Mutants of Rhizobium trifolii strain 7012 defective in C4-dicarboxylate transport were isolated by using a selective procedure based on pH indicator media. The mutant strains CR7098 and CR7099 failed to grow on or transport succinate, fumarate, or malate, but grew at wild-type rates on several other carbon sources.The C4-dicarboxylate transport system was inducible in strain 7012, but was expressed constitutively in four out of five succinate-positive revertants of strain CR7098. In the fifth CR7098 revertant (strain CR8008) the system was inducible. However, in contrast to strain 7012, strain CR8008 failed to use the C4-dicarboxylates in the presence of a second carbon source. Revertants of strain CR7099 were similar to strain 7012. Both strains CR7098 and CR7099 nodulated white and red clover at a rate similar to that ofstrain 7012, but nodules formed by the mutant strains were white and ineffective. Microscopic examination showed that the pattern of development of white clover nodules formed by strain CR7098 was similar to that observed with nodules formed by strain 7012, except that large amounts of starch accumulated in bacteroid-filled cells and senescence occurred earlier. Revertant strains were effective, except for strain CR8008, which formed ineffective nodules. The results show that a supply of C4-dicarboxylates to bacteroids is essential for nitrogen fixation in clover nodules. However, rhizobia within plant cells must also utilize other carbon sources to support growth and division.

SUMMARYWhen Streptococcus lactis was starved in phosphate buffer the intracellular amino acid pool was rapidly released into the external medium from the onset of starvation whereas lactic dehydrogenase and DNA appeared in the suspending buffer only as organisms began to lose viability. Addition of spermine enhanced survival and suppressed the release of ultraviolet-absorbing material.Thin sections of Streptococcus lactis were examined by electron microscopy at intervals during starvation in a number of environments. Ribosome particles rapidly disappeared from organisms starved in the absence of Mg2+ and nonviable organisms remained structurally intact; in the presence of Mg2+ ribosomes were retained but, after prolonged starvation, some organisms autolysed soon after the death rate increased. Addition of a suitable energy source maintained cell structures intact for a much longer period but again lysis occurred as the death rate increased. Starvation led to unfolding or extrusion of the mesosomes and dislocation of the nuclear material. The death rate of starved S. lactis organisms in phosphate buffer partly depended on the presence of Mg2+, which probably acted by promoting polymer stability, particularly RNA. In this environment, a suitable exogenous energy source further enhanced survival which may ultimately be a function of cell wall and/or membrane stability.

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Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1–153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.