No abstract

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.

Successful growth of Mycobacterium lepraemurium was observed in cultures of mouse peritoneal macrophages. The optimal host cell maintenance medium was composed of 40% horse serum, 50% of the chemically defined medium NCTC 109, and 10% of a 1: 5 dilution of beef embryo extract, supplemented with both liver extract and ferric nitrate. Multiplication of the bacilli was observed in 1 week and maximal growth in 6 to 7 weeks. All macrophages were filled with tens to hundreds of the organisms in cultures showing maximal growth. Glycerol caused an increase in the normal length of M. lepraemurium, without a corresponding increase in the number of the bacilli. Elongation of M. lepraemurium was observed 3 or 4 days after infection. Rapid and uniform growth of M. lepraemurium was achieved in serially transferred cultures (subcultures). The cumulative increase of the number of intracellular bacilli was 1.4 X 1020-fold in 14 transfers over a period of 68 weeks in one series, and 1017-fold in 12 transfers over a period of 56 weeks in another series. The generation time of M. lepraemurium was 7 days, a growth rate which approximates the fastest growth of the organisms in vivo. Organisms harvested from cultures at various stages of growth produced murine leprosy in mice, but showed no growth in bacteriological media. The present model offers an opportunity for studies on the host-parasite relationship without the complication of extracellular growth of the parasites.

of penicillin-induced spheroplasts of Salmonella typhimurium were examined by electron and light microscopy. The process of spheroplast formation was followed for a period of 20 hr from its inception. Flagella were found to be confined to those areas of the spheroplast where cell-wall fragments remained. Flagella disappeared as the spheroplasts aged. Spheroplasts produced from nonflagellated organisms were found incapable of synthesizing flagella. Upon inactivation of the penicillin, however, flagella again were synthesized by spheroplasts during reversion to their original rod form. Flagella formation, it is suggested, is dependent on prior synthesis of the normal cell wall. The morphology of the microorganisms at the time of appearance of new flagella is described.

described the relationship between flagellation and motility in penicillin-induced spheroplasts of Salmonella typhimurium, and they concluded that the formation of flagella was dependent upon prior cell wall synthesis. This conclusion has now been confirmed with Escherichia coli (ATCC 13070), the results found being identical to those reported for S. typhimurium. Further, spheroplasts made by deprival of diaminopimelic acid (DAP; P. Meadow et al., Biochem. J. 66:270, 1957) were investigated by use of a DAP-requiring mutant of E. coli (ATCC 13071). The process of spheroplast formation differs in that no "rabbit-eared" stage was present as seen in the penicillin method. Upon DAP deprival, the cells become oval and continue to swell, becoming large spheroplasts. This mode of spheroplast formation is thought to be responsible for the general distribution of flagella around the resulting spheroplasts when observed optically by use of flagellar stains, or with an electron microscope. This is strikingly different from the flagella distribution found in penicillin-induced spheroplasts, where they are confined to areas of "rabbit-eared" fragments of the parent rodshaped cell. Spheroplasts (formed either by DAP deprival or penicillin induction) from nonflagellated cells grown at 44 C, upon transfer to 37 C, do not regenerate flagella as do organisms possessing cell walls. We conclude that the inability of spheroplasts to form flagella is not due to one of the multiple effects of penicillin (J. T. Park, Antimicrobial Agents and Chemotherapy-1963, p. 366, 1964), since DAP deprival affects only formation of rigid mucopeptide in the cell wall. E. coli, when exposed to ethylenediaminetetraacetic acid (EDTA)-lysozyme atpH 8.0 (R. G. E. Murray et al., Can. J. Microbiol. 11:547, 1965), also became nonmotile upon loss of its rod shape. When Trypticase Soy broth (BBL), with 15% (w/v) sucrose and 0.1 M MgSO4, was added after 5-min exposure of this organism to EDTAlysozyme, reversion of approximately 40% of nonmotile spheroplasts to motile normal cells was observed. After prolonged EDTA-lysozyme