Role of Autolysins in the Killing of Bacteria by Some Bactericidal Antibiotics H

Home , Autolysin, Benzylpenicillin

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 synthesis. 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. licheniformis 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 mucopeptide 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 chloramphenicol 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 chloramphenicol did not affect the rate of mucopeptide synthesis 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 harvested 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 disrupted 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. 108, 1971 ROLE OF AUTOLYSINS 1 237 complete lysis (Fig. 1). Both of these latter anti- staphylococcal cultures to inhibit protein syn- biotics stopped mucopeptide synthesis (Table 1). thesis by 95%, lysis due to the presence of methi- At lower concentrations of the antibiotics, either cillin was prevented even when the chloramphen- partial lysis or a lag in growth occurred, but the icol was added after the methicillin, providing culture recovered and then grew, though usually at a slower rate than the control. With both anti- 100 biotics and at all concentrations up to 10 ,ug/ml for vancomycin and 50 ,g/ml for cycloserine, \'; * Walls . Iichoniformis there was a short period of time, decreasing with 80 A a.* Walls I subtilis increasing concentration of the antibiotic, during which the optical density of the cultures continued to increase at a normal rate. Similar : 60 though less dramatic lysis was obtained during ._ ! the action of vancomycin on B. subtilis 168 trp, at somewhat higher concentrations of the antibiotic. The exponential growth rates of these two 40 organisms were almost exactly the same in the minimal salts-glucose medium, but the slopes of ,~~~~~ the exponential part of the lysis curves, after the 201 addition of vancomycin, indicated half-lives for the population of bacteria of 57 to 60 min for B. licheniformis with 0.5 ,ug or more of vancomycin per ml and 136 min for B. subtilis with 1 gg/ml; ~0 10 20 30 40 50 at increased concentrations of 5 or 10 ,ug of van- Minutea comycin per ml, the half-life of B. subtilis was FIG. 2. Rates of autolysis of walls prepared without reduced a little, but only to 102 and 104 min. treatment with sodium dodecyl sulfate from B. licheni- The relation of these rates of lysis to each other formis NCTC 6346 his and B. subtilis trp. The walls was similar to the relative rates of lysis of walls were isolated, suspended in buffer of pH 9.5 at 0 C, two sus- raised to 35 C, and incubated. Turbidity was measured from the bacteria of the species when by the extinction of the suspension at 450 nm. Autol- pended in buffer (Fig. 2). ysis of the walls from B. licheniformis was so fast that When enough chloramphenicol was added to the zero time value was determined by extrapolation.

TABLE 1. Inhibition ofmucopeptide synthesis by vancomycin and cycloserine in Bacillus licheniformis his and B. licheniformis his lyt--Sa 6346 his his-lyt--5 Antibiotic Concn (fsg/ml) Radioactivity Inhibition Radioactivity Inhibition incorporated % incorporated (counts/min) () (counts/min) (%) Vancomycin 0 96 214 0.25 81 15 213 <5 0.50 45 53 158 27 1.00 21 79 24 88 5.00 19 81 20 91 10.00 18 82 18 92 Cycloserine 0 327 1,157 0.50 262 20 1,028 10 1.00 256 22 1,104 5 5.00 188 43 935 20 10.00 144 56 773 33 20.00 107 67 700 39 50.00 93 72 550 53 100.00 93 72 397 66' aMucopeptide synthesis was measured by the incorporation of [U-'4C]glucose as described in Materials and Methods. Chloramphenicol (50 ug/ml) was first added, and 30 min later the 'IC-glucose and other antibiotics were added. The purpose of adding the chloramphenicol first was to prevent lysis of the parent which otherwise occurs when the wall inhibitors are added. 1238 ROGERS AND FORSBERG J. BACTERIOL. this was within the time corresponding to an in- untreated crease in growth equivalent to about one-fifth of 3.0 * 10Jug/ml Vanc a generation time (17). When this experiment CAP added at T=O. Vanc at was done with B. licheniformis using vancomycin 2.0 30 min and chloramphenicol, no effect of chloramphen- 120 min icol was demonstrable, even when this antibiotic was added at the same time as the vancomycin. With B. subtilis 168 trp, as with B. licheniformis, to. the effect of chloramphenicol on the lysis caused 0.8 by vancomycin was rather slight. This was not oa due to any special effect of vancomycin other <0 X than its effect on wall synthesis, as results with f methicillin acting on B. subtilis 168 are similar 0 (Fig. 3 and 4). The effect of methicillin could not be examined with B. licheniformis 6346 his because this organism is an active producer of f,- 0.2 \- ---A lactamase (penicillinase), and the presence of methicillin in minimal salts medium, even at 10 ,ug/ml, caused only a lag followed by resumption oL ,_,_ ,_ , \ of growth at a normal exponential rate. In casein 0' 2 3 4 hydrolysate medium the effect of methicillin was Hours slightly greater, but again, with 10 ug of the anti- FIG. 4. Effect of adding vancomycin (10 yg/ml) to biotic per ml, exponential growth resumed after exponentially growing cultures of B. licheniformis a period of 2 hr during which some lysis oc- NCTC 6346 his at various times after the addition of curred. Acting on B. subtilis 168 trp, the lysis chloramphenicol (50 1lg/ml). Growth of an untreated culture and one receiving vancomycin (10 itg/ml) alone are shown for comparison. 3.0' * Untreated caused by methicillin appeared to be somewhat 0 50 ug/ml CAP more influenced by the simultaneous presence of 5.O.6,g/mIa Moth chloramphenicol than that caused by vanco- 2.0 A 5.0pg/mIMoth&50Mg/miCAP mycin. Comparative testing of closer ranges of concentrations of antibiotics would be necessary to establish this. Nevertheless, a perceptible ef- A fect of the addition of chloramphenicol to cul- 1.0.-., tures of B. subtilis was seen when its addition 0.88-. p was delayed up to 30 min after the addition of 0a methicillin, whereas with vancomycin any delay 0.6 / abolished the slight effect it had. < /- When the antibiotics that inhibit cell wall syn- */' thesis are added some time after chloramphen- 0.4- / O o0 ° icol, lysis does not occur for a long period of 4. ...rd time (Fig. 3 and 4). For this to happen, wall synthesis has to proceed in the presence of chloramphenicol for more than I hr before vancomycin is 02. added, during which time an amount of wall is . 8; *M formed that is similar to that normally added during a generation of growth in the absence of antibiotics. When the vancomycin is added ear- 03 . . . . \ . . lier, lysis is not prevented. 0 1 2 3 4 5 6 Action of antibiotics on lysis of lyt- strains. Hours When vancomycin alone was added to the lytic- mutants, were found to be rela- FIG. 3. Effe t of allowing chloramphenicol (SO negative they jsg/ml) to act on exponentially growing cultures of B. tively resistant to lysis. A concentration of 1.0 subtilis 168 trp for 30 min before adding methicillin (5 Ag/ml inhibited growth slightly; in some experi- /ug/ml). The optical densities of this culture are com- ments there was slight initial lysis followed by pared with those of an untreated culture and cultures growth at the normal rate. At 5.0 gg/ml, slow treated with chloramphenicol (50 ,g/ml) and methi- continuous lysis occurred. The rate and extent of cillin (5 ,g/ml) alone. this lysis was not increased by raising the con- VOL. 108, 1971 ROLE OF AUTOLYSINS 1239 centration of antibiotic to 25 gg/ml (Fig. 5). The exponential rate of lysis in the presence of these larger concentrations of vancomycin was about 10% of that of the parent treated with 1 Ag of the antibiotic per ml. This slow lysis is nevertheless faster than would be expected simply on the basis of the relative rate of autolysis of the mutant and the parent. The mutant lyses at about I to 2% of the rate of the parent (2). Similar resistance to cycloserine was shown by the lyt- mutant (Fig. 6). It was little affected by either 5 or 10 ,ug/ml concentrations of the antibiotic, and even with 20 ,ug/ml growth was stopped but very little lysis occurred; however, at 50 ug/ml, lysis occurred. Table 2 summarizes results with three other lyt- mutants. In general they required 5 to 10 times the concentration of either antibiotic to stop growth or to cause persistent lysis. Viability of bacteria in cultures in the lyt- mutants after treatment with antibiotics. A concentration of either vancomycin or cycloserine can be chosen such that the total mass of lyt- bacteria in the culture neither increases by growth nor decreases by lysis. This could be the result of either the death of a proportion of the organisms or the fact that the antibiotics act as bacteriostatic rather than bactericidal substances. FIG. 6. Effect of various concentrations of D-cyclo- Viable counts were made at various times after serine added to exponentially growing cultures of B. licheniformis NCTC 6346 his lyt--5. TABLE 2. Effects of vancomycin and cycloserine on 3.0 * Untroated lyt- mutants of Bacillus licheniformis 6346 his o 0.25jug/mlVane of Concn showing C showing lysis A 1.0 *I growth inhibition f 2.04 a 2.0 I II gCo Il) A 5.0 B t Strain without lysis (Ag/m1) (g/ml) * 25.0 Is Is Vanco- Cyclo- Vanco- Cyclo- mycin serine mycin serine 1.c ,go,,13 Parent (6346 his) 0.25-0.50 1.0-5.0 0.5 5-10 /, lyt--3 1-5 20 about 20 50 1 lyt-4 >1 <5 20 5 >50 ,-7,^/,04 lyt--5 2-5 20 5-10 50 00.8 lyt--6 I a 10 5 20-50 aConsiderable lysis during first 3 hr after addition of antibiotic, followed by recovery. - A the addition of I ,ug of vancomycin per ml to the parent B. licheniformis 6346 his. After 2 hr the counts were only 0.1% of those from a control 0.21 culture containing no antibiotic and had fallen a further fivefold after 4 hr. Unfortunately this technique could not be used reliably to study viability of the lyt- mutants because they grow as very long chains of unseparated bacteria (2). 0 1 2 3 4 5 6 7 Recourse was had, therefore, to a more indirect Hours technique. If bacteria are transferred from a rap- FIG. 5. Effect of various concentraltions of vanco- idly growing culture to a suitable, warmed, fresh mycin added to exponentially growingg cultures of B. growth medium and no lag in exponential growth licheniformis NCTC 6346 his lyt-S. occurs, then a very high proportion of the cells in 1240 ROGERS AND FORSBERG J. BACTERIOL. the original culture must have been viable. Cycloserine (20 ,g/ml) was added to an expo- 3.0 * Untreated nentially growing culture of lyt--5. At intervals * Treated & samples filtered after this, samples (10 ml) were removed and After 2 30 min rapidly filtered through 0.45-sm membrane fil- 2.0- "1o 60 it ters (Millipore) kept at 35 C. The micro- a so 120 t o organisms were washed once with 10 ml of sterile medium prewarmed to 35 C, the filters were aseptically transferred to 10 ml of prewarmed 1.0 . / medium contained in sterile tubes, and the tubes were agitated strongly on a Whirlimix (Fisons, 0b8 -/ 11 Loughborough, England). The suspension was then poured into a warm 100-ml side arm flask, 0~~~~~~~~~.8 and incubation was continued at 35 C with shaking. The organisms still grew exponentially, a2 immediately and without lag, after 2 hr of treatment with 20 Ag of cycloserine per ml (Fig. 7). There was a reproducible tendency for the cul- 60) tures incubated longest with the antibiotic to 30120~~~~~~~~~1 grow the fastest. This may have been due to the accumulation of precursors during the period of inhibition of wall synthesis. 01~~~~~2 some With vancomycin, the bacteria suffered 0 2 4 6 8 10 12 '24 Hours FIG. 8. A similar experiment to that shown in Fig. e 7, but with vancomycin (I gg/ml) used in the parent 3.0 Untreated culture. The subsidiary cultures, started from the fil- * Treated & samples fiterbred tered samples, were compared with the behavior of o After 0 min the original culture containing vancomycin (A) and a 2.0 a I 30 is culture containing no antibiotic (0). Both the culture o It 60 " started from the sample filtered after 120 min and the * " 120 " parent antibiotic-containing culture showed an initial period ofslight lysis but grew again. By 18 hr they had reached the same density as the culture containing no 1.0 ,4 antibiotic. O.8 0 /o/@ damage when incubated for longer than 30 min 0 08 o*//^/S with I gg of the antibiotic per ml (Fig. 8). The vancomycin-treated culture itself showed a small rise and fall in optical density before leveling 0.41 off, presumably corresponding to the lysis of a proportion of the bacteria in the culture. When the sample was taken 1 hr after the addition of vancomycin, at the point where the period of some lysis was just beginning, a period of accel- A0160 120 /' ~30 erating growth lasting about 2 hr occurred before reaching the same exponential rate as that of the sample taken before the addition of antibiotic. ni . , The bacteria in the sample taken when lysis was 0 1 2 3 4 5 6 7 proceeding (that is, the 2-hr sample) continued Hours in the fresh antibiotic-free medium with exactly FIG. 7. Recovery of bacteria from a culture of B. the same initial pattern of growth as those in the licheniformis NCTC 6346 his lytS5 which had been original culture containing vancomycin. In other treated with cycloserine (20 pg/ml). A t each of the experiments no lysis of the primary or reinocu- with an arrow, of the original points marked samples lated cultures was but incubation with culture containing cycloserine were ftl observed, teria were washed and then resuspe tndedfrom the vancomycin for longer than I hr led to a lag of membrane filter into fresh medium wi,thout antibiotic. about 2 hr, indicating some damage by the anti- Growth of these cultures was then followed in the usual biotic. way alongside the parent culture. Similar experiments were not possible with the VOL. 108, 1971 ROLE OF AUTOLYSINS 1241 parent bacteria because filtering or centrifuging even antibiotic-free cultures leads to variable pe- riods of lysis and extended lags before growth. Effect of vancomycin and cycloserine on wall synthesis by lyt- mutants. The results so far show that the lyt- mutants do not lyse or lyse only slowly when cultures are treated with 5 to 10 times the concentration of vancomycin or cycloserine that causes lysis of the parent, and that exponentially growing cultures can be treated for some time with relatively high concentrations of the antibiotics without being killed. It is impor- tant to show whether or not wall formation by the mutants is as sensitive as that of the parent 0 to inhibition by the antibiotics. Figure 9 shows that incorporation of radioactivity from [U-14C]glucose into mucopeptide continues in a linear fashion for some 3 hr after the addition of 50 Ag of chloramphenicol per ml and that this can be almost completely inhibited by a sufficient concentration of vancomycin. A more critical examination (Table 1) of the concentration of vancomycin required to inhibit mucopeptide formation by one of the mutants and by the parent microorganism showed that Hours wall synthesis was 50% inhibited by 0.6 gg/ml in FIG. 9. A comparison of the incorporation of 14C the parent and 0.7 ,g/ml in the lyt- mutant. from [U- 4C]glucose into mucopeptide of B. licheni- These concentrations are in reasonable agree- formis his lyt- by a culture treated with chloramphen- ment with each other and with the minimum icol (50 Ag/ml) with one containing vancomycin (10 concentration of antibiotic leading to lysis of the ,ug/ml) as well as chloramphenicol (SO tg/ml). The parent. About 3 to 4 times the concentration was mucopeptide was isolated, and its radioactivity was determined. Vancomycin and chloramphenicol were required to stop growth of the mutant. This con- added to exponentially growing cultures, and when centration of vancomycin caused 80 to 90% inhi- both antibiotics were used they were added simultane- bition of wall synthesis. Wall synthesis by the ously. lyt--5 mutant was, however, significantly less sensitive to cycloserine than that of the parent. The methicillin. With bacilli the situation is some- mutant required about 35 gg/ml to cause 50% what different. Lysis occurs unless protein syn- inhibition of wall synthesis compared with a con- thesis is shut off some time before wall synthesis centration of 9 to 10 Ag/ml for the parent. is inhibited. During a period of incubation without protein synthesis occurring, more wall is DISCUSSION laid down over the whole area of the cells of ba- The death of bacteria in cultures of B. licheni- cilli (4). Presumably this new wall material will formis treated with vancomycin or cycloserine is contain no significant amount of the autolysin not due to the inhibition of wall synthesis alone. because no lysin is being formed under these Almost complete inhibition of synthesis by van- conditions. One possibility would then seem to comycin only stops growth, and subsequent lysis be that in bacilli, autolytic enzyme sufficient to and death are due to the action of the autolytic lyse the cells is already present at the time pro- enzymes which continue to be formed, presum- tein synthesis is stopped. Lysis cannot recur ably at an exponential rate. These enzymes break when enough new wall which does not contain down the wall already existing when synthesis lysin is formed underneath the old wall. In staph- was stopped and lead to bacterial lysis. This ex- ylococci, the amount of enzyme present at the planation of cell death was predicted earlier (17) time when protein synthesis is stopped is presum- on the basis of results with staphylococci acted ably not sufficient to cause rapid damage. Alter- upon by methicillin. With these organisms, autol- natively, differences of location and export of the ysis of the bacteria and cell death could be pre- existing lytic enzyme may account for the dif- vented by the addition of antibiotics, such as ferent types of behavior. For example, if the chloramphenicol, that stop protein synthesis, export of active enzyme from the cytoplasm re- providing they were added fairly soon after the quires protein synthesis in staphylococci but not 1242 ROGERS AND FORSBERG J. BACTERIOL. in bacilli, this could also provide an adequate terial cell walls, presumably with D-alanyl-D-ala- explanation for the continued lysis of bacilli after nine sequences in unfinished mucopeptide, with the cessation of protein synthesis. The substrate nucleotide precursors (14, 15), and with disac- specificity of the autolytic enzymes present in B. charide-pentapeptide formed by the isoprenoid licheniformis, B. subtilis, and S. aureus would intermediate in the membrane. More time may seem to be similar. All three organisms have an be required to synthesize enough of this range of amidase cleaving the L-alanyl-N-acetylmuramic precursors of mucopeptide to allow synthesis to acid bond and a glycosidase splitting the I - 4 proceed again. N-acetylglucosaminido-N-acetylmuramic acid bond in the mucopeptides (1, 3, 21). Staphylo- ACKNOWLEDGMENTS in a cocci, addition, contain peptidase attacking Our thanks are due to Cheryl Davis, Gillian Hallas, and the glycyl cross-bridge. In these three species of Marian Kusel for excellent technical assistance during this microorganism, the amidase is the dominant work. enzyme in autolysis, at least in some strains. Part of this work was conducted while C. W. Forsberg held Wall synthesis is about equally susceptible to the a postdoctoral fellowship award from the National Research inhibition by vancomycin in the parent and mu- Council of Canada. tants studied here, although synthesis by the mutant is somewhat resistant to the action of LITERATURE CITED cycloserine, for reasons unknown. The failure of 1. Brown, W. C., and F. E. Young. 1970. Dynamic interac- tions between cell wall polymers, extra-cellular proteases the antibiotics to lyse and kill the mutant is thus and autolytic enzymes. Biochem. Biophys. Res. not likely to be due to differences in suscepti- Commun. 38:564-568. bility of wall synthesis, which might exist, as the 2. Forsberg, C. W., and H. J. Rogers. 1971. Autolytic en- walls differ chemically from those of the parent zymes in growth of bacteria. Nature (London) 229:272- 273. (2). 3. Hughes, R. C. 1970. Autolysis of isolated walls of Bacillus These conclusions agree with the results (23, licheniformis N.C.T.C. 6346 and Bacillus subtilis Mar- 24) obtained for pneumococci which were made burg strain 168. Biochem. J. 119:849-860. 4. Hughes, R. C., P. J. Tanner, and Elaine Stokes. 1970. resistant to autolysins by the incorporation of Cell-wall thickening in Bacillus subtilis. Comparison of ethanolamine in their walls in place of choline or thickened and normal walls. Biochem. J. 120:155-170. which were deficient in lytic enzyme. These bac- 5. Jarvetz, E., J. B. Gunnison, R. S. Speck, and V. R. teria were also killed much more slowly by peni- Coleman. 1951. Studies on antibiotic synergism and an- tagonism. The interference of chloramphenicol with the cillin G, D-cycloserine, and phosphonomycin. action of penicillin. Arch. Intern. Med. 87:349-359. Thus, the bacterial activity of antibiotics whose 6. Kaufman, W., and K. Bauer. 1958. Some studies on the mode of action is to stop wall synthesis is likely anaerobic autolysis of Bacillus subtilis. J. Gen. Micro- to be due to the action of the bacterial lytic en- biol. 18:XI. 7. Mandelstam, J., and H. J. Rogers. 1959. The incorpora- zymes. Otherwise these antibiotics, like those tion of amino acids into the cell-wall mucopeptide of stopping protein synthesis, would be bacterio- staphylococci and the effect of antibiotics on the process. static. The extent of action of the lysins neces- Biochem. J. 72:654-662. sary to lead to bacterial death presumably de- 8. Neuhaus, F. C. 1962. The enzymatic synthesis of D-alanyl- D-alanine. I. Purification and properties of D-alanyl-D- pends upon the cellular organization of the par- alanine synthetase. J. Biol. Chem. 237:778-786. ticular species. For example, the tolerable level 9. Neuhaus, F. C. 1962. The enzymatic synthesis of D-alanyl- of damage to the membranes may differ from D-alanine. II. Kinetic studies on D-alanyl-D-alanine syn- species to species, and the amount of membrane thetase. J. Biol. Chem. 237:3128-3135. 10. Neuhaus, F. C., and J. L. Lynch. 1962. Studies on the in- damage resulting from wall damage may also hibition of D-alanyl-D-alanine synthetase by the anti- differ. biotic D-cycloserine. Biochem. Biophys. Res. Commun. Vancomycin appears to be significantly more 8:377-382. damaging to the mutant than cycloserine. After 11. Nomura, M., and J. Hosoda. 1956. Nature of the primary action of the autolysin of Bacillus subtilis. J. Bacteriol. about I hr of exposure to vancomycin, growth 72:573-581. lagged, and temporary slight lysis was encoun- 12. Nomura, M., J. Hosoda, and H. Yoshikawa. 1958. Studies tered when the antibiotic was removed. This in amylase formation by Bacillus subtilis. VI. 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