Induction of autolysis in Bacillus subtilis by ochratoxin A U. SINGER AND R. R ~ ~ S C H E N T H A L E R ' Irrstitrrte ~ ~ ~ ' M i c ~ ~ o l ~ iU~lii,e,lrity o l o g y . of M r i e r ~ i t o . ,Mr~c.rlster,Gerrr~co~y
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Accepted February 9, 1978 U., and R. ROSCHENTHALER. 1978. Induction of autolysis in Bacill~rss1rhrilis by ochSINGER, ratoxin A. Can. J . Microbiol. 24: 563-568. Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 pg/ml caused autolysis of Btrcillris s~rhtilis.Optical density of cultures decreased. and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10pg/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property ofalysis inhibitor. The relevance of thisfraction in respect to autolysisisdiscussed. SINGER, U., et R. ROSCHENTHALER. 1978. Induction of autolysis in Bucill~rss~rbrilisby ochratoxin A. Can. J. Microbiol. 24: 563-568. L'addition d'ochratoxine A (OTA) au cours de la phase exponentielle de croissance i une concentration supkrieure ?I 12 pg/ml produit I'autolyse de Bacillrrssrrhtilis. Ladensiteoptique des culturesdecroit, et i des concentrations plus Clevees les cultures deviennent steriles. L'optimum d'induction d e lyse par OTA est B un pH approximatif de 5. Aux concentrations inferieures B 10 pglml la synthese proteique est inhibee plus fortement que la synthese d'ARN. La synthese de la paroi est aussi fortement inhibee. Une fraction extraite du lysat i lapropriete d'inhiber la lyse. L'iniportance de cette fraction vis-A-vis de I'autolyse et discutee. [Tnlduit par lejournal]
Introduction Autolytic enzymes are regarded as essential in the growth of bacteria. A balance between the hydrolytic action of these enzymes and the synthesis of new cell wall material has been postulated (Holtje and Tomasz 1975), thus involving these enzymes in the replication and growth of the cell wall. Also, autolytic enzymes are believed to play a role in cell separation and genetic transformation (Holtje and Tomasz 1975). The bactericidal action of penicillin and other cell wall synthesis inhibiting antibiotics is due to the action of autolytic enzymes. They destroy the cell wall when the synthesis of the cell wall is inhibited and thus render the cell osmotically fragile, causing death in hypotonic environment (Rogers 1968; Rogers and Forsberg 1971). In a pl-evious paper (Heller et crl. 1975) we showed that ochratoxin A (OTA), a mycotoxin isolated from Aspergillus oclzrcrcells and other fungi, inhibits the growth of Streptococcus.ficecalis. The mode of action involved inhibition of protein and RNA synthesis; even at very high concentrations the bacteria were not killed by OTA. In Bacillils slrhtilis, however, OTA induced autolysis in growing cells when the concentration was higher than 12 Fg/ml. Thus, it was bactericidal to this species. These findings led us to investigate its mode of action in B. srrbtilis more closely. IAuthor to whom reprint requests should be acidressed.
Materials and Methods Brrcteriol Srrrrirls
The experiments were carried out with B . srrhtilis 168 1(which required L-tryptophan or indole) or with a derivative of this strain. A spontaneously occurring mutant was isolated which had lost the property of being lysed in the presence of more than 12pglml of OTA. Even at concentrations as high a s 40 pg/ml this strain could not be lysed and therefore was designated lyt-. Thismutant remainedstableafter subculturing more than 20 times in the absence of OTA. Gro\vrll Corlditiorls orltl Measrrrernents
The minimal medium of Anagnostopoulos and Spizizen (1960) was used with a supplement of 0.5% glucose. Furthermore, it contained per litre: arginine, 80mg; leucine, 250rng; isoleucine, 100mg. sodium glutamate, 500mg; tryptophan, 80mg; valine, 150 mg, and the remaining 14 amino acids in quantitiesof 40mg. The concentration of MgSO, X 7H10 in the salts medium was halved to O.lg per litre a s Mg2+ counteracts OTA inhibition (Heller et 01. 1975). This reduction did not alter the growth rate. The pH of the medium was adjusted to 5.85 with HCI, if not otherwise indicated. For growing protoplasts the hypertonic minimal medium of Hirokawa and Ikeda (1966). adjusted to pH 6.0, was used. Liquid cultures in 100-ml Erlenmeyer flasks with a fused-on measuring tube and two baffles were incubated with aeration at 37°C in a gyrotory water bath shaker (New Brunswick). The Erlenrneyerflasks usually contained 3 ml of the cultu~~ernedium. Optical densities (OD) of cultures were measured in a n Eppendorf photometer at 546nm after allowing the culture to run into the measuring tube. When a comparison was necessary after a given time of incubation, the inocula for the different flasks were taken from the same culture at a n ODof0.400. The growth late (p) was estimated by dividing60min by the doubling time (in minutes). The doubling time was determined according to d = (t , - t o ) log ?/log E , - log E,, where E, and E , are the extinctions at times 1 , and t , , respectively. To obtain values for autolysis rates that were comparable to growth rates,
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the same fol.mul;i wns:ipplied to the autolysis rate. tlesignated as -p. This methotl gives agross estimation o f the al~tolysisrate as al~tolysis~,oughlyfollowetl an exponentigil function for about 2 h >ifteradtlitiono f O T A .
Can. J. Microbiol. 1978.24:563-568. Downloaded from www.nrcresearchpress.com by UNIV VICTORIA on 11/19/14. For personal use only.
Proto/)ltr.\li/l# For protoplasting, the method o f Hirokawa and Iketla (1966) was followed. Mrtr.srrrc,t,rc~trr cj/'Pro/eit~c r t r t l RNA S.vt~llrc,.si.s The incorporation o f [14C]ul-acilinto l.ibonucleic acid (RNA) and ['"Clglycine into protein was measured as previoi~slydescribecl (Hellere/ (11. 1975). T o compare the extent of inhibition of protein and R N A synthesis in relation to the time afteratltlition of O T A . the ~xtlioactivity(cpm) o f the respective preclrrsors incol.porated per optical density unit of the OTA-treated c i ~ l t i ~ was r e tlivitlecl by that o f the control c i ~ l t l ~ r e . Mc,tr.srrrc,t~~,tr/ ofMrrcopc~p/itIc~ Sytr//rc,.si.s
The method o f Rogel-s and Forsbelg (1971) was followetl. except that we usetl 7Okglml o f chlol-amphenicol insteutl o f SOpglml. The values for incorporation o f [14C]glucoseinto mi~copepticleswas divided by the O D o f the inhibited cultllres to obtain an idea ofcell wall synthesis per;lvel.:ige cell. I.soltr/iot~~f'L.v,si.s It~lrihi/or'fi.ot~r Bac/rricrl Lystrte.~ I n the isolation o f a macromoleci~lw.fraction from B. .srrbtili.s capable o f inhibiting lysis, the isolation methotl o f Holt.je and Tomasz (1975) was followed. Cells were g~.ownwith aeration to :in OD, o f 0.600. Then 4 mg O T A (36pg/mI) 01.2.5 mg penicillin (22 p g / ~ i i l )or 2mg cycloserine (18pg/ml) were nclcled to re. shaking at 37°C f o ~ 2.5 . h, the O D tlespective c i ~ l t l ~ r e sAfter creased to less than 0.1 in all cult~rres.The lysates were centrifi~geclat 4°C ancl IOOOOrpm for l j m i n . The col-responding supernatants were tlialyzed for 48 h against distillecl waiter with three changes o f water. Aclsorption o f O T A to the lysate was evident when it was ir~-adi[rtedwith U V light :it 350nm. Therefore, IOmg/ml o f bovine serum albumin was atlcled to the water again\t which the lysate wasclialyzecl. As bovine serum albumin strongly binds O T A (Chu 1971: own observ:itions). the O T A content o f the lysate could be recluced from 36 pg/mI to less than 0.4pg/ml by thismethotl. Thisshows that bovine serum albumin bintls O T A more strongly than the lysate. After dialysis, the preparations were frozen at -20°C ant1 lyophilized. The lyophilizates were weighed (about 5 mg), dissolvetl in I ml o f distilled water, and heated at 100°C for 20min. The precipitate was centrifuged down oncl tliscarded. Portions o f the supernatant were iiddetl to culti~res.
FIG. I . Effect o f O T A on B . .sr~h/ilis 168 :it p H 6.0. I n the exponential phase of growth, a cultl~rewas divided into nine subcultures which received O T A (arrow) at the concentrations indicated (pg/ml ofgrowth medium).
lysis occurred at pH 5.1. After OTA addition, autolytic activity could be immediately and significantly increased by a shift to a lower pH (e.g., froni 5.7 to 5.3, data not shown). The temperature optimal for lysis was 36°C; at higher and lower temperatures less autolysis was observed; below 30°C nolysis occurred at all (data not shown). In the course of the work, a mutant was isolated which had lost the property of being lysed by OTA. Growth of this strain, designated lyt-, was still inhibited. OTA did not lyse protoplasts of B. srihtilis (Fig. 3) in a hypertonic mediurn containing 20% of sucrose (Hirokawa and Ikeda 1966). Also, bacillary forms were not lysed in this niediuni. The increase in mass, however, was somewhat reduced in both forms.
Itlhihitiotz of Proteitt cirttl Cell Weill Syr~tl~esis In B. slibtilis OTA inhibits protein synthesis more strongly than RNA synthesis. Figure 4 shows Ochrtrr~aitrA t r t r t l Cl~rttric~trls O T A was isolateil ant1 purifiecl from wheat kernels infected the time dependence of the inhibition of incorpol-awith A. oclirtrcerr.~,as previously described (Heller t r l . 1975). tion of labelled glycine and ~ ~ r a c iinto l acidThe radioactive materials were a generous gift o f Dr. P. precipitable material. Incorporation of labelled Fromageot, C. E. A,, Saclay, France. Chlor~imphenicolwas thyniidine into deoxyribonucleic acid (DNA) was purchased from Serva, Heidelberg. and cycloserine fromsigma, not affected by OTA (data not shown). Munich. Inhibition of mucopeptide synthesis was also demonstrated. Figure 5 shows incorporation of Results [14C]glucoseinto the mucopeptide fraction of the Aritoly~isCcirisecl b y OTA cells in the presence and in the absence of OTA. OTA is a potent growth inhibitor in B. slihtilis. In OTA strongly inhibits niucopeptide synthesis. defined growth medium, at low pH, and in concenChloramphenicol was added to avoid lysis in the trations above 10-20Fglml ailtolysis of the cells presence of mucopeptide synthesis inhibition occ~11.s(Fig. 1). Above 2 0 ~ g l m lthe cultures may (Rogers and FOI-sberg 1971). No inhibition of become sterile. If autolysis is incomplete, cultures m~~copeptide synthesis by this drug was observed. resume growth with a slower growth rate after a delay of several hours. The pH range in which OTA Isoleition cir~tlActiorz of an Inhibitor. c!f'Cell Lysis causes lysis is between 6.0 and 4.0 (Fig. 2). Optimal A heat-resistant, non-dialyzable fraction from B.
S I N G E R A N D ROSCHENT'HALER cpm OTA-wlture I cDm contrd
[%]uracil
incorporation
Can. J. Microbiol. 1978.24:563-568. Downloaded from www.nrcresearchpress.com by UNIV VICTORIA on 11/19/14. For personal use only.
0.6
A
C
FIG. 4. Inhibition ofprotein ant1 RNA synthesisin B . s ~ ~ h / i l i . s . Two exponentially g r . o w i n g c ~ ~ l t ~ received ~res 0.6 p C i / m l ( l Ci = 37 GBq) o f [ ' J C ] ~ ~ r a cand i l 0.8pCi/ml o f [lJC]glycine. respectively. An amoLlnt of 10Fglml of O T A was addetl (arrow). ;lncl the inco~.pou~tion o f the precursors into acid-precipi table material was measured after clifferent time intervals. The ratio o f incorpol-ution into OTA-treated cells to that o f the control was c>~lculated.
-3,p
of OTA-treated culture ot controlculture
FIG. 2. p H dependence of the O T A effect in B . .s~,h/ili.s168. ( t r ) Closed triangles :we growth rates o f B. .srlh~ili.sat different pH, open triangles growth rates and lysis rates in the pl-esence of 20 pglml o f O l ' A . (b)The p H range in which O T A is bactericidal is obtained by calc~~lating the ratio between growth rates (p) or lysis rates(- aftertr treatment with OTA,nnd growth rate ( p ) o f the control c ~ ~ l t ~The ~ r concentl.ation e. o f OTA was 20 pglrnl.
I
30
60
90
120
150 min
FIG. 5. Inhibition by O T A o f incorporation ofglucoseinto the peptidoglycan fraction. [14C]gl~~cose was added at time 0. The incorpolxtion was measured in the prebence of 70pglrnI o f chlou~mphenicol(Rogers and Fousberg 197 1). Squares indicate control culture; triangles, after the addition o f 15 pglmlofOl'A.
FIG. 3. Effect o f O T A on bacill;~ry forms and on growing protoplasts in hypertonic medium. Open squares are bacillary form control cultures. closed squares are bacillary form cultures in the presence o f 15 pglml o f 01'A. Open triangles are growing c o n t ~ uprotoplasts, l and closecl triangles growing protoplasts in the pl-esence of 15 pglml o f O T A .
s~~brilis autol ysates was isolated, which inhibited OTA-induced lysis. The autolysis of cultures was brought about by OTA, penicillin, o r cycloserine. OTA-induced lysis was strongly inhibited by this fraction (Fig. 6a). Figure 66 shows the dose dependence of the autolysis rate of bacterial cultures in the presence of 23 Fg/ml of OTA upon concentration of the 'lysis inhibitor.'
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FIG.6. Inhibition ofOTA-induced autolytic activity by a hot waterextract of lysates ('lysis inhibitor'). (0)Circles designate control cultures; open triangles, after the addition of 23 kglrnl of OTA (arrow); and squares, after the addition of 23 ~ g / r nof l OTA and 200kg/ml of 'lysis inhibitor.' ( h ) Relation of concentration of 'lysis inhibitor' to autolysis. Cultures in the exponential growth phase received 23 kglml of OTA each.
Discussion OTA displays antibiotic properties at a pH of about 6 against many gram-positive bacteria. In S . faecalis it is a potent inhibitor of growth, but does not kill the cells, even at concentrations as high as 1 mglml (Heller et al. 1975). In B. subtilis, however, a bactericidal action of OTA is observed under conditions described in this papel-. This effect is due to autolysis of the cells as evident from a decrease of OD, microscopical observations, and especially from the observation that metabolizing protoplasts are not lysed by OTA in hypertonic medium. The finding of a mutant which could not be lysed by OTA but which was still sensitive in respect t o growth inhibition supports the idea that the bactericidal action of OTA is due to the activity of autolytic enzymes. A direct stimulation or inhibi-
tion of the cell wall associated autolysins in a cellfree preparation according to Fan (1970) was not observed (data not shown). Therefore, it can be concluded that OTA does not interfere with activity of autolysin (s). In B. sribtilis, two autolytic enzymes have been found, an amidase and a p-glycosidase (Brown and Young 1970; Fan and Beckman 1972). This is in contrast to S.Jirecnlis, for which only a single enzyme, a muramidase, has been reported (Cleveland et 01. 1976). The enzyme that is likely to be active in OTA-induced autolysis of B. sllbtilis is the p-glycosidase and not the amidase. The reason for this suggestion is that the glycosidase has a pH optimum between 5 and 6; the p H optimum of the amidase is at pH 8.6 (Fan and Beckman 1972). The lysis induced by OTA is optimal at pH5.1. No lysis
Can. J. Microbiol. 1978.24:563-568. Downloaded from www.nrcresearchpress.com by UNIV VICTORIA on 11/19/14. For personal use only.
SINGER A N D ROSCHENTHALER
,
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or growth inhibition is observed at pH 8.6. As de- 1971). Since Holtje and Tomasz (1975) have demscribed for penicillin, the in vitro pH optimum of onstrated a lipoteichoic acid-like lysis inhibitor in the major autolysin is in agreement with the pH pneumococci, it was recognized that activity of optimum of the lytic response in vivo (Tomasz and autolysins is under negative control. In B. slrbtilis, Holtje 1977). Also, the temperature dependence of the amidase activity was also found to be inhibited OTA-induced a~~tolysis supports this suggestion. by a lipoteichoic acid-type inhibitor (Cleveland et The activity of the amidase has been reported (Fan 01. 1975). The 'lysis inhibitor' which we have isoand Beckman 1972) to be only a little lower at 30°C lated from autolysates of B. slrhtilis was obtained than at 37°C. Even at 24°C this enzyme is still by the method of Holtje and Tomasz (1975). As this active. However, no lysis is caused by OTA below fraction was heated for 20 min at 100°C, most of the 30°C. The temperatul-e dependence of OTA-in- protein was denatured and discarded. Therefore, it duced a ~ ~ t o l y sdiffers is from that of the amidase. is very likely that this fraction contained the As no direct action of OTA on the autolysin lipoteichoic acid-like material described by the activity could be demonstrated, a similar mech- authors mentioned. This would mean that the inanism for the bactericidal action of OTA had to hibitor for the P-glycosidase is the same as for the be considered as that described for other lysis- amidase in B. slrhtilis. Cleveland et al. (1976) have inducing agents. Rogers and Forsberg (1971) have shown that the lytic activity of S. , f ~ ~ e c a l i s I-eported that inhibitors of mucopeptide synthesis, muramidase (i.e., a different enzyme than amidase) such as penicillin or cycloserine, are able to induce can be inhibited by a lipoteichoic acid fraction from autolytic activ~tyin bacterial cells. It was dem- a hot water extract, and this observation supports onstrated that in B. s~rhtilis,OTA causes inhibi- our suggest ion. The difference in the sensitivity to OTA-induced tion of cell wall synthesis under the conditions described (Fig. 5). But even at concentrations lower autolysis between S . f~iecalisand B. s~lhtilismay b e than those necessary for autolysis, OTA is a strong due to the inhibition by OTA of a protein necessary inhibitor of protein synthesis. This is quite a unique to activate the latent muramidase of S. ,fhecalis property, as in general protein synthesis inhibitors described by Pooley and Shockman (1970). Cells of (e.g. chloramphenicol) are used to prevent lysis S . fuecalis become very rapidly resistant t o au(Rogers and Forsberg 1971 ). Preliminary experi- tolysis when protein synthesis is inhibited (Pooley ments pel-formed to find an explanation for this and Shockman 1970). This is in contrast toB. sllbdiscrepancy showed that OTA prevented cyclo- tilis which requires the addition of protein synthesis serine-induced lysis, but stimulated autolysis inhibitors approximately 30 min before addition of cell wall inhibitors t o prevent autolysis (Rogers and caused by penicillin or vancomycin. In a recent investigation, we have shown that Forsberg 1971; own observations). No observations were made that would justify after addition of OTA to B. suhtilis, the pools of the notion that a specific and strong binding of O T A ppGpp and pppGpp increase about four times. Simultaneously, a decrease in the adenosine 5'-tri- to the lysis inhibitor would occur with a simultanephosphate (ATP) and guanosine 5'-triphosphate ous inactivation of the biological activity of OTA. (GTP) pools is o b s e r v e d . q f t e r the cells were Also, no nonspecific improvement of growth condipreincubated with chloramphenicol and then ex- tions by this fraction was found. Therefore, it is posed to OTA, only the pool of ppGpp increased conceivable that in S .fkecalis the protein synthesis while the level of pppGpp remained as low as in the inhibition caused by OTA prevents autolysis, while untreated culture (data not shown). Ishiguro and in B. suhtilis it triggers a regulatory mechanism, Ramey (1976) found a stringent control of the involving the MS-nucleotides, leading to an inhibimucopeptide synthesis in Escherichia coli and an tion of mucopeptide synthesis, release of lysis ininhibition by ppGpp of in vitro mucopeptide syn- hibitor into the medium (Tomasz and Hijltje 1977), thesis. The same mechanism probably operates in and finally to lysis. the inhibition of mucopeptide synthesis by OTA in B. sllhtilis. The ability of cell wall inhibitors to induce autolAcknowledgements ysis was originally thought to be due to an imbalWe are very much obliged to Dr. J . Harwig, ance between synthesis and the degradative action Health and Welfare Canada, for his patient reviewof autolysins (Rogers 1968; Rogers and Forsberg ing of our manuscript and for suggestion of many 'U S ~ n g eand ~ R Roschenthaler. 1978. Inhib~tionof p ~ o t e ~ n valuable corrections. We also thank Deutsche Forschungsgemeinschaft for supporting this prosynthesis by o c h ~ a t o x ~An In B o ~ ~ l l ~ s~lbtllls. rs Eur. J. Toxic01 (In p ~ e s s ) . ject, A. Wegner for performing some control exper-
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Can. J. Microbiol. 1978.24:563-568. Downloaded from www.nrcresearchpress.com by UNIV VICTORIA on 11/19/14. For personal use only.
iments, and Dr. K. Heller for typing the manuscript.
HELLER, K., C. Sc.1-IULZ,R. LOSER,and R. ROSCHENIHALER. 1975. The inhibition ofbacterialgrowth by ochratoxin A. Can. J . Microbial. 21: 972-979. H I R O K A WH., A , and Y. IKEDA.1966. Genetic recombination of transforming DNA molecules with recipient genome and ANAGNOS-IOPOULOS. C . , and J . S ~ r z r z t 1960. ~ . Requirements :inlong themselves in protoplasts of Btrc~illiissrrbti1i.s. J . Bacfor transformation in Btrcil1ri.s srr6tili.s. J . Bacteriol. 81: teriol. 92: 455-463. 741-716. BROWN,W. C.. and F. E. YOUNG.1970. Dynamic interaction HOLTJE,J. V., ilncl A. TOMASZ.1975. Lipoteichoic acid: a between cell wall polymers, ext~.acellular proteases, ant1 specific inhibito~.ofa~~tolysin activity in pneumococcus. Proc. autolytic enzynies. Biocheni. Biophys. Res. Commun. 38: Natl. Acacl. Sci. U.S.A. 72: 1690-1694. ISHIGURO, E., and W. D. RAMEY.1976. Stringent control of 546-568. C H U .F. S . 1971. Interaction of ochratoxin with bovine serum peptitloglycan synthesis in Escherichitr coli K-12. J . Bacnlb~~niin. Arch. Biocheni. Biophys. 147: 359-366. teriol. 127: 1 1 19-1 127. 1970. . Relationship C L E V L L A NR. I ) ,F., J. V. HOLIJE.,A. J . W I C K E NA. , TOMASZ, POOLEY,M. H.. ancl G. D. S H O C K M A N cell wall synthesis, ant1 debetween location of a~~tolysin, L. DANEO-MOORL;, and G. D. S H O C K M A1975. N . lnhibition of bacterial wall lysins by lipoteichoic acids and related conivelopment of resistance to cellular autolysis in Strrptoc~occrr.~ pounds. Biochem. Biophys. Res. Commun. 67: 1128-1 135. ~f'ircctrlisafter inhibition ofprotein synthesis. J . Bacteriol. 103: C L E V E L A N R. D , F., A. J . W I C K E NL.. DANEO-MOORE, and 457-466. G. D. S H O C K M A1976. N . Inhibition ofwall a~rtolysisin Strop- ROGERS,H. J. 1968. Killing of staphylococci by penicillin. tococc.rrsftr~c.trlisby lipoteichoic acicls ancl lipids. J . Bacterial. Nature (London), 213: 31-33. ROGERS, H . J..ilndC. W. FORSBERG. 1971. Roleofa~~tolysinsin 126: 192-197. the killing of bacteria by some bactericidal antibiotics. J . FAN, D. P. 1970. Cell wall binding properties of the Bncil1rr.s .sirhtili.s a~~tolysin (s). J. Bacteriol. 103: 488-493. Bacteriol. 108: 1235- 1243. F A N ,D. P., and M. M. BECKMAN. 1972. New centrifugation TOMASZ, A.,ilncl J . V. HOL-IJE. 1977. Murein hydrolasesand the technique for isolating enzymes from large cell structures: lytic killing action of penicillin. It1 Microbiology-1977. Etlitctl by D. Schlessinger. American Society for Microbiolisolation ant1 characterization of two Bacil1rr.s slrbti1i.s autolysins. J. Bacteriol. 109: 1258-1265. ogy, Washington, D.C. pp. 209-215.
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Accepted February 9, 1978 U., and R. ROSCHENTHALER. 1978. Induction of autolysis in Bacill~rss1rhrilis by ochSINGER, ratoxin A. Can. J . Microbiol. 24: 563-568. Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 pg/ml caused autolysis of Btrcillris s~rhtilis.Optical density of cultures decreased. and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10pg/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property ofalysis inhibitor. The relevance of thisfraction in respect to autolysisisdiscussed. SINGER, U., et R. ROSCHENTHALER. 1978. Induction of autolysis in Bucill~rss~rbrilisby ochratoxin A. Can. J. Microbiol. 24: 563-568. L'addition d'ochratoxine A (OTA) au cours de la phase exponentielle de croissance i une concentration supkrieure ?I 12 pg/ml produit I'autolyse de Bacillrrssrrhtilis. Ladensiteoptique des culturesdecroit, et i des concentrations plus Clevees les cultures deviennent steriles. L'optimum d'induction d e lyse par OTA est B un pH approximatif de 5. Aux concentrations inferieures B 10 pglml la synthese proteique est inhibee plus fortement que la synthese d'ARN. La synthese de la paroi est aussi fortement inhibee. Une fraction extraite du lysat i lapropriete d'inhiber la lyse. L'iniportance de cette fraction vis-A-vis de I'autolyse et discutee. [Tnlduit par lejournal]
Introduction Autolytic enzymes are regarded as essential in the growth of bacteria. A balance between the hydrolytic action of these enzymes and the synthesis of new cell wall material has been postulated (Holtje and Tomasz 1975), thus involving these enzymes in the replication and growth of the cell wall. Also, autolytic enzymes are believed to play a role in cell separation and genetic transformation (Holtje and Tomasz 1975). The bactericidal action of penicillin and other cell wall synthesis inhibiting antibiotics is due to the action of autolytic enzymes. They destroy the cell wall when the synthesis of the cell wall is inhibited and thus render the cell osmotically fragile, causing death in hypotonic environment (Rogers 1968; Rogers and Forsberg 1971). In a pl-evious paper (Heller et crl. 1975) we showed that ochratoxin A (OTA), a mycotoxin isolated from Aspergillus oclzrcrcells and other fungi, inhibits the growth of Streptococcus.ficecalis. The mode of action involved inhibition of protein and RNA synthesis; even at very high concentrations the bacteria were not killed by OTA. In Bacillils slrhtilis, however, OTA induced autolysis in growing cells when the concentration was higher than 12 Fg/ml. Thus, it was bactericidal to this species. These findings led us to investigate its mode of action in B. srrbtilis more closely. IAuthor to whom reprint requests should be acidressed.
Materials and Methods Brrcteriol Srrrrirls
The experiments were carried out with B . srrhtilis 168 1(which required L-tryptophan or indole) or with a derivative of this strain. A spontaneously occurring mutant was isolated which had lost the property of being lysed in the presence of more than 12pglml of OTA. Even at concentrations as high a s 40 pg/ml this strain could not be lysed and therefore was designated lyt-. Thismutant remainedstableafter subculturing more than 20 times in the absence of OTA. Gro\vrll Corlditiorls orltl Measrrrernents
The minimal medium of Anagnostopoulos and Spizizen (1960) was used with a supplement of 0.5% glucose. Furthermore, it contained per litre: arginine, 80mg; leucine, 250rng; isoleucine, 100mg. sodium glutamate, 500mg; tryptophan, 80mg; valine, 150 mg, and the remaining 14 amino acids in quantitiesof 40mg. The concentration of MgSO, X 7H10 in the salts medium was halved to O.lg per litre a s Mg2+ counteracts OTA inhibition (Heller et 01. 1975). This reduction did not alter the growth rate. The pH of the medium was adjusted to 5.85 with HCI, if not otherwise indicated. For growing protoplasts the hypertonic minimal medium of Hirokawa and Ikeda (1966). adjusted to pH 6.0, was used. Liquid cultures in 100-ml Erlenmeyer flasks with a fused-on measuring tube and two baffles were incubated with aeration at 37°C in a gyrotory water bath shaker (New Brunswick). The Erlenrneyerflasks usually contained 3 ml of the cultu~~ernedium. Optical densities (OD) of cultures were measured in a n Eppendorf photometer at 546nm after allowing the culture to run into the measuring tube. When a comparison was necessary after a given time of incubation, the inocula for the different flasks were taken from the same culture at a n ODof0.400. The growth late (p) was estimated by dividing60min by the doubling time (in minutes). The doubling time was determined according to d = (t , - t o ) log ?/log E , - log E,, where E, and E , are the extinctions at times 1 , and t , , respectively. To obtain values for autolysis rates that were comparable to growth rates,
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the same fol.mul;i wns:ipplied to the autolysis rate. tlesignated as -p. This methotl gives agross estimation o f the al~tolysisrate as al~tolysis~,oughlyfollowetl an exponentigil function for about 2 h >ifteradtlitiono f O T A .
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Proto/)ltr.\li/l# For protoplasting, the method o f Hirokawa and Iketla (1966) was followed. Mrtr.srrrc,t,rc~trr cj/'Pro/eit~c r t r t l RNA S.vt~llrc,.si.s The incorporation o f [14C]ul-acilinto l.ibonucleic acid (RNA) and ['"Clglycine into protein was measured as previoi~slydescribecl (Hellere/ (11. 1975). T o compare the extent of inhibition of protein and R N A synthesis in relation to the time afteratltlition of O T A . the ~xtlioactivity(cpm) o f the respective preclrrsors incol.porated per optical density unit of the OTA-treated c i ~ l t i ~ was r e tlivitlecl by that o f the control c i ~ l t l ~ r e . Mc,tr.srrrc,t~~,tr/ ofMrrcopc~p/itIc~ Sytr//rc,.si.s
The method o f Rogel-s and Forsbelg (1971) was followetl. except that we usetl 7Okglml o f chlol-amphenicol insteutl o f SOpglml. The values for incorporation o f [14C]glucoseinto mi~copepticleswas divided by the O D o f the inhibited cultllres to obtain an idea ofcell wall synthesis per;lvel.:ige cell. I.soltr/iot~~f'L.v,si.s It~lrihi/or'fi.ot~r Bac/rricrl Lystrte.~ I n the isolation o f a macromoleci~lw.fraction from B. .srrbtili.s capable o f inhibiting lysis, the isolation methotl o f Holt.je and Tomasz (1975) was followed. Cells were g~.ownwith aeration to :in OD, o f 0.600. Then 4 mg O T A (36pg/mI) 01.2.5 mg penicillin (22 p g / ~ i i l )or 2mg cycloserine (18pg/ml) were nclcled to re. shaking at 37°C f o ~ 2.5 . h, the O D tlespective c i ~ l t l ~ r e sAfter creased to less than 0.1 in all cult~rres.The lysates were centrifi~geclat 4°C ancl IOOOOrpm for l j m i n . The col-responding supernatants were tlialyzed for 48 h against distillecl waiter with three changes o f water. Aclsorption o f O T A to the lysate was evident when it was ir~-adi[rtedwith U V light :it 350nm. Therefore, IOmg/ml o f bovine serum albumin was atlcled to the water again\t which the lysate wasclialyzecl. As bovine serum albumin strongly binds O T A (Chu 1971: own observ:itions). the O T A content o f the lysate could be recluced from 36 pg/mI to less than 0.4pg/ml by thismethotl. Thisshows that bovine serum albumin bintls O T A more strongly than the lysate. After dialysis, the preparations were frozen at -20°C ant1 lyophilized. The lyophilizates were weighed (about 5 mg), dissolvetl in I ml o f distilled water, and heated at 100°C for 20min. The precipitate was centrifuged down oncl tliscarded. Portions o f the supernatant were iiddetl to culti~res.
FIG. I . Effect o f O T A on B . .sr~h/ilis 168 :it p H 6.0. I n the exponential phase of growth, a cultl~rewas divided into nine subcultures which received O T A (arrow) at the concentrations indicated (pg/ml ofgrowth medium).
lysis occurred at pH 5.1. After OTA addition, autolytic activity could be immediately and significantly increased by a shift to a lower pH (e.g., froni 5.7 to 5.3, data not shown). The temperature optimal for lysis was 36°C; at higher and lower temperatures less autolysis was observed; below 30°C nolysis occurred at all (data not shown). In the course of the work, a mutant was isolated which had lost the property of being lysed by OTA. Growth of this strain, designated lyt-, was still inhibited. OTA did not lyse protoplasts of B. srihtilis (Fig. 3) in a hypertonic mediurn containing 20% of sucrose (Hirokawa and Ikeda 1966). Also, bacillary forms were not lysed in this niediuni. The increase in mass, however, was somewhat reduced in both forms.
Itlhihitiotz of Proteitt cirttl Cell Weill Syr~tl~esis In B. slibtilis OTA inhibits protein synthesis more strongly than RNA synthesis. Figure 4 shows Ochrtrr~aitrA t r t r t l Cl~rttric~trls O T A was isolateil ant1 purifiecl from wheat kernels infected the time dependence of the inhibition of incorpol-awith A. oclirtrcerr.~,as previously described (Heller t r l . 1975). tion of labelled glycine and ~ ~ r a c iinto l acidThe radioactive materials were a generous gift o f Dr. P. precipitable material. Incorporation of labelled Fromageot, C. E. A,, Saclay, France. Chlor~imphenicolwas thyniidine into deoxyribonucleic acid (DNA) was purchased from Serva, Heidelberg. and cycloserine fromsigma, not affected by OTA (data not shown). Munich. Inhibition of mucopeptide synthesis was also demonstrated. Figure 5 shows incorporation of Results [14C]glucoseinto the mucopeptide fraction of the Aritoly~isCcirisecl b y OTA cells in the presence and in the absence of OTA. OTA is a potent growth inhibitor in B. slihtilis. In OTA strongly inhibits niucopeptide synthesis. defined growth medium, at low pH, and in concenChloramphenicol was added to avoid lysis in the trations above 10-20Fglml ailtolysis of the cells presence of mucopeptide synthesis inhibition occ~11.s(Fig. 1). Above 2 0 ~ g l m lthe cultures may (Rogers and FOI-sberg 1971). No inhibition of become sterile. If autolysis is incomplete, cultures m~~copeptide synthesis by this drug was observed. resume growth with a slower growth rate after a delay of several hours. The pH range in which OTA Isoleition cir~tlActiorz of an Inhibitor. c!f'Cell Lysis causes lysis is between 6.0 and 4.0 (Fig. 2). Optimal A heat-resistant, non-dialyzable fraction from B.
S I N G E R A N D ROSCHENT'HALER cpm OTA-wlture I cDm contrd
[%]uracil
incorporation
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0.6
A
C
FIG. 4. Inhibition ofprotein ant1 RNA synthesisin B . s ~ ~ h / i l i . s . Two exponentially g r . o w i n g c ~ ~ l t ~ received ~res 0.6 p C i / m l ( l Ci = 37 GBq) o f [ ' J C ] ~ ~ r a cand i l 0.8pCi/ml o f [lJC]glycine. respectively. An amoLlnt of 10Fglml of O T A was addetl (arrow). ;lncl the inco~.pou~tion o f the precursors into acid-precipi table material was measured after clifferent time intervals. The ratio o f incorpol-ution into OTA-treated cells to that o f the control was c>~lculated.
-3,p
of OTA-treated culture ot controlculture
FIG. 2. p H dependence of the O T A effect in B . .s~,h/ili.s168. ( t r ) Closed triangles :we growth rates o f B. .srlh~ili.sat different pH, open triangles growth rates and lysis rates in the pl-esence of 20 pglml o f O l ' A . (b)The p H range in which O T A is bactericidal is obtained by calc~~lating the ratio between growth rates (p) or lysis rates(- aftertr treatment with OTA,nnd growth rate ( p ) o f the control c ~ ~ l t ~The ~ r concentl.ation e. o f OTA was 20 pglrnl.
I
30
60
90
120
150 min
FIG. 5. Inhibition by O T A o f incorporation ofglucoseinto the peptidoglycan fraction. [14C]gl~~cose was added at time 0. The incorpolxtion was measured in the prebence of 70pglrnI o f chlou~mphenicol(Rogers and Fousberg 197 1). Squares indicate control culture; triangles, after the addition o f 15 pglmlofOl'A.
FIG. 3. Effect o f O T A on bacill;~ry forms and on growing protoplasts in hypertonic medium. Open squares are bacillary form control cultures. closed squares are bacillary form cultures in the presence o f 15 pglml o f 01'A. Open triangles are growing c o n t ~ uprotoplasts, l and closecl triangles growing protoplasts in the pl-esence of 15 pglml o f O T A .
s~~brilis autol ysates was isolated, which inhibited OTA-induced lysis. The autolysis of cultures was brought about by OTA, penicillin, o r cycloserine. OTA-induced lysis was strongly inhibited by this fraction (Fig. 6a). Figure 66 shows the dose dependence of the autolysis rate of bacterial cultures in the presence of 23 Fg/ml of OTA upon concentration of the 'lysis inhibitor.'
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FIG.6. Inhibition ofOTA-induced autolytic activity by a hot waterextract of lysates ('lysis inhibitor'). (0)Circles designate control cultures; open triangles, after the addition of 23 kglrnl of OTA (arrow); and squares, after the addition of 23 ~ g / r nof l OTA and 200kg/ml of 'lysis inhibitor.' ( h ) Relation of concentration of 'lysis inhibitor' to autolysis. Cultures in the exponential growth phase received 23 kglml of OTA each.
Discussion OTA displays antibiotic properties at a pH of about 6 against many gram-positive bacteria. In S . faecalis it is a potent inhibitor of growth, but does not kill the cells, even at concentrations as high as 1 mglml (Heller et al. 1975). In B. subtilis, however, a bactericidal action of OTA is observed under conditions described in this papel-. This effect is due to autolysis of the cells as evident from a decrease of OD, microscopical observations, and especially from the observation that metabolizing protoplasts are not lysed by OTA in hypertonic medium. The finding of a mutant which could not be lysed by OTA but which was still sensitive in respect t o growth inhibition supports the idea that the bactericidal action of OTA is due to the activity of autolytic enzymes. A direct stimulation or inhibi-
tion of the cell wall associated autolysins in a cellfree preparation according to Fan (1970) was not observed (data not shown). Therefore, it can be concluded that OTA does not interfere with activity of autolysin (s). In B. sribtilis, two autolytic enzymes have been found, an amidase and a p-glycosidase (Brown and Young 1970; Fan and Beckman 1972). This is in contrast to S.Jirecnlis, for which only a single enzyme, a muramidase, has been reported (Cleveland et 01. 1976). The enzyme that is likely to be active in OTA-induced autolysis of B. sllbtilis is the p-glycosidase and not the amidase. The reason for this suggestion is that the glycosidase has a pH optimum between 5 and 6; the p H optimum of the amidase is at pH 8.6 (Fan and Beckman 1972). The lysis induced by OTA is optimal at pH5.1. No lysis
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SINGER A N D ROSCHENTHALER
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or growth inhibition is observed at pH 8.6. As de- 1971). Since Holtje and Tomasz (1975) have demscribed for penicillin, the in vitro pH optimum of onstrated a lipoteichoic acid-like lysis inhibitor in the major autolysin is in agreement with the pH pneumococci, it was recognized that activity of optimum of the lytic response in vivo (Tomasz and autolysins is under negative control. In B. slrbtilis, Holtje 1977). Also, the temperature dependence of the amidase activity was also found to be inhibited OTA-induced a~~tolysis supports this suggestion. by a lipoteichoic acid-type inhibitor (Cleveland et The activity of the amidase has been reported (Fan 01. 1975). The 'lysis inhibitor' which we have isoand Beckman 1972) to be only a little lower at 30°C lated from autolysates of B. slrhtilis was obtained than at 37°C. Even at 24°C this enzyme is still by the method of Holtje and Tomasz (1975). As this active. However, no lysis is caused by OTA below fraction was heated for 20 min at 100°C, most of the 30°C. The temperatul-e dependence of OTA-in- protein was denatured and discarded. Therefore, it duced a ~ ~ t o l y sdiffers is from that of the amidase. is very likely that this fraction contained the As no direct action of OTA on the autolysin lipoteichoic acid-like material described by the activity could be demonstrated, a similar mech- authors mentioned. This would mean that the inanism for the bactericidal action of OTA had to hibitor for the P-glycosidase is the same as for the be considered as that described for other lysis- amidase in B. slrhtilis. Cleveland et al. (1976) have inducing agents. Rogers and Forsberg (1971) have shown that the lytic activity of S. , f ~ ~ e c a l i s I-eported that inhibitors of mucopeptide synthesis, muramidase (i.e., a different enzyme than amidase) such as penicillin or cycloserine, are able to induce can be inhibited by a lipoteichoic acid fraction from autolytic activ~tyin bacterial cells. It was dem- a hot water extract, and this observation supports onstrated that in B. s~rhtilis,OTA causes inhibi- our suggest ion. The difference in the sensitivity to OTA-induced tion of cell wall synthesis under the conditions described (Fig. 5). But even at concentrations lower autolysis between S . f~iecalisand B. s~lhtilismay b e than those necessary for autolysis, OTA is a strong due to the inhibition by OTA of a protein necessary inhibitor of protein synthesis. This is quite a unique to activate the latent muramidase of S. ,fhecalis property, as in general protein synthesis inhibitors described by Pooley and Shockman (1970). Cells of (e.g. chloramphenicol) are used to prevent lysis S . fuecalis become very rapidly resistant t o au(Rogers and Forsberg 1971 ). Preliminary experi- tolysis when protein synthesis is inhibited (Pooley ments pel-formed to find an explanation for this and Shockman 1970). This is in contrast toB. sllbdiscrepancy showed that OTA prevented cyclo- tilis which requires the addition of protein synthesis serine-induced lysis, but stimulated autolysis inhibitors approximately 30 min before addition of cell wall inhibitors t o prevent autolysis (Rogers and caused by penicillin or vancomycin. In a recent investigation, we have shown that Forsberg 1971; own observations). No observations were made that would justify after addition of OTA to B. suhtilis, the pools of the notion that a specific and strong binding of O T A ppGpp and pppGpp increase about four times. Simultaneously, a decrease in the adenosine 5'-tri- to the lysis inhibitor would occur with a simultanephosphate (ATP) and guanosine 5'-triphosphate ous inactivation of the biological activity of OTA. (GTP) pools is o b s e r v e d . q f t e r the cells were Also, no nonspecific improvement of growth condipreincubated with chloramphenicol and then ex- tions by this fraction was found. Therefore, it is posed to OTA, only the pool of ppGpp increased conceivable that in S .fkecalis the protein synthesis while the level of pppGpp remained as low as in the inhibition caused by OTA prevents autolysis, while untreated culture (data not shown). Ishiguro and in B. suhtilis it triggers a regulatory mechanism, Ramey (1976) found a stringent control of the involving the MS-nucleotides, leading to an inhibimucopeptide synthesis in Escherichia coli and an tion of mucopeptide synthesis, release of lysis ininhibition by ppGpp of in vitro mucopeptide syn- hibitor into the medium (Tomasz and Hijltje 1977), thesis. The same mechanism probably operates in and finally to lysis. the inhibition of mucopeptide synthesis by OTA in B. sllhtilis. The ability of cell wall inhibitors to induce autolAcknowledgements ysis was originally thought to be due to an imbalWe are very much obliged to Dr. J . Harwig, ance between synthesis and the degradative action Health and Welfare Canada, for his patient reviewof autolysins (Rogers 1968; Rogers and Forsberg ing of our manuscript and for suggestion of many 'U S ~ n g eand ~ R Roschenthaler. 1978. Inhib~tionof p ~ o t e ~ n valuable corrections. We also thank Deutsche Forschungsgemeinschaft for supporting this prosynthesis by o c h ~ a t o x ~An In B o ~ ~ l l ~ s~lbtllls. rs Eur. J. Toxic01 (In p ~ e s s ) . ject, A. Wegner for performing some control exper-
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iments, and Dr. K. Heller for typing the manuscript.
HELLER, K., C. Sc.1-IULZ,R. LOSER,and R. ROSCHENIHALER. 1975. The inhibition ofbacterialgrowth by ochratoxin A. Can. J . Microbial. 21: 972-979. H I R O K A WH., A , and Y. IKEDA.1966. Genetic recombination of transforming DNA molecules with recipient genome and ANAGNOS-IOPOULOS. C . , and J . S ~ r z r z t 1960. ~ . Requirements :inlong themselves in protoplasts of Btrc~illiissrrbti1i.s. J . Bacfor transformation in Btrcil1ri.s srr6tili.s. J . Bacteriol. 81: teriol. 92: 455-463. 741-716. BROWN,W. C.. and F. E. YOUNG.1970. Dynamic interaction HOLTJE,J. V., ilncl A. TOMASZ.1975. Lipoteichoic acid: a between cell wall polymers, ext~.acellular proteases, ant1 specific inhibito~.ofa~~tolysin activity in pneumococcus. Proc. autolytic enzynies. Biocheni. Biophys. Res. Commun. 38: Natl. Acacl. Sci. U.S.A. 72: 1690-1694. ISHIGURO, E., and W. D. RAMEY.1976. Stringent control of 546-568. C H U .F. S . 1971. Interaction of ochratoxin with bovine serum peptitloglycan synthesis in Escherichitr coli K-12. J . Bacnlb~~niin. Arch. Biocheni. Biophys. 147: 359-366. teriol. 127: 1 1 19-1 127. 1970. . Relationship C L E V L L A NR. I ) ,F., J. V. HOLIJE.,A. J . W I C K E NA. , TOMASZ, POOLEY,M. H.. ancl G. D. S H O C K M A N cell wall synthesis, ant1 debetween location of a~~tolysin, L. DANEO-MOORL;, and G. D. S H O C K M A1975. N . lnhibition of bacterial wall lysins by lipoteichoic acids and related conivelopment of resistance to cellular autolysis in Strrptoc~occrr.~ pounds. Biochem. Biophys. Res. Commun. 67: 1128-1 135. ~f'ircctrlisafter inhibition ofprotein synthesis. J . Bacteriol. 103: C L E V E L A N R. D , F., A. J . W I C K E NL.. DANEO-MOORE, and 457-466. G. D. S H O C K M A1976. N . Inhibition ofwall a~rtolysisin Strop- ROGERS,H. J. 1968. Killing of staphylococci by penicillin. tococc.rrsftr~c.trlisby lipoteichoic acicls ancl lipids. J . Bacterial. Nature (London), 213: 31-33. ROGERS, H . J..ilndC. W. FORSBERG. 1971. Roleofa~~tolysinsin 126: 192-197. the killing of bacteria by some bactericidal antibiotics. J . FAN, D. P. 1970. Cell wall binding properties of the Bncil1rr.s .sirhtili.s a~~tolysin (s). J. Bacteriol. 103: 488-493. Bacteriol. 108: 1235- 1243. F A N ,D. P., and M. M. BECKMAN. 1972. New centrifugation TOMASZ, A.,ilncl J . V. HOL-IJE. 1977. Murein hydrolasesand the technique for isolating enzymes from large cell structures: lytic killing action of penicillin. It1 Microbiology-1977. Etlitctl by D. Schlessinger. American Society for Microbiolisolation ant1 characterization of two Bacil1rr.s slrbti1i.s autolysins. J. Bacteriol. 109: 1258-1265. ogy, Washington, D.C. pp. 209-215.
