Demonstration of the Physiological Role of Autolysis by a Comparative Study with a Wild‐Type and its Non‐Autolytic Mutant of Micrococcus lysodeikticus (luteus) Cultivated with Externally Added Proteolytic Enzymes*

Abstract

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 Wall‐Bridge Maintaining Three Dimensional Structure of Cell Packets Formed by the Localized Suppression of Cell Separation of a Micrococcus lysodeikticus (luteus) Mutant1

“…Bergey's Manual (8th ed.) notes that M. lysodeikticus has no visible capsule, and we have not so far discerned any capsule-like material around individual cells, as reported previously (17). However, the present study has revealed that the bridge formed by the fluffy materials and a small void space at the junction of the cross wall with peripheral wall were observed in thin sections of wild-type IFO 3333 cells (Fig.…”

“…When grown in the liquid medium with proteolytic enzymes added, the mutant tetrads form cell packets arranged regularly in three dimensions (17). Figure lab shows thin sections of the trypsin-induced cell packets.…”

“…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.…”

“…However, a comparison of the analytical data in Table 1 with those in Table 2 in the previous paper (17) indicated that the yield (49%) of MT-T-CE (solubilized cell envelopes prepared from stationary phase cell packets of the mutant MT grown with trypsin) was definitely higher than that (38%) of MT-T-LCW (cell walls isolated from log phase cell packets of the MT strain grown with trypsin) and the glutamic acid content (0.55 ,umole/mg) in MT-T-CE was somewhat higher than that (0.47 pmole/mg) in MT-T-LCW, while there were no detectable differences in the yield and glutamic acid contents between MT-LCW (cell walls from the log phase MT cells) and MT-CE (solubilized cell envelopes from the stationary phase MT cells). These findings (Figs.…”

“…Strains of wild-type IFO 3333 and mutant MT were used (17,19). Cell packets were formed by cultivation of mutant MT in the presence of trypsin (5 pg/ml, at final concentration, unless otherwise specified).…”