Cytoplasmic membrane of Rhizobium meliloti bacteroids. II. Functional differentiation and generation of membrane potentials

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Abstract
The expression of the nitrogenase enzyme system in the alfalfa symbiont R. meliloti is associated with a process of functional differentiation of the bacterial cytoplasmic membrane. Morphological changes are complemented by an increase in cellular volume and enclosure of the bacteroids in a plant-derived peribacteroidal membrane. The average volume of bacteroids isolated in isotonic media was 3 times that of air-grown cells and the average protein content increased by 3- to 4-fold. The induction of transmembrane electrical potentials was observed on oxygenation of anaerobic cells in the presence of succinate or sucrose. Membrane potentials were measured with radioactive probes in static experiments or by flow dialysis. Ca2+ enhanced bacteroid membrane potentials which were greater in magnitude (negative inside) than those of air-grown cells. Bacteroids exhibited weak H+ transport activity in response to respiration and had no measurable pH gradient as determined by 31P NMR of intracellular phosphate ion. This finding contrasted with free-living R. meliloti which developed a strong, directly measurable H+ pumping action on oxygenation. A fluorescent probe indicated that both air-grown cells and isolated bacteroids became energized in the same manner upon oxygenation, even though probes of membrane potential other than 86Rb+ indicated reverse polarity of the electrical potential of the former. The Ca2+ effect on the bacteroid cytoplasmic membrane was also qualitatively demonstrable with the fluorescent probe. Ca2+ caused respiration-supported bacteroid membrane potentials to increase. This effect was accompanied by increases in membrane fluidity and nitrogenase activity in the absence of external reducing agents or added ATP.