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The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, A&) and either dimethyloxazolidinedione or methylamine (osmotic component, ApH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by A,). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0. Vibrio costicola is a moderately halophilic bacterium which requires 1 M NaCl for optimal growth (13) and lyses in media of low osmotic strength (9). Lysis can be prevented by salts other than NaCl, but there is a specific Na+ requirement for carrier-mediated transport (24).Understanding the specific requirement for Na+ in transport requires knowledge of the electrochemical ion gradients in V. costicola.A transmembrane proton motive force (APIH+) may be established in various microorganisms by proton efflux by using respiratory energy, light energy in photosynthetic organisms or extreme halophiles, or the energy from ATP hydrolysis (15,36), resulting in a membrane potential (A+i, interior negative) and an osmotic component (ApH, interior alkaline) such that in millivolts, AP.H+ = A+ -60 ApH, at 30°C (30).Energy may be released from the osmotic component by proton-symport mechanisms in medium more acidic than the internal pH (pHi); at a higher pH of the medium (pHo), the cytosol is more acidic than the medium, so A4 must compensate for the inverse pH gradient.In certain bacteria, a sodium motive force may be formed by several mechanisms, resulting from the outward-directed movement of Na+. t National Research Council of Canada paper no. 22690.First, the energy of the proton motive force may be converted to a sodium motive force through Na+/H+ antiport activity as found in Alteromonas haloplanktis (33), Halobacterium halobium (26), alkalophilic bacilli (29), Mycoplasma mycoides (6), and others, including Escherichia coli (4, 44) (for a review, see reference 25). In addition to a role in energy coupling, antiport activity may be involved in regulation of cytosolic pH (34). Second, in Klebsiella aerogenes, efflux of Na+ th...

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The moderately halophilic bacterium Vibrio costicola accumulates a-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na+ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCI as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCI) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCI. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.

The importance of proton movements was assessed in the moderate halophile Vibrio costicola. When anaerobic cells in acidic buffer (pH 6.5) were given an 02 pulse, protons were extruded regardless of the presence of Na+. At pH 8.5, however, V. costicola produced an acidic response to an 02 pulse in the absence of Na+ and an alkaline response when Na+ was present. An Na+/H+ antiport activity was confirmed at pH 8.5. All of these effects were prevented by protonophores or butanol treatment. Growth in complex medium at pH 8.5 was prevented by a high concentration (50 FLM) of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or a low concentration (5 ,uM) of another protonophore, 3,3',4',5-tetrachlorosalicylanilide (TCS). The relative ineffectiveness of the former protonophore was caused by the proteose peptone and tryptone ingredients of the complex medium, since 5 ILM completely prevented growth in their absence. The results are explained by a primary respiratory-linked proton efflux coupled to a secondary Na+/H+ antiport operating at alkaline pH. Evidence was seen for a role of Na+ in stimulatin.g proton influx at alkaline pH, presumably via the pH homeostasis mechanism.