Relationship between formate hydrogen lyase and proton-potassium pump under heterolactic fermentation in Escherichia coli: functional multienzyme associations in the cell membrane

Anaerobically grown glucose-fermenting E. coli cells produce molecular hydrogen, acidify the medium and uptake potassium ions. It was shown that the H2 release and the proton-potassium exchange with the fixed (2H+/K+) stoichiometry of the initial DCC-sensitive fluxes were lost in mutants with the deleted fdhF gene or the hycA-H operon responsible for the biosynthesis of formate dehydrogenase H (FDH,H) or hydrogenase 3 (H3), respectively, which are the main components of the formate hydrogen lyase FHL(H). However, both processes occurred in mutants with the deleted hycE, hycF or hycG genes encoding the major and minor components of H3, respectively. The K+ uptake was sensitive to the osmotic shock resulting from glucose addition to the medium and decreased significantly in the presence of valinomycin. The H2 release and the 2H+/K+ exchange were absent in the mutant with the deleted hycB gene encoding the corresponding minor component of H3. This mutant acidified the medium and uptook K+ with Km typical for TrkA, but the stoichiometry of the DCC-inhibited fluxes was variable, and the K+ gradient between the cytoplasm and the medium in this mutant was lower than in the mutants lacking other minor components of H3. The results obtained suggest that the hycB gene product, FdhF and HycE, form probably the FHL(H) complex that directly interacts with the H+-ATPase complex F0F1 and the TrkA(H) system of K+ uptake. Such a multienzyme association is responsible for the H2 production and 2H+/K+ exchange. The major and other minor components of H3 have probably no direct role in the H2 production and 2H+/K+ exchange. H2 production by precursor's or hycE mutant's protoplasts treated with toluene was shown to occur upon addition of the thiol reagent dithiothreitol to the medium containing ATP, potassium ions, NAD+, and NADH. H2 production was inhibited by DCC. The quantity of available thiol groups in membrane vesicles of the precursor or the hycE, hycF or hycG mutants, in which the H2 production and 2H+/K+ exchange were observed, was larger than in other mutants. The number of SH groups decreased in the presence of DCC. These results indicate a significance of the thiol groups for the function of the proposed association.     

Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli

Proton translocation, coupled to formate oxidation and hydrogen evolution, was studied in anaerobically grown fermenting Escherichia coli JW136 carrying hydrogenase 1 (hya) and hydrogenase 2 (hyb) double deletions. Rapid acidification of the medium by EDTA-treated anaerobic suspension of the whole cells or its alkalization by inverted membranes was observed in response to application of formate. The formate-dependent proton translocation and 2H(+)-K(+) exchange coupled to H(2) evolution were sensitive to the uncoupler, carbonylcyanide-m-chlorophenylhydrazone, and to copper ions, inhibitors of hydrogenases. No pH changes were observed in a suspension of formate-pulsed aerobically grown ("respiring") cells. The apparent H(+)/formate ratio of 1.3 was obtained in cells oxidizing formate. The 2H(+)-K(+) exchange of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes does take place in JW136 cell suspension. Hydrogen formation from formate by cell suspensions of E. coli JW136 resulted in the formation of a membrane potential (Deltapsi) across the cytoplasmic membrane of -130 mV (inside negative). This was abolished in the presence of copper ions, although they had little effect on the value of Deltapsi generated by E. coli under respiration. We conclude that the hydrogen production by hydrogenase 3 is coupled to formate-dependent proton pumping that regulates 2H(+)-K(+) exchange in fermenting bacteria.     

Formate increases the F0F1-ATPase activity in Escherichia coli growing on glucose under anaerobic conditions at slightly alkaline pH

Escherichia coli growing on glucose under anaerobic conditions at slightly alkaline pH carries out a mixed-acid fermentation resulting in the production of formate among the other products that can be excreted or further oxidized to H(2) and CO(2). H(2) production is largely dependent on formate dehydrogenase H and hydrogenases 3 and 4 constituting two formate hydrogen lyases, and on the F(0)F(1)-ATPase. In this study, it has been shown that formate markedly increased ATPase activity in membrane vesicles. This activity was significantly (1.8-fold) stimulated by 100mM K(+) and inhibited by N,N(')-dicyclohexylcarbodiimide and sodium azide. The increase in ATPase activity was absent in atp, trkA, and hyf but not in hyc mutants. ATPase activity was also markedly increased by formate when bacteria were fermenting glucose with external formate (30mM) in the growth medium. However this activity was not stimulated by K(+) and absent in atp and hyc but not in hyf mutants. The effects of formate on ATPase activity disappeared when cells were performing anaerobic (nitrate/nitrite) or aerobic respiration. These results suggest that the F(0)F(1)-ATPase activity is dependent on K(+) uptake TrkA system and hydrogenase 4, and on hydrogenase 3 when cells are fermenting glucose in the absence and presence of external formate, respectively.     

Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness.

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F0F1 ATP-synthase and formate oxidation by formate hydrogen lyase (FHL) has been established in inverted membrane vesicles of Escherichia coli JW 136 mutant with double deletions (delta hya/ delta hyb) of hydrogenase 1 and 2 grown anaerobically on glucose in the absence of external electron acceptors (pH 6.5). ATP synthesis was suppressed by H+ -ATPase inhibitors N,N'-dicyclohexylcarbodiimide (DCCD) and sodium azide as well as by the protonophore carbonyl cyanide-m-chlorophenyhydrazone (CCCP). Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of vesicles. The maximal rate of ATP synthesis (0.83 microM/min x mg protein) stimulated by K+ ions was determined when sodium formate, ADP and inorganic phosphate were applied simultaneously. The results confirm the assumption about the dual role of hydrogenase 3, formate hydrogen lyase subunit, which is able to couple the reduction of protons to H2 and their translocation through a membrane with chemiosmotic synthesis of ATP.     

The pH dependence of proton-deuterium exchange, hydrogen production and uptake catalyzed by hydrogenases from sulfate-reducing bacteria

Different patterns have been found in the pH dependence of hydrogenase activity with enzymes purified from different species of Desulfovibrio. With the cytoplasmic hydrogenase from Desulfovibrio baculatus strain 9974, the pH optima in H2 production and uptake were respectively 4.0 and 7.5 with a higher activity in production than in uptake. The highest D2-H+ exchange activity was found also at pH 4.0 but the optima differed for the HD and the H2 components. Both similarly rose when the pH decreased from 9.0 to 4.5, but the rate of H2 evolution slowed whereas the HD evolution continued rising till pH values around 3.0 were reached. The H2 to HD ratio at pH above 4.5 was higher than one. With the periplasmic hydrogenase from Desulfovibrio vulgaris Hildenborough, the highest exchange activity was near pH 5.5, the same value as in hydrogen production. The periplasmic hydrogenase from Desulfovibrio gigas had in contrast the same pH optimum in the exchange (7.5-8.0) as in the H2 uptake. The ratio of H2 to HD was below one for both enzymes. These different patterns may be related to functional and structural differences in the three hydrogenases so far studied, particularly in the composition of their catalytic centers.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Cation exchanges of yeast in the absence of magnesium.

Environmental Mg2+ was found to influence the K+/Na+ exchange rate of metabolizing yeast. Addition of EDTA increased the exchange rate and Mg2+ reversed the effect of EDTA. Yeast starved in the absence of Mg2+ exchanged cellular K+ or Na+ for external H+ when maintained at acidic pH. The exchange rate depended on cellular pH and showed the same kinetics for both K+ and Na+. At acidic pH, the presence of external cations neither inhibited H+ absorption nor changed the cation/H+ 1 : 1 stoichiometry. At neutral pH, external cations inhibited H+ influx but did not change the cation efflux. The K+/Na+ exchange is discussed as electrically coupled and the K+/H+ and Na+/H+ exchanges as electroneutral antiports.     

Escherichia coli membrane proton conductance and proton efflux depend on growth pH and are sensitive to osmotic stress

The dependence of Escherichia coli membrane H+ conductance (Gm H+) with a steady-state pH in the presence and absence of an external source of energy (glucose) was studied, when cells were grown under anaerobic and aerobic conditions, with an assay pH of 7.0. Energy-dependent H+ efflux by intact cells growing at pH of 4.5-7.5 was also measured. The elevated H+ conductance and lowered H+ flux were shown for cells growing in acidic pH and under anaerobic conditions, when bacteria were fermenting glucose. The atp mutant, which is deprived of the F0F1- adenosine triphosphatase, had less Gm H+ independent of growth conditions. In contrast with wild-type or precursor strain, a remarkable difference in Gm H+ for atp mutant was observed between aerobic and anaerobic conditions; such a difference was significant at pH 4.5. These results could indicate distinguishing pathways determining Gm H+ under anaerobic conditions after the fermentation of glucose at different pH and an input of the F0F1-adenosine triphosphatase in Gm H+. In addition, the effect of osmotic stress was demonstrated with grown cells. Gm H+ and H+ efflux both were increased after hyperosmotic stress at pH 7.5, and these changes were inhibited by N,N\'-dicyclohexylcarbodiimide, whereas these changes were lower in atp mutant. A role of the F0F1-adenosine triphosphatase in osmo-sensitivity of bacteria was confirmed under fermentative conditions.     

Relation between H2 production and energy-dependent metabolism of H+ and K+ ions in E.coli

Anaerobically grown E. coli escape H2 into the medium during the operation of H(+)-K(+)-pump exchanging 2H+ from a cell for one K+ of the medium. Anaerobic cells grown in the nitrate medium as well as the aerobically grown bacteria possessed neither 2H+/K+ exchange system, nor the ability for H2 production. Introduction of N,N'-dicyclohexylcarbodiimide into the medium, the removal of external K+ or the decrease of external osmotic pressure blocked both the functioning of H(+)-K(+)-pump and H2 production. The substitution of glucose by lactate reduced the activity of bacteria without change in pump operation and H2 production. It is assumed that formate-hydrogen lease and H(+)-K(+)-pump are working in collaboration.     

The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH

The hyc operon of Escherichia coli encodes the H2-evolving hydrogenase 3 (Hyd-3) complex that, in conjunction with formate dehydrogenase H (Fdh-H), constitutes a membrane-associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2-evolving hydrogenase (Hyd-4) was identified in E. coli. In this study the roles of the hyc- and hyf-encoded systems in formate-dependent H2 production and Fdh-H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd-3- and Fdh-H-dependent, and Fdh-H activity was also mainly Hyd-3-dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd-4, Fdh-H and F0F1-ATPase-dependent, and Fdh-H activity was partially dependent on Hyd-4 and F0F1-ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh-H activity are dependent on both the F0F1-ATPase and a novel FHL, designated FHL-2, which is composed of Hyd-4 and Fdh-H, and is driven by a proton gradient established by the F0F1-ATPase.     

NMR studies on Na+ transport in Synechococcus PCC 6311.

The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.     

Interaction of membrane proton conductivity,membrane and oxidation-reduction potential in Escherichia coli

It was shown that the proton conductivity of Escherichia coli membranes depends on pH and other conditions of bacterial growth. It is considerably lower in cells fermenting glucose and accomplishing the nitrate-nitrite respiration compared with cells accomplishing the oxygen respiration. Proton conductivity increases substantially with decreasing pH of medium. It was found that proton conductivity is related to the redox and membrane potentials of cells. The energy-dependent flux of protons from cells and the ATPase activity of membrane vesicles considerably vary depending on whether bacteria are grown under aerobic or anaerobic conditions. The H+ flux from cells fermenting glucose (pH 7.5) was 1.7 times greater than the H+ flux from cells that accomplish the nitrate-nitrite and oxygen respiration. The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity increased 2.5 times as K+ concentration increased to 100 mM (including residual K+ in potassium-free medium). The DCCD-sensitive ATPase activity considerably decreased with decreasing pH of medium, whereas the ATPase activity that was not suppressed by DCCD was stimulated. These results can be used for establishing the relationship between membrane proton conductivity and the energy-dependent H+ flux and ATPase activity.     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H2) when grown on glucose. H2 formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H2 was found to be dependent on Hyd-4 and the F0F1-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate, H2 production dependent on pH was investigated. In both types of cells, H2 production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N'-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F0F1-ATPase. The results indicate that Hyd-3 has a major role in H2 production at acidic pH independently on the F0F1-ATPase.     

Oxidation of Carbon Monoxide in Cell Extracts of Pseudomonas carboxydovorans

Extracts of aerobically, CO-autotrophically grown cells of Pseudomonas carboxydovorans were shown to catalyze the oxidation of CO to CO(2) in the presence of methylene blue, pyocyanine, thionine, phenazine methosulfate, or toluylene blue under strictly anaerobic conditions. Viologen dyes and NAD(P)(+) were ineffective as electron acceptors. The same extracts catalyzed the oxidation of formate and of hydrogen gas; the spectrum of electron acceptors was identical for the three substrates, CO, formate, and H(2). The CO- and the formate-oxidizing activities were found to be soluble enzymes, whereas hydrogenase was membrane bound exclusively. The rates of oxidation of CO, formate, and H(2) were measured spectrophotometrically following the reduction of methylene blue. The rate of carbon monoxide oxidation followed simple Michaelis-Menten kinetics; the apparent K(m) for CO was 45 muM. The reaction rate was maximal at pH 7.0, and the temperature dependence followed the Arrhenius equation with an activation energy (DeltaH(0)) of 35.9 kJ/mol (8.6 kcal/mol). Neither free formate nor hydrogen gas is an intermediate of the CO oxidation reaction. This conclusion is based on the differential sensitivity of the activities of formate dehydrogenase, hydrogenase, and CO dehydrogenase to heat, hypophosphite, chlorate, cyanide, azide, and fluoride as well as on the failure to trap free formate or hydrogen gas in coupled optical assays. These results support the following equation for CO oxidation in P. carboxydovorans: CO + H(2)O --> CO(2) + 2 H(+) + 2e(-) The CO-oxidizing activity of P. carboxydovorans differed from that of Clostridium pasteurianum by not reducing viologen dyes and by a pH optimum curve that did not show an inflection point.     

The number of accessible SH-groups in Escherichia coli membrane vesicles is increased by ATP or by formate

The number of accessible SH-groups was determined in membrane vesicles prepared from Escherichia coli growing in fermentation conditions at slightly alkaline pH on glucose with or without added formate. Addition of ATP or formate to the vesicles caused a approximately 1.4-fold increase in the number of accessible SH-groups. The increase was inhibited by treatment with N-ethylmaleimide or the presence of the F(0)F(1)-ATPase inhibitors N,N(')-dicyclohexylcarbodiimide or sodium azide. The increase in accessible SH-groups was also absent in strains with the ATP synthase operon deleted or with the single F(0) domain cysteine Cysb21 changed to Ala. Using hyc and hyf mutants, it was shown that the increase was also largely dependent on hydrogenase 4 or hydrogenase 3, main components of formate hydrogen lyase, when bacteria were grown in the absence or presence of added formate. These results suggest a relationship between the F(0)F(1)-ATP synthase and hydrogenase 4 or hydrogenase 3 under fermentation conditions.     

Electrochemical study of proton translocation function of hydrogenase 3

The oxidation of formate associated with fast acidification of medium by whole Escherichia coli cells lacking both hya and hyb hydrogenases was studied. The extent of acidification was dependent on the amount of formate added. An average H+/formate ratio of 1.3 was obtained. The proton release was inhibited by carbonyl cyanide m-chlorophenylhydrazone. Inverted vesicles of E. coli were found to translocate protons upon oxidation of formate at pH 6.5. The extent of alkalization was also dependent on the amount of formate added. The maximum H+/formate ratio for this reaction was close to 0.6. Formate oxidation by inverted vesicles from E. coli (delta hya delta hyb) was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. It was supposed that the hydrogenase 3 (hyc) component of E. coli formate hydrogen lyase is responsible for the translocation of protons at low pH.     

The pha2 gene cluster involved in Na+ resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter

Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.