Genetic transformations of pneumococcus mediated by streptomycin-induced lysates were studied to gain some insight into the nature of freshly released transforming principle, and the influence of the physiologic state of the donor population on the transformation process. It was found that streptomycin could make the DNA of sensitive cells available for transformation of other cells. Living cultures of pneumococcus growing exponentially in ordinary media were also found to discharge significant quantities of genetically active DNA. Such cultures, not treated with any drug, showed no evidence of concomitant cell disintegration or death. Both single markers and small linkage groups could be transferred in transformations mediated by drug-induced lysates and by filtrates of living cultures. The quantity of DNA liberated is small (less than 0.1 µg per ml), but these transformations are at least as efficient as transformations mediated by purified DNA, when compared on the basis of total DNA available. Up to 1 per cent of the cells in an average recipient culture can be transformed by a small quantity of culture fluid. Both in drug-treated and in untreated cultures the amount of transforming activity increased and then decreased during growth of the culture. Although the source of transforming DNA in growing cultures could not be established, the decline in the transforming activity of aging drug-treated or untreated cultures was attributed to the presence of deoxyribonuclease. The release of this nuclease by pneumococcal cultures midway in exponential growth is sufficient to result in a mild degradation of the low concentration of freshly released transforming agent present. Maximal release of active transforming agent by a living culture coincided in time with the development of maximal receptivity to exogenous DNA by that culture. As a result, recombinants could be recovered from appropriately genetically marked strains growing in each other's presence. In view of these results, it seems possible that DNA-mediated transformations might provide, or might have provided, a mechanism of genetic recombination in nature for some bacterial species in which sexual mechanisms may not be available.

A B S T R A C TThe fine structure of an unencapsulated strain of Diplococcus pneumoniae is described. A striking feature of thcsc bacteria is an intracytoplasmic m e m b r a n e system which appears to be an extension of septa of dividing bactcria. The possible function of these structures and their relationship to the plasma m e m b r a n e and other types of intracytoplasmic membranes found in pncumococcus is discussed. I N T R O D U C T I O NO u r main interest in the fine structure of Diplococcus pneumoniae stems from the fact that these bacteria readily undergo genetic transformation. Prior to undertaking electron microscope studies on this process, the fine structure of pneumococcal cells in thin sections was examined. During the preliminary stage of these studies on a transformable strain, we observed some unique membranous structures which, to the best of our knowledge, have not previously been described in bacteria. M A T E R I A L S A N D M E T H O D SUnencapsulated strains of Diplocoecus pneumoniae R6, R1, and some nutritional mutants derived from R6 were used in these studies. These strains were derived from Diplovoceus pneumoniae R36A (1) which has been used as a laboratory strain for over 20 years. Forty-ml batches of bacteria were grown in a modified Adams-Roe medium (2) (medium A) or in a semisynthetic medium (3) (medium B) at 37°C in 25 x 150 mm tubes, without aeration. Small inocula (105 cells/ml) of cells from the exponential phase of growth were used and the cultures were harvested in the late exponential phase. In these media, stationary phase cultures undergo spontaneous autolysis, the first recognizable stage of which is a quantitative conversion of the cocci to fragile spherical bodies of fairly uniform size which are deficient in their cell walls. Throughout this paper, such preparations will be referred to as "spheroplasts."The bacteria were fixed and stained according to the method of Ryter and Kellenberger (4), embedded in cross-linked methacrylate, and sectioned with a Porter-Blum mlcrotome using a diamond knife. The sections were stained with lead according to the method of Karnovsky (5) (method B) and were examined in the RCA electron microscopes models 2B, 3F, or in the Siemens Elmiskop I. R E S U L T SThe nuclear region of pneumococcus resembles that of other bacteria prepared by the method of Ryter and Kellenberger (4). It is located centrally in the cell, is devoid of a nuclear membrane, and has a density lower than that of the surrounding cytoplasm (Fig. 1). The nuclear region is filled with more or less tightly packed uniform fibrils 25 to 30 A wide (Fig. 2).Differences in the internal structure of the nuclear region could be observed from one experiment to another or even in adjacent cells within the same section, in spite of the fact that the preparative procedure was the same throughout.These variations were essentially of three types: variation in the degree of interpenetration of the nuclear region and the cytoplasm which ranged 453 on

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Growing populations of Pneumococcus were found to release into the culture medium deoxyribonucleate-containing material with genetic transforming activity. Active material was maximally produced at the time when the culture was most responsive to added deoxyribonucleate. Since mixed cultures thus give rise to recombinants, it may be that transformation provides a natural mechanism of genetic recombination for Pneumococcus .