Distribution of Na+-dependent respiration and a respiration-driven electrogenic Na+pump in moderately halophilic bacteria

Abstract

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.

Correlation between the respiration-driven Na+ pump and Na+-dependent amino acid transport in moderately halophilic bacteria

“…halophilus (ATCC 21971) showed nearly the same activity with either NaCl or KCl, exhibiting no requirement for Na+ for the active uptake of AIB. In our previous paper (Unemoto et al, 1992), the above six Gram-negative, but not the two Gram-positive, halophiles were demonstrated to have a respiration-driven Na+ pump. Therefore, these results strongly suggest that moderate halophiles that have a respiration-driven Na+ pump require Na+ for the active uptake of AIB.…”

“…The moderately halophilic bacteria used in this study and their culture conditions were described previously (Unemoto et al, 1992). Each bacterium was grown in the presence of 1.0 M-NaC1 and harvested at the late exponential phase of growth by centrifugation.…”

“…Therefore, halophilic bacteria that require Na+ for optimal growth are generally considered to utilize A p N a + as the driving force of amino acid uptake. Recently, we have demonstrated that among moderately halophilic bacteria from diverse origins, Gram-negative, but not Gram-positive, halophiles have a respiration-driven Na+ pump (Unemoto et al, 1992). At present, amino acid transport has been investigated.…”

Existence of Na+‐translocating NADH‐quinone reductase in Haemophilus influenzae

“…The suspension was passed through a French pressure cell (Aminco) at 8000 lbf.in -2. The membrane fraction was isolated by ultracentrifugation as described in [11], and was finally suspended in 10 mM HEPES-Tris (pH 7.5)/5 mM MgSO4/10% (w/v) glycerol at about l0 mg protein per ml. It was stored at -80°C.…”

“…The membrane potential generated by Na+-dependent oxidation of NADH is not completely dissipated by the proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP) in marine and moderately halophilic bacteria [11].…”