, Mg21 and pH. Membrane It is noted that considerable ATPase activity was retained vesicles (1 mg protein mLÀ1) were incubated in 20 mM Tris-HCl buffer pH 7.6 containing 100 mM NaCl, 5 mM MgCl2 and indicated concentra- It was also checked whether salt stress could affect Na1- tions of ATP (a), or containing 100 mM NaCl, 4 mM ATP and indicatedconcentrations of MgCl stimulated ATPase activity of the plasma membrane. Na1- 2 (b), or containing 100 mM NaCl, 4 mM ATP, 5 mM MgCl2, at indicated pH values (c) of 6.0–7.0 (, 20 mM Mes-KOH stimulated ATPase was present in high amounts in cells buffer), 7.0–8.5 (m, 20 mM Hepes-KOH) and 7.5–9.0 (’, 20 mM Tris- grown at low salt and stimulated slightly in cells grown at HCl). Inset in (a) shows a double-reciprocal plot of the activity against the  2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Na1-stimulated ATPase in Aphanothece halophytica Table 1. Effect of inhibitors on the Na1-ATPase of plasma membrane ÃATP hydrolytic activity was assayed as described in ‘Materials andmethods’. Each inhibitor was added to the reaction mixture 10 min Fig. 4. Effect of salt stress during growth on Na1-ATPase. Membrane before the start of the reaction. One hundred percent activity corre- vesicles were prepared from cells grown under nonstress condition (m) with 0.5 M NaCl or under salt-stress condition () with 2.0 M NaCl. TheATPase assay was carried out as described in ‘Materials and methods’ atindicated concentrations of NaCl.
cells (Incharoensakdi & Laloknam, 2005), suggesting thatglucose could be utilized as energy source by A. halophytica.
The Na1 extrusion is likely due to the contribution by ATPase because glucose can yield ATP during its metabo- N-Ethylmaleimide, KNO3 and KSCN, which are inhibitors lism. The decreased ATP levels inside the cells during the of V-type ATPase, had little or no effect on ATPase (Table 1).
course of Na1 uptake (Fig. 1b, inset) also support the Similarly, no inhibition was caused by either azide, an contention that ATP was hydrolyzed by ATPase to provide inhibitor of F-type ATPase, or the protonophore CCCP.
a driving force for Na1 uptake and Na1 extrusion. This was Ouabain, an animal Na1/K1-ATPase inhibitor, did not further substantiated by Na1 uptake experiments using inhibit ATPase. Amiloride, a potent inhibitor of many membrane vesicles (Fig. 1c). Inhibition of ATPase by Na1-coupled transport systems including Na1/H1 antipor- orthovanadate resulted in strong inhibition of Na1 uptake.
ter, had no significant effect on ATPase. However, orthova- Na1-stimulated ATPase activity was observed in plasma nadate, an inhibitor of P-type ATPase, and DCCD could membrane vesicles (Fig. 2). This ATPase was specific for inhibit ATPase. Na1-gradient dissipators, gramicidin D and Na1 with no activity toward K1, Li1 and Ca21. The ATPase monensin, caused some stimulation of ATPase. All the was inhibited by orthovanadate and DCCD, whereas no results suggested that A. halophytica contains a P-type inhibition was observed by CCCP, amiloride, KNO N-ethylmaleimide, azide and ouabain (Table 1). On theother hand, both gramicidin D and monensin, which can dissipate electrochemical sodium ion gradient, were able tostimulate Aphanothece ATPase. This is to be expected This study demonstrated that A. halophytica could take up because the rate of ATP hydrolysis is controlled by ion Na1 in a concentration-dependent fashion (Fig. 1a). The potential, i.e. the lower the potential, the higher the activity.
uptake of Na1 by Aphanothece cells could be strongly Taken together, it is thus likely that the Na1-stimulated inhibited by the protonophore CCCP, suggesting the invol- ATPase in A. halophytica is a P-type ATPase.
vement of electrochemical proton gradient in the uptake The results in Fig. 1c provide strong evidence for the system (Fig. 1a, inset). Furthermore, Aphanothece cells also coupling of ATPase and Na1 transport. An increased uptake had Na1 extrusion capacity when energized with glucose of 22Na1 occurred upon the addition of ATP. The hydrolysis (Fig. 1b). The utilization of glucose for growth has been of ATP by ATPase is the driving force for Na1 transport.
reported in some photoautotrophic cyanobacteria, i.e. Ana- Furthermore, the abolition of Na1 transport observed under baena variabilis (Ohki & Katoh, 1975), Aphanocapsa 6714 conditions when ATP was added in the presence of either the (Pelroy et al., 1972) and Plectonema boryanum (White & Na1 ionophore or gramicidin D or monensin suggested that Shilo, 1975). Previously, the uptake of nitrate in the presence Na1 gradient might be involved in the transport. Strong of glucose was shown to increase in the starved Aphanothece inhibition of Na1 uptake by CCCP (Fig. 1c) indicated that  2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved mH1-dependent Na1/H1 antiporter might be involved in study using certain defined mutants such as the Na1/H1 the ATP-dependent Na1 transport. The participation of the antiporter deficient mutant is needed.
P-type ATPase in Na1 transport is also suggested bythe strong suppression of Na1 uptake by orthovanadate(Fig. 1c).
Membrane vesicles prepared from Aphanothece cells This work was supported by the Thailand Research Fund grown under stress condition (2.0 M NaCl) showed higher (BRG 4880004) to A. I. and partly by the scholarship from Na1-ATPase activity than that from vesicles of nonstressed the Faculty of Graduate Studies of Chulalongkorn Univer- (0.5 M NaCl) cells (Fig. 4). Similar observation was reported in a marine cyanobacterium Spirulina subsalsa subject toprolonged adaptation to hypersaline conditions (Gabbay-Azaria et al., 1994).
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