JOURNAL OF BACTERIOLOGY, Dec. 1971, p. 1235-1243 Vol. 108, No. 3 Copyright 0 1971 American Society for Microbiology Printed in U.S.A. Role of Autolysins in the Killing of Bacteria by Some Bactericidal Antibiotics H. J. ROGERS AND C. W. FORSBERG National Institute for Medical Research, Mill Hill, London, N. W.7, England Received for publication 7 June 1971 The rapid lysis of Bacillus licheniformis NCTC 6346 and B. sublilis 168 trp caused by vancomycin and D-cycloserine can be inhibited by stopping protein syn- thesis. Protein synthesis must be stopped for more than one doubling time of the cells before addition of wall inhibitors. Poorly lytic mutants (lyt-) of B. licheni- formis required 10 to 20 times the concentration of vancomycin or cycloserine to be added to growing cultures to cause even slow lysis. At lower concentrations growth of the mutants is stopped, but the bacteria remain fully viable. Sensitivity of mucopep- tide synthesis to vancomycin is the same in both mutants and parent. Sensitivity to the action of D-cycloserine is slightly less in the mutant than in the parent. The penicillins and other antibiotics which in- tide synthesis has now been examined upon auto- hibit the biosynthesis of the cell walls of bacteria lysin-deficient mutants isolated from a strain of may ultimately kill sensitive microorganisms Bacillus licheniformis. Cell walls of these mu- because the autolytic enzymes continue to be tants autolyse only very slowly; therefore the formed and destroy more mucopeptide than can strains have been called lyt- (2). It will be shown be made (17, 24). The wall becomes too weak to that these mutants are relatively resistant to the restrain the osmotic forces within the cells, and bactericidal action of antibiotics such as cyclo- even if cell lysis is not apparent the membranes serine and vancomycin, although of course their are damaged. This hypothesis (17) was originally growth is stopped. In other words, the antibiotics formulated on the basis of the well known phe- become bacteriostatic rather than bactericidal. nomenon that chloramphenicol in the presence of the penicillins is able to prevent the death of MATERIALS AND METHODS some microorganisms (5, 16). Chloramphenicol Microorganisms. The parent was B. licheniformis added shortly after methicillin or benzylpenicillin his, isolated from NCTC 6346 in this department by prevented the lysis of Staphylococcus aureus (17). Moyra McConnell. The lytic-deficient mutants were As the concentration of methicillin was in- isolated as previously described (2). Both the parent creased in the presence of a constant amount of and lyt-3 were kept as spore suspensions; the re- chloramphenicol, a point was reached at which mainder of the mutants were maintained on nutrient mucopeptide synthesis and breakdown were ex- agar plates. Some experiments were also done with B. subliis 168 trp, which was kept in a spore suspension. actly balanced. At higher penicillin concentra- In all cases, an overnight shaken culture was grown in tions, when mucopeptide biosynthesis was more Spizizen salts medium (19) supplemented by 100 ,gg of completely inhibited, there was loss of mucopep- histidine per ml or 20 gg of tryptophan per ml and tide, and eventually lysis occurred. This was pre- 0.3% sodium glutamate contained in a flask five times sumably due to autolysin already present in the the volume of the medium and fitted with a side arm. bacteria at the time of addition of the chloram- This was then inoculated at such a rate as to give an phenicol which stopped protein biosynthesis. optical density of 0.07 to 0.10. Incubation was con- Further evidence in favor of the role of the tinued at 35 C with shaking, until the density reached lytic enzymes in killing microorganisms has been 0.200 to 0.300. At this point the culture was divided into 20-ml amounts and placed in similarly designed produced from the effect of penicillin, cycloser- flasks containing the necessary amounts of antibiotics. ine, and phosphonomycin on pneumococci (24). The extinction of the cultures at 675 nm was measured When the choline normally present in the walls every 20 min by running part of the cultures into the of these organisms is replaced by ethanolamine, side arms and using a Unicam SP-600 spectro- the walls become resistant to the action of the photometer; the readings were corrected by the so- autolysin (23), and the bacteria are killed much called AOD adjustment of Toennies (22). less readily by the antibiotics. Antibiotics. Chloramphenicol was from Parke-Davis, The action of antibiotics that inhibit mucopep- Hounslow, Middlesex, England; vancomycin was from 1235 1236 ROGERS AND FORSBERG J. BACTERIOL. Eli Lilly & Co., Indianapolis, Ind., U.S.A., cycloserine tical densities were read at 450 nm in a Unicam SP- from the Sigma London Chemical Co., London, 600 spectrophotometer. S.W.6, England, and methicillin from Beecham's Re- search Laboratories, Brentford, Middlesex, England. RESULTS Measurement of mucopeptide synthesis. Cultures in the medium already described were allowed to reach an Effect of vancomycin and cyloserine on the par- extinction value of about 0.200. ent, B. licheniformis 6346 his-. B. licheniformis At this time 50 yg of chloramphenicol per ml was 6346 is particularly prone to lysis when the cells added. After a further 30 min of incubation, 2.0 are harvested and suspended in buffer. When iuCi/ml of [U- "Cjglucose and the required concentra- they were growing rapidly in casein hydrolysate tion of either vancomycin or cycloserine were added. medium containing glucose, it proved almost im- Samples were then taken at intervals into enough tri- possible to prepare and work with suspensions of chloroacetic acid to give a concentration of 5%. Vol- the bacteria. Earlier work (6, 11, 12), as well as umes (0.1 ml) of these samples were then filtered onto glass fiber discs which were washed with 5% trichloro- our own, suggests that this autolysis results from acetic acid containing 1 mg of glucose per ml and then the changes during the anaerobiosis suffered by successively heated with 5% trichloroacetic acid at 90 the cells during harvesting and resuspension of C for 6 min, washed with 0.1 M Na-K phosphate buffer the cell paste. Figure I shows that growth of aer- (pH 7.5), incubated for 7 hr with 2 ml of a solution of ated exponentially-growing cultures can be I mg of crystallised trypsin per ml in 0.1 M Na-K phos- stopped by chloramphenicol without lysis; a slow phate buffer (pH 7.5), and finally washed with absolute increase in extinction continues. This increase, alcohol and ether. The discs were dried for 1 hr at 105 like that in staphylococci (7, 16) and B. subtilis C. The radioactivity they contained was estimated by (4), is largely due to the increase that takes place immersion in scintillation fluid and counting with a Packard counter. More than 2,000 counts above back- in the amount of wall material. The addition to ground were collected. This method for measuring the cultures of vancomycin above a concentra- mucopeptide synthesis was designed by I. G. Mathison tion of 0.25 ,ug/ml, or cycloserine above a con- of this institute. In the presence of chloramphenicol, it centration of 5 ,ug/ml, led to rapid and almost gives results which agree within i5 to 10% with those from the more orthodox Park and Hancock (13) method for mucopeptide isolation. The reason for adding chlor- amphenicol to the cultures first was to avoid the mas- sive lysis which otherwise occurred with the parent strain when vancomycin or cycloserine was added (see below). Separate experiments showed that chlor- amphenicol did not affect the rate of mucopeptide syn- thesis by either the parent or the mutant bacteria. Preparation of walls containing autolysin. Cultures (1 liter in 5-liter flasks) in the logarithmic phase of growth (0.25 to 0.30 mg/ml), in Spizizen's minimal medium supplemented with 0.3% glutamate, were har- vested rapidly by centrifugation at room temperature. They were washed once in 0.1 volume of cold water (4 C). All subsequent operations were performed at 4 C. The cells in water (10 to 20 mg/ml in 25 ml of water) were disrupted in a Braun tissue homogenizer by shaking with 20 ml of no. 12 Ballotini beads for 3.5 min with cooling by vaporization of CO2. The dis- rupted cell suspensions were filtered through a no. 2 sintered glass filter to remove the glass beads and cen- trifuged at 1,000 x g for 10 min to remove any re- sidual whole cells. The walls were then sedimented by centrifugation at 36,000 x g for 20 min and washed five times in water. The cell walls were lyophilized and stored at -5 C. There was no apparent difference in the rate of autolysis of fresh and lyophilized walls of either B. subtilis or B. licheniformis. Cell wall autolysis. A cell wall suspension (1.0 ml at 2 mg/ml) in water was added to matched tubes im- Hours mersed in an ice bath. Sodium carbonate buffer (1 ml FIG. 1. Effect of chloramphenicol (50 ,g/ml) and of 0.2 ionic strength and pH 9.5) and water (2 ml) of various concentrations of vancomycin and D-cyclo- were added. The final turbidity was 0.30 to 0.45 optical serine when added to exponentially growing cultures of density units. The tubes were placed in a water bath B. licheniformis NCTC 6346 his. Antibiotics were maintained at 35 C and removed at intervals. The op- added at zero time on the graph. VOL.