Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 M NaCl to exponentially growing cultures at 30 degrees C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.

Mycobacteria which are usually penicillin resistant are known to produce f3-lactamases (I, 2). Kasik and his coworkers (3, 5) investigated some properties of f3-lactamases produced by Mycobacterium smegmatis, M. phlei, and M.fortuitum by using a crude enzyme from bacterial cells. The f3-lactamases from these rapid-growing mycobacteria showed broad substrate specificity. However, the nature of the purified enzyme from mycobacterial cells has not been studied. This paper describes the purification and some of the properties of an enzyme from M. smegmatis cells.M. smegmatis ATCC 607 was grown for three days at 37 C in a Sauton medium which contained (per liter): K 2HP04, 0.5 g; MgS0 4· 7H20, 0.5 g; L-asparagine, 4.0 g; sodium citrate, 3.0 g; ferric ammonium citrate, 0.05 g; and glycerol, 50.0 g (final pH 7.0). The cells were harvested on filter paper in a funnel, washed with water, and then suspended in ice-cold 0.07 M phosphate buffer, pH 6.6, at a concentration of 0.2 to 0.3 g (wet weight) per ml. The cell suspensions were disrupted by sonic treatment at 20,000 Hz for 10 min in an ice-water bath. The suspensions were centrifuged at 20,000 xg for 30 min at 0 C, and the supernatants were retained as the cell-free enzyme preparation. The enzyme preparation was brought to 15% saturation by the addition of solid (NH4)2S04. After 30 min the precipitate formed was removed by centrifugation and solid (NH 4hS04 was again added to the supernatant to 30% saturation. The resulting precipitate was collected by centrifugation and dissolved in 0.07 M phosphate buffer (pH 6.6). After dialysis against distilled water at 2 C, the enzyme solution was applied to a Sephadex G-75 column (3.2 X 50 em) previously equilibrated with 0

Staphylococcus aureus produces multiple bacteriolytic enzymes (autolysins) and grows usually as a mixture of single cells, pairs, short chains, and irregular clusters. Autolysin-defective mutants that form cubic cell packets (Pa4A and PaH13) or grape-like clusters (Cu9S and CuD10) were isolated from S. aureus FDA 209P after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The Pa4A mutant grown in nutrient broth formed cell packets consisting of 8-64 cells that appeared regularly arranged in three dimensions. Thin-section electron micrographs revealed that the packet cells were encased in an orderly manner within a thick peripheral wall and that their septa failed to split. Zymographic analysis of enzyme extracts from mutant Pa4A showed that it lacked the 33-kDa autolytic enzyme band present in the parent strain. Another mutant, Cu9S, formed grape-like clusters and showed a single autolytic enzyme band (33-kDa). The possibility that the 33-kDa autolytic enzyme is involved in splitting of the septum prior to cell separation inS. aureus is discussed.

Penicillin-susceptible mutants and p-lactamase-negative

The penicillin‐binding proteins (PBPs) of Mycobacterium smegmatis were studied. Five PBPs ranging in Mr from approx. 114000 to 25000 were detected in the cytoplasmic membrane. The affinities of the PBPs for selected β‐lactam antibiotics were determined. Most of the antibiotics bound to PBPs 3 and 4 at low concentrations. A penicillin‐susceptible mutant and a cefmetazole‐resistant mutant were isolated by selection in vitro. The PBPs of these mutants were identical to those of the parent strain. The affinity of cefmetazole for the individual PBPs was identical in each mutant.