Respiratory activities of moderately halophilic bacteria from diverse origins were studied with respect to the requirement for Na+, the site of Na+-dependent reactions in the respiratory chain and the presence of a redoxdriven Na+ pump. All the moderately halophilic bacteria examined required 1.0-2.0 M-Naa for optimum growth.The NADH oxidase activity of the respiratory chain was influenced by the method of membrane preparation. When the cells were disrupted by a French press, the presence of 0.2 M-Na2So4 (or 0.4 M-NaCl) was required to protect against loss of Na+-dependent NADH oxidase activity. Membranes prepared by osmotic lysis retained high Na+-dependent NADH oxidase activity. Of eight moderate halophiles investigated, the six Gram-negative bacteria possessed Na+-dependent NADH oxidase activity. The site of Na+-dependent activation in the respiratory chain was located on the NADH : quinone reductase segment in all these halophiles. Other activities, such as succinate oxidase and the terminal oxidase, showed no specific requirement for Na+. Using inverted membrane vesicles prepared from these halophiles, it was found that membrane potential generation linked to NADH oxidation was not completely dissipated by addition of the protonophore carbonyl cyanide rnchlorophenylhydrazone (CCCP), but was entirely sensitive to CCCP plus the Na+-conducting ionophore monensin. A Na+-dependent NADH oxidase was not detected in membranes from the two Gram-positive halophiles, Mavinococcus halophilus and Micrococcus varians subsp. halophilus, that we investigated. Since the membrane potential generated by oxidation of NADH was completely dissipated by CCCP, these halophiles did not have any respiration-driven Na+ pumps.

The effect of Na+ on amino acid uptake by moderately halophilic bacteria was examined using a-aminoisobutyric acid (AIB) as a nonmetabolizable amino acid analogue. Of the eight moderate halophiles investigated, the six Gram-negative bacteria that have a respiration-driven Na+ pump specifically required Na+ for AIB uptake. On the other hand, the two Gram-positive bacteria that have no respiration-driven Na+ pump showed no requirement for Na+ for AIB uptake. Thus, the mode of energy coupling to amino acid transport was quite different between the Gram-negative and Gram-positive moderate halophiles tested : the former, but not the latter, utilized Na+ circulation for the active uptake of AIB.