Bacteroids of Rhizobium leguminosarum were formed rapidly in media containing 0.35% yeast extract, but not in media containing 0.1% yeast extract. Bacteroid production in the former media could be prevented by increasing the oxygen tension, suggesting that the limited oxygen supply within root nodules may be the principal factor initiating the natural formation of bacteroids. The artificially produced bacteroids were shorter and possessed a more irregular external morphology than bacteroids extracted from red root nodules of field pea, although the internal organization of both types of bacteroids was similar.During the development of bacteroids in laboratory media there was a rapid decrease in viable count and a concurrent increase in nephelos, although this increase was at a reduced rate compared to that exhibited by the normal small rod forms of rhizobia. These data indicate an inhibition of cellular division and a continued synthesis of at least certain protoplasmic constituents. In this connection the capacity to synthesize nucleic acids was similar in both artificially produced bacteroids and the small, rod types of the test organism, although some minor differences were found with bacteroids in situ within the nodules when nodule age varied.All types of bacteroids, whether they were within the nodule, had been extracted from the nodule, or were produced in laboratory media, suffered an impairment in the synthesis of protein and cell wall. Therefore, in several important respects, artificially produced and naturally produced bacteroids are physiologically similar. The alteration in wall-synthesizing capacity and the possible formation of limited amounts of abnormal wall material incapable of employing certain ions, such as Mg2+, for maintenance of rigidity is a likely cause of both the inhibition of cellular division and of the abnormal external morphology of these rather unusual cells.

Electron microscopy of infection threads and bacteria in young root nodules of Medicago sativa. J. Bacteriol. 86:125-137. 1963.-Ultrathin sections of alfalfa nodules (Medicago sativa L.

A relatively small but consistent increase in the frequency of spore formation by an oligosporogenous strain of Bacillus popilliae (NRRL B-2309M) was obtained by adding 0.1% sodium pyruvate to the sporulation medium. The frequency of spore formation was essentially the same when a low level of glucose, trehalose, or glucose-6-phosphate or a high level of a-methyl-D-mannoside was added as the carbon and energy source. Many other variations in the cultural medium and cultural conditions failed to enhance spore formation of 2309M, and no spores were found in four asporogenic strains under any of the conditions tried. There were no significant differences between the 2309M strain and three nonsporeforming cultures with respect to (i) the rate and extent of growth, (ii) the rates of glucose utilization, or (iii) volatile acid production and utilization. None of the cultures tested was found to produce detectable levels of extracellular protease or an antibiotic. The only consistent marker found associated with spore formation was the development of catalase activity, and this activity was stimulated by heating at 80 C for 10 min. This was not found unless morphological evidence of spore formation was observed. The germination of the spores formed by 2309M in vitro was stimulated by heat shock and by the addition of pyruvate to the germination medium. ' Journal article no. 5553, Michigan Agricultural Experiment Station.

A relatively small but consistent increase in the frequency of spore formation by an oligosporogenous strain of Bacillus popilliae (NRRL B-2309M) was obtained by adding 0.1% sodium pyruvate to the sporulation medium. The frequency of spore formation was essentially the same when a low level of glucose, trehalose, or glucose-6-phosphate or a high level of α-methyl- d -mannoside was added as the carbon and energy source. Many other variations in the cultural medium and cultural conditions failed to enhance spore formation of 2309M, and no spores were found in four asporogenic strains under any of the conditions tried. There were no significant differences between the 2309M strain and three nonsporeforming cultures with respect to (i) the rate and extent of growth, (ii) the rates of glucose utilization, or (iii) volatile acid production and utilization. None of the cultures tested was found to produce detectable levels of extracellular protease or an antibiotic. The only consistent marker found associated with spore formation was the development of catalase activity, and this activity was stimulated by heating at 80 C for 10 min. This was not found unless morphological evidence of spore formation was observed. The germination of the spores formed by 2309M in vitro was stimulated by heat shock and by the addition of pyruvate to the germination medium.

Barbituric acid is required at a level of 0.1% for maximum growth of Bacillus popilliae in a synthetic medium. However, only a trace amount of that added (~2%) disappeared from the medium during growth. A general distribution of the 14C from 2-14C-barbituric acid among cell components was observed, but a small amount of the radioactivity from this isotope was consistently associated with both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The 14C found in RNA was uniformly distributed throughout the 4 S, 16 S, and 23 S RNA fractions, and control experiments indicated that the association of 14C from 2-14C-barbituric acid with the nucleic acids may occur by nonspecific absorption. The presence of barbituric acid resulted in very significant stimulation of both nucleic acid and protein synthesis. The mechanism of this stimulation is unresolved.