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The log phase cells of autolytic Micrococcus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced.A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was arranged in tetrads or in clusters consisting of regular tetrads, in contrast to the wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology : large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells.The possibility that there are two kinds or phases of autolytic enzymes, "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.In the previous paper (21), we reported that the log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 were autolyzed by the action of an N-acetyl-muramyl-L-alanine amidase and this cellular autolysis was depressed by the addition of trypsin to the autolyzing buffer.To investigate the physiological role or significance of the cell wall autolytic enzyme(s), we isolated a non-autolytic mutant of the above strain and compared the autolytic ability, cell morphology and cell wall compositions of this mutant MT with those of the wild-type IFO 3333, particularly by cultivating the mutant and wildtype cells in the presence of externally added proteolytic enzymes.

Cell packets of Micrococcus lysodeikticus (luteus) mutant strain MT grown in medium supplemented with trypsin consisted of a tetrad as the unit structure. An interstice was observed between the unit-tetrads, and a three dimensional structure of cell packets was maintained by the cell wall-bridge along the rim of the cell packets which linked each unit-tetrad. This unique structure of strain MT cell packets seemed to occur when the cell separation was suppressed locally, i.e., when the cross wall inside the initial site of cell separation was cut off, while the wall outside the initial site of separation was not cut off but remained as a joint of the daughter cells.The mechanism of cell wall-bridge formation is discussed in connection with cell separation.Bacteria in general multiply via the three overlapping stages of cell surface extension, cell division, and cell separation. The suppression of only the last stage brought about by various external factors results in long cell chaining in bacteria which have only one plane of cell division (4-6, 20, 22-24) and in cell packeting in bacteria with three planes of division perpendicular to one another (15 , 17, 27). Electron microscopic studies have revealed that individual cells in the long chains are connected by cross walls (6,20). In cell packets, however, the connecting point of individual cells has not been well defined, except that anaerobic sarcinae are known to have a strong tendency to form cell packets (Bergey's Manual, 8th ed.) in which connecting materials of individual cells are cross walls or cellulose (2).We found that Micrococcus lysodeikticus (luteus) mutant strain MT occurring in tetrads formed cell packets when grown in medium with proteolytic enzymes added (17). This paper reveals that the cell wall-bridge is a material maintaining a regular arrangement of the above mentioned cell packets and discusses how this wall-bridge is formed.