Journal of General Microbiology (1976), 96,87-94 Printed in Great Britain
Autolysis in Strains of Viridans Streptococci By M A R I E - L O U I S E S U N D A N D L. L I N D E R Department of Oral Microbiology, Karolinska Institute, Stockholm, Sweden (Received I 9 December I 975) SUMMARY
Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na,HPO,/ NaH,PO, buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCCI5909) was maximal in 0.05 to 0-2M buffer, while the two S. mutans strains and S. mitis ATCCI5912 showed maximal autolysis in 0.5 and 1.0M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 "C than at 45 "Cand 10"C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, A T C C I ~ ~and O ~ ATCCI5912) was maximal at pH 7.0 and 7.5 and in 1.0M buffers. Streptococcus mitis A T C C I ~ ~ O ~ also showed maximal lysis in 0.01 M and 0.5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCCI5912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quantity.
INTRODUCTION
Autolysins, that is, bacterial enzymes or enzyme systems which can cause bacterial lysis by the hydrolysis of various bonds in the peptidoglycan of the wall, have an important physiological role in, for example, division, cell separation and remodelling of shape (Ghuysen & Shockman, I 973 ; Rogers, 1970; Fan, 1970b). In addition, autolytic enzymes may be involved in the final stages of peptidoglycan synthesis in at least one species (Pooley et al., 1972;Pooley & Shockman, 1969). Linder (1974~) showed that autolysis of some of the cells in a population may release cell-bound enzymes specific for substrates unable to penetrate the cell. Autolytic enzymes are suitable for preparing spheroplasts and protoplasts (Mohan et al., 1965;Joseph & Shockman, 1974)and for isolating bacterial membranes (Salton & Freer, 1965). Autolysis under controlled conditions is also an effective and gentle method for disintegration of cells to extract cell products (Linder, 19746;Davis & Salton, 1975). The viridans streptococcus, S. mitis ATCC903,has been investigated for autolysis (Linder, 19746, c). Whole cells of this strain revealed unusual lysis behaviour compared with other strains of the Lactobacillaceae (Coyette & Shockman, I 973 ; Coyette & Ghuysen, I 970; Shockman et al., 1961; Neujahr & Logardt, 1973): (i) the rate of autolysis was maximal in buffers of high molarity ;(ii) stationary-phase cells autolysed as readily as exponential-phase cells; and (iii) lysis was maximal at pH values above 7. The aim of the present investigation was to determine optimum conditions for autolysis
88
M-L.S U N D A N D L. LINDER
of whole cells and walls of S. sanguis, S. mutans and S. mitis. The mode of action of the I~, autolytic system in the strain with the highest autolytic activity, S.mitis A T C C I ~ ~was investigated. METHODS
Bacterial strains. Streptococcus sanguis strains 804 (Carlsson, I 965) and OPCI (Carlsson, ~968),S.mutans strains KPSK2 (Carlsson, 1968)and IB (Krasse, 1966), and S. mitis strains ATCCI 5909, ATCCI59I2 and ATCCg03 were used. Cultivation technique and nutrient medium. Bacteria were grown anaerobically at 37 "C and pH 6.5 in a stirred fermenter (FG 500,Biotec, Stockholm, Sweden) with automatic pH control. The medium, PPI,contained proteose peptone and 2% (w/v) glucose (Linder, Holme & Frostell, 1974). Lysis of whole cells. Bacteria were harvested in the late-exponential growth phase (exp. cells) and in the stationary phase about 30 min after glucose exhaustion (stat. cells). They were washed twice in cold (5 to 7 "C) 0.01M-sodium phosphate buffer pH 6.8, resuspended in appropriate incubation buffers, homogenized with a tissue grinder (A. H. Thomas, Philadelphia, U.S.A.), and incubated at 37 "Cfor 20 h. Preparation of walls. Bacteria harvested in the late-exponential growth phase were washed twice in cold 0.01M-sodium phosphate buffer pH 6.8, resuspended in 20 ml of the same buffer at a concentration equivalent to about 80 mg dry wt ml-l, and disintegrated in the X-press by the method of Edebo (1960). To the resulting suspension, deoxyribonuclease (20 pg ml-l) and ribonuclease (2 pg ml-l) were added. After 10min at 20 "C, the suspension was centrifuged (3000g; 20 min) to remove the remaining whole cells. The supernatant fraction, carefully decanted, was centrifuged (10000g; 20 min) to sediment the walls, which were then washed four times in cold 0.01M-sodium phosphate buffer pH 6.8. All experiments were made with freshly prepared walls. The degree of contamination of the wall preparations by whole cells was estimated by viable count. Wall preparations of strains of S. mitis suspended in 20 ml 0.01M-sodium phosphate buffer pH 6.8 yielded less than 102viable cells per ml. Preparation of walls free from autolytic activity. Wall-bound autolytic enzymes can be inactivated to yield autolysin-free walls that are suitable as substrate(s) for isolated autolysins (Fan, 1g7oa;Coyette & Shockman, 19-73) Walls of s.mitis ATCCI5gI2, free from autolytic activity, were prepared by heating to IOO "Cfor 15 min. Those of S.mitis ATCCg03, however, could not be completely inactivated by heating to IOO "C for 15 to 30 min or by treatment with sodium dodecylsulphate (SDS) I % (w/v; final concentration) at 37 "C for 3 h, although lysis was decreased by 70 to 80% compared with controls. For these walls, exposure to low pH gave autolysin-free walls. Walls incubated at pH 4-9in 0.05 M-sodium phosphate buffer at 20 "Cfor 15 min showed no lysis during this period, and no lysis during subsequent incubation for 20 h in 0.01M-sodium phosphate buffer pH 7-5. Autolysin-free walls were used as controls in experiments where autolysis was followed by thin-layer chromatography of dinitrophenylated products of wall autolysates. Measurement of autolysis. Lysis was determined by the decrease in extinction at 550 nm, using a Zeiss PMQ I1 spectrophotometer, and is expressed as the percentage decrease in the initial extinction. All readings were made in the range 0.100to 0.800 by diluting the sample in the same buffer or in distilled water. Analytical procedures. The autolysis of walls was followed chemically by measuring the solubilization of N-terminal amino groups and reducing groups. Free amino groups were determined by the reaction with 2,4-dinitrofluorobenzene (DNFB) as described by Ghuysen,
Autolysis of streptococci
89
Tipper & Strominger (1966).The liberation of reducing groups was assayed by the sensitive method of Park & Johnson (1949).Samples of walls were incubated for autolysis for various periods of time and then centrifuged at 20000 g for 15 min. The supernatants were stored frozen until assayed for free amino groups and reducing groups. The dinitrophenylated amino acids were also estimated by thin-layer chromatography on silica-gel plates (Merck, Darmstadt, Germany) according to Ghuysen et al. (I 966). Chemicals. DNase I, RNase A type I-A, DNFB and DNFB-amino acids were all obtained from Sigma. RESULTS
Autolysis of whole cells Whole cells of S.mitis ATCC903 were not studied as their autolysis has already been reported (Linder, 19743).With the other strains, samples were harvested from the exponential and stationary growth phases, washed twice and suspended in Na,HPO,/NaH,PO, buffer to give an extinction of about 12,equivalent to approximately 4 mg dry wt ml-l. The effect of pH and salt concentration was studied. The results (Figs I and 2) show that all strains autolysed in phosphate buffer, the extent of lysis being affected by pH and salt concentration. The pH optimum for autolysis of these six strains, as for the previouslystudied S. mitis ATCC903 (Linder, 1974b),was above pH 7. One strain, S.sanguis 804, also had a high autolytic activity at pH 4-5(Fig. I a). Streptococcus mitis ATCCI5909 autolysed to a high degree over a broad range of pH values above 5-5 (Fig. re). Exp. cells exhibited higher autolytic activity than stat. cells except for two strains, S. mutans IB and S.mutans KPSK2 (Figs ~ d c), , which showed maximal lysis in stat. cells. The pH curves for exp. cells and stat. cells were almost parallel in the other strains, with differences in lysis of 5 to 20 yo. Salt concentration had a more varied affect on lysis than pH. Some strains (Figs 26, e) showed maximal lysis in 0.05 to 0.2M buffers, while S. mitis ATCCI5912 and exp. cells of S.mutans IB showed increasing autolysis with increasing salt concentration (Figs 2f, d ) . As can be seen from Figs I and 2, S.mitis ATCCI5912 and S.mitis ATCCI5909 exhibited the highest autolytic activity, being at maximum more than 80 and 60 % respectively. Whole cells (both exp. and stat.) were also tested for autolysis at 45 "C and J O "C at various pH values and at various salt concentrations, as in the experiments at 37 "C. Optimal autolysis at 45 "C occurred at the same values of pH and salt molarity as in the experiments at 37 "C.Maximum autolysis was o to 5 yoless at 45 "Cthan at 37 "Cfor both exp. and stat. cells. Autolysis also occurred at 10"Cbut at a reduced rate: the maximum autolytic activity was 40 to 50% of that at 37 "C. Autolysis of walls To study autolysis of walk, three strains of s. mitis, ATCCI59 I 2, ATCCI 5909 and ATCC903, were chosen because of their high autolytic activity as demonstrated here and previously (Linder, rg74b). Walls were incubated for 20 h in Na,HPO,/NaH,PO, buffer under the same conditions of pH, salt molarity and temperature as for whole cells. The pH optimum for strain A T C C I ~was ~ IpH ~ 7.0,and for strains ATCCI5909 and ATCC903, pH 7.5 (Fig. 3a). Salt concentration affected lysis of the walls differently in the three strains (Fig. 3b). Walls ~I~ maximum lysis at high salt concentration (1.0M), while those of strain A T C C I ~exhibited of strains A T C C ~ Oand ~ ATCCI5909 showed two maxima, one at 0.01M and the other at 0.5 to 1.0M. In experiments performed at 45 "Cand at 10 "C(Figs 4a,b, c), wall lysis was reduced by
90
M-L.S U N D A N D L. L I N D E R
I
I
I
I
60
.3" 40 h
.-
v) v)
3 20
? 7.0
5.0
9.0
5.0
7.0 PH
9.0
5.0
7.0
9.0
Fig. I . Autolysis of whole cells of six strains of viridans streptococci in 0-2M - S O ~ ~ phosphate U ~ buffer of different pH values. (a) Streptococcus sanguis 804; (b) S. sanguis OPCI; (c)S. mutans K P S K ~ ;(d) S. mu tans^^; (e)S. mitis A T C C I ~ ~(f) O ~S.; mitis ~ ~ ~ ~ 1 5 Closed 9 1 2 .symbols, exp. cells; open symbols, stat. cells.
yo
10 to 20 at 45 "C and by 30 to 40% at 10"C compared with lysis at 37 "C.In strain ATCCg03, lysis at 10"C was reduced by about 80 % and remained at a practically constant
level over the pH range studied.
Mode of action of the autolytic enzyme system of S. mitis A T C C I ~ ~ I ~ Figures I to 3 show clearly that, of all the strains tested, S. mitis A T C C I ~had ~ Ithe ~ highest autolytic activity for both whole cells and walls. Autolysis of this strain was characterized by a decrease in extinction and by release of soluble N-terminal amino groups. From an initial extinction of the wall suspensions of about 21 (corresponding to g mg dry wt ml-l), autolysis started without lag (Fig. 5) and was accompanied by a progressive release of soluble amino groups that was proportional to the decrease in extinction. No release of reducing groups was detected. Dinitrophenylated amino acids were identified by comparison with known standards of DNFB-derivatives of amino acids on thin-layer plates of silica gel (Ghuysen et al., 1966). Walls were autolysed at 37 "Cfor I, 3 and 20 h in 0.2 M-Na,HPO,/NaH,PO, buffer, pH 7-5. Two controls were used : a zero-time autolysis supernatant, and the supernatant of autolysinfree walls incubated in the same buffer as the test samples for 20 h at 37 "C.Dinitro-
9=
Autolysis of streptococci
70
50
30 0.2
1.0
0.6
0.2
0-2
1.0
0.6
0.6
Phosphate concn (M) Fig. 2. Autolysis of whole cells of six strains of viridans streptococci in sodium phosphate buffer pH 7.5 of different molarities. Strains and symbols as in Fig. I . 80
60
-
I
I
(6)
(a)
I
I
I
+
-
-
v
40
-
7.0 9.0 0.2 0.6 1.0 PH Phosphate concn (M) Fig. 3. Autolysis of walls of three S. mitis strains in sodium phosphate buffer at different values, (b) molarities. m, A T C C ~ ;O ~ ,ATCCI5909 ; A, ATCCI 5912, 5.0
1.0
M-L. S U N D A N D L. L I N D E R
92
80
60
40
20 7.0 9.0 5.0 PH Fig. 4. Effect of temperature on autolysis of walls from three S. mitis strains in 0-2M-sodium ; ATCCI5909; (c) ~~~(215912. A, 10"c: phosphate buffer of different pH values. (a) A T c C ~ O ~(6) OY37 "C; a 4 5 "C.
7.0
5.0
9.0
6-
I
I
I
1
-
0
.d
3 .r
I
I
I
60 Incubation time (min)
7 :14
.I I20
Fig. 5. Kinetics of autolysis of S. mitis A T C C I ~ walls, ~ I ~ incubated in 0.2 M-sodium phosphate buffer pH 7-5at 37 "C.Extinction ( 0 )was measured at 550 nm, and free:amino groups(.) were determined with I-fluor0-2~4-dinitrobenzene. Alanine was used as the standard.
Table
I.
Amino acids released into the soluble fraction during autolysis of walls 01 S. mitis ATCCI5912 Walls (initial concentration, g mg dry wt ml-l) were incubated for 5 h at 37 "C.
Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline GIycine
Concn (,urn01ml-l)
Amino acid Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
0.868 trace ND
0.297 0.379 0.658 0.8 I 2
0.305
0.684 ND,
Not detected.
Concn (pmol mi-l)
1.134 trace 0.726 0.135 0.476 0.700 0.185 0.285
Autolysis of streptococci 93 phenylated amino groups of alanine, glutamic acid, lysine, valine, serine, glycine and leucine occurred in all three test supernatants (1,3 and 20 h). All spots which were visible in the 20 h tests were also seen at I and 3 h, although they were fainter. No dinitrophenylated products were obtained from the two controls. The release of free amino acids during autolysis of walls at 37 "C for 5 h was investigated using an amino-acid analyser. Autolysin-free walls were incubated in parallel as a control. Between 14 and 16 amino acids were released into the soluble fraction during autolysis (Table I), alanine, lysine and glutamic acid being released in greatest quantity. .
DISCUSSION
It was suggested in a previous paper that autolysis in S. mitis is a physiologically useful In this strain (ATCC~O~), lysis in sodium mechanism for release of enzymes (Linder, 1974~~). phosphate buffer is regulated by the salt concentration. A striking characteristic of autolysis of this strain is that a constant proportion of the cells is resistant to autolysis regardless of whether they are harvested in the exponential growth phase or in the stationary (autolytic) phase. The potentiating effect of high salt concentration on lysis in S.mitis ATCCI5912 and S.mutans IB is similar to that in S.mitis ATCC903 but has not been reported with other bacteria. However, in S.faecalis ~ ~ ~ ~ 9autolysis 7 9 0 ,of exp. cells is most rapid at phosphate buffer concentrations of 0.01M and 0.3 M (Shockman et al., 1961).The present investigation has shown that all strains of viridans streptococci tested autolyse in phosphate buffer and that cells harvested in the stationary phase possess almost the same autolytic activity as exponential cells. This contrasts with the autolytic behaviour of S.faecalis ATCC9790 (Shockman et al., 1961)and strains of Lactobacillus (Coyette & Ghuysen, 1970;Neujahr & Logardt, 1973). Many of the amino acids released during autolysis of the walls of S.mitis ATCCI5912 were probably not part of the peptidoglycan portion of the wall and may represent a polypeptide or protein. The observation that autolysin-free walls did not release free amino groups or amino acids indicates that the peptide or protein may have been a wall protein rather than an intracellular or membrane contamination of the wall preparation. It appears that the autolytic system of S.mitis A T C C I ~contains ~ I ~ endopeptidases which hydrolyse bonds both within the tetrapeptide of the peptidoglycan and within the wall protein. Bacteriolytic enzymes splitting bonds within the tetrapeptide are extremely rare; only two reports on such enzymes have appeared (Neujahr & Logardt, 1973;Welker, 1971).These enzymes are of considerable physiological importance and of experimental and taxonomic interest. We have attempted to isolate the autolytic enzymes of S.mitis ATCCI5912 but without success. Soluble enzyme activity could not be recovered from the supernatant fluid after wall lysis, perhaps because the autolytic enzymes are firmly bound to the walls, as in S. faecalis (Shockman, Pooley & Thompson, 1967),and are released only at unusually high concentrations of salt (Pooley, Porres-Juan & Shockman, 1970). It is also possible that the enzymes are rapidly inactivated after release from the wall. Thus Coyette & Shockman (1973)found that only 20 % of the original wall lytic activity could be recovered in the supernatant after lysis of walls of Lactobacillus acidophilus for 16h at 37 "C. This investigation was supported by grants from the Faculty of Odontology, Karolinska Institute, from Patentmedelsfonden for odontologisk profylaxforskning and from The Swedish Dental Society. We wish to thank Dr G. Samuelsson, Uppsala, for performing the amino-acid analyses.
94
M-L. S U N D A N D L. LINDER REFERENCES
CARLSSON, J. (1965). Zooglea-forming streptococci resembling Streptococcus sanguis isolated from dental plaque in man. Odontologisk Revy 16, 348-358. CARLSSON, J. (1968). A numerical taxonomic study of human oral streptococci. Odontologisk Revy 19, 137-160. COYETTE, J. & GHUYSEN, J-M. (1970). Wall autolysin of Lactobacillus acidophilus strain 63 AM Gasser. Biochemistry, New York 9, 2952-2955. COYETTE, J. & SHOCKMAN, G. D. (1973). Some properties of the autolytic N-acetylmuramidase of Lactobacillus acidophilus. Journal of Bacteriology 114, 34-41. DAVIS,R. H. & SALTON, M. R. J. (1975). Some properties of a ~-alaninecarboxypeptidase in envelope fractions of Neisseria gonorrhoeae. Infection and Immunity 12, I 065-1 078. EDEBO, L. (1960). A new press for the disruption of micro-organismsand other cells. Journal of Biochemical and Microbiological Technology and Engineering 2,453-479. FAN,D. P. (1970~).Cell wall binding properties of the Bacillus subtilis autolysin(s).Journal of Bacteriology 103,488-493. FAN,D. P. (197ob). Autolysin(s) of Bacillus subtilis as dechaining enzyme. Journal of Bacteriology 103, 494-499. GHUYSEN, J-M. & SHOCKMAN, G. D. (1973). Biosynthesis of peptidoglycan. In Bacterial Membranes and Walls, pp. 37-130. Edited by L. Leive. New York: Marcel Dekker. GHUYSEN, J-M., TIPPER, D. J. & STROMINGER, J. L. (1966). Enzymes that degrade bacterial cell walls. In Methods in Enzymology, vol. 8, pp. 685-699. Edited by S. P. Colowick and N. 0. Kaplan. New York : Academic Press. JOSEPH, R. & SHOCKMAN, G. D. (1974). Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis 9790 : release of cell wall, autolysin and formation of stable autoplasts. Journal of Bacteriology 118, 735-746. KRASSE, B. (1966). Human streptococci and experimental caries in hamsters. Archives of Oral Biology 119429-436. LINDER, L. ( 1 9 7 4 ~ ~Formation ). and release of hyaluronidase and aminopeptidase in growing cultures of Streptococcus mitis A T C C ~ O Acta ~ . pathologica et microbiologica scandinavica B 82, 608-6 14. LINDER, L. (I974 b). Extraction of cell-bound hyaluronidase and aminopeptidase from Streptococcus mitis A T C C ~ Acta O ~ . pathologica et microbiologica scandinavica B 82, 593-601. LINDER, L. (I 974 c). Formation and release of hyaluronidase and aminopeptidase in non-growing cells of Streptococcus mitis ATCC903. Acta pathologica et microbiologica scandinavica B 82, 61 5-624. LINDER,L., HOLME, T. & FROSTELL, G. (1974). Hyaluronidase and aminopeptidase activity in cultures of Streptococcus mitis ATCC903. Acta pathologica et microbiologica scandinavica B 82, 52 1-526. MOHAN,R. R., KRONISH, D. P., PIANOTTI, R. S., EPSTEIN, R. L. & SCHWARTZ, B. S. (1965). Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. Journal of Bacteriology 90, 1355-1 364. NEUJAHR, H. Y. & LOGARDT, I. M. (1973). Autolytic enzyme system from Lactobacillus fermentii. Biochemistry, New York 12, 2578-2583. PARK,J. T. & JOHNSON,M. J. (1949). A method for determination of reducing power. Journal of Biological Chemistry 181, 149-151. POOLEY, H. M. & SHOCKMAN, G. D. (1969). Relationship between the latent form and the active form of the autolytic enzyme of Streptococcus faecalis. Journal of Bacteriology 100,617-624. POOLEY, H. M., PORRES-JUAN, J. M. & SHOCKMAN, G. D. (1970). Dissociation of an autolytic enzyme-cell wall complex by treatment with unusually high concentrations of salt. Biochemical and Biophysical Research Communications 38, I 134-1 140. POOLEY, H. M., SHOCKMAN, G. D., HIGGINS,M. L. & PORRES-JUAN, J. M. (1972). Some properties of two autolytic-defectivemutants of Streptococcus faecalis ~ ~ C C 9 7 9Journal 0. of Bacteriology 109, 423-43 I . ROGERS, H. J. (1970). Bacterial growth and the cell envelope. Bacteriological Reviews 34, 194-214. SALTON, M. R. J. & FREER,J. H. (1965). Composition of the membranes isolated from several Grampositive bacteria. Biochimica et biophysica acta 107, 530-538. SHOCKMAN, G. D., CONOVER, M. J., KOLB,J. J., PHILLIPS, P. M., RILEY, L. S. & TOENNIES, G. (1961). Lysis of Streptococcus faecalis. Journal of Bacteriology 81, 36-43. SHOCKMAN, G. D., POOLEY, H. M. & THOMSON, J. S. (1967) Autolytic enzyme system of Streptococcus faecalis. 111. Localisation of the autolysin at the sites of cell wall synthesis. Journal of Bacteriology 94, 1525-1530. WELKER, N. E. (1971). Structure of the cell wall of Bacillus stearothermophilus:mode of action of a thermophilic bacteriophage lytic enzyme. Journal of Bacteriology 107, 697-703.
Autolysis in Strains of Viridans Streptococci By M A R I E - L O U I S E S U N D A N D L. L I N D E R Department of Oral Microbiology, Karolinska Institute, Stockholm, Sweden (Received I 9 December I 975) SUMMARY
Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na,HPO,/ NaH,PO, buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCCI5909) was maximal in 0.05 to 0-2M buffer, while the two S. mutans strains and S. mitis ATCCI5912 showed maximal autolysis in 0.5 and 1.0M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 "C than at 45 "Cand 10"C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, A T C C I ~ ~and O ~ ATCCI5912) was maximal at pH 7.0 and 7.5 and in 1.0M buffers. Streptococcus mitis A T C C I ~ ~ O ~ also showed maximal lysis in 0.01 M and 0.5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCCI5912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quantity.
INTRODUCTION
Autolysins, that is, bacterial enzymes or enzyme systems which can cause bacterial lysis by the hydrolysis of various bonds in the peptidoglycan of the wall, have an important physiological role in, for example, division, cell separation and remodelling of shape (Ghuysen & Shockman, I 973 ; Rogers, 1970; Fan, 1970b). In addition, autolytic enzymes may be involved in the final stages of peptidoglycan synthesis in at least one species (Pooley et al., 1972;Pooley & Shockman, 1969). Linder (1974~) showed that autolysis of some of the cells in a population may release cell-bound enzymes specific for substrates unable to penetrate the cell. Autolytic enzymes are suitable for preparing spheroplasts and protoplasts (Mohan et al., 1965;Joseph & Shockman, 1974)and for isolating bacterial membranes (Salton & Freer, 1965). Autolysis under controlled conditions is also an effective and gentle method for disintegration of cells to extract cell products (Linder, 19746;Davis & Salton, 1975). The viridans streptococcus, S. mitis ATCC903,has been investigated for autolysis (Linder, 19746, c). Whole cells of this strain revealed unusual lysis behaviour compared with other strains of the Lactobacillaceae (Coyette & Shockman, I 973 ; Coyette & Ghuysen, I 970; Shockman et al., 1961; Neujahr & Logardt, 1973): (i) the rate of autolysis was maximal in buffers of high molarity ;(ii) stationary-phase cells autolysed as readily as exponential-phase cells; and (iii) lysis was maximal at pH values above 7. The aim of the present investigation was to determine optimum conditions for autolysis
88
M-L.S U N D A N D L. LINDER
of whole cells and walls of S. sanguis, S. mutans and S. mitis. The mode of action of the I~, autolytic system in the strain with the highest autolytic activity, S.mitis A T C C I ~ ~was investigated. METHODS
Bacterial strains. Streptococcus sanguis strains 804 (Carlsson, I 965) and OPCI (Carlsson, ~968),S.mutans strains KPSK2 (Carlsson, 1968)and IB (Krasse, 1966), and S. mitis strains ATCCI 5909, ATCCI59I2 and ATCCg03 were used. Cultivation technique and nutrient medium. Bacteria were grown anaerobically at 37 "C and pH 6.5 in a stirred fermenter (FG 500,Biotec, Stockholm, Sweden) with automatic pH control. The medium, PPI,contained proteose peptone and 2% (w/v) glucose (Linder, Holme & Frostell, 1974). Lysis of whole cells. Bacteria were harvested in the late-exponential growth phase (exp. cells) and in the stationary phase about 30 min after glucose exhaustion (stat. cells). They were washed twice in cold (5 to 7 "C) 0.01M-sodium phosphate buffer pH 6.8, resuspended in appropriate incubation buffers, homogenized with a tissue grinder (A. H. Thomas, Philadelphia, U.S.A.), and incubated at 37 "Cfor 20 h. Preparation of walls. Bacteria harvested in the late-exponential growth phase were washed twice in cold 0.01M-sodium phosphate buffer pH 6.8, resuspended in 20 ml of the same buffer at a concentration equivalent to about 80 mg dry wt ml-l, and disintegrated in the X-press by the method of Edebo (1960). To the resulting suspension, deoxyribonuclease (20 pg ml-l) and ribonuclease (2 pg ml-l) were added. After 10min at 20 "C, the suspension was centrifuged (3000g; 20 min) to remove the remaining whole cells. The supernatant fraction, carefully decanted, was centrifuged (10000g; 20 min) to sediment the walls, which were then washed four times in cold 0.01M-sodium phosphate buffer pH 6.8. All experiments were made with freshly prepared walls. The degree of contamination of the wall preparations by whole cells was estimated by viable count. Wall preparations of strains of S. mitis suspended in 20 ml 0.01M-sodium phosphate buffer pH 6.8 yielded less than 102viable cells per ml. Preparation of walls free from autolytic activity. Wall-bound autolytic enzymes can be inactivated to yield autolysin-free walls that are suitable as substrate(s) for isolated autolysins (Fan, 1g7oa;Coyette & Shockman, 19-73) Walls of s.mitis ATCCI5gI2, free from autolytic activity, were prepared by heating to IOO "Cfor 15 min. Those of S.mitis ATCCg03, however, could not be completely inactivated by heating to IOO "C for 15 to 30 min or by treatment with sodium dodecylsulphate (SDS) I % (w/v; final concentration) at 37 "C for 3 h, although lysis was decreased by 70 to 80% compared with controls. For these walls, exposure to low pH gave autolysin-free walls. Walls incubated at pH 4-9in 0.05 M-sodium phosphate buffer at 20 "Cfor 15 min showed no lysis during this period, and no lysis during subsequent incubation for 20 h in 0.01M-sodium phosphate buffer pH 7-5. Autolysin-free walls were used as controls in experiments where autolysis was followed by thin-layer chromatography of dinitrophenylated products of wall autolysates. Measurement of autolysis. Lysis was determined by the decrease in extinction at 550 nm, using a Zeiss PMQ I1 spectrophotometer, and is expressed as the percentage decrease in the initial extinction. All readings were made in the range 0.100to 0.800 by diluting the sample in the same buffer or in distilled water. Analytical procedures. The autolysis of walls was followed chemically by measuring the solubilization of N-terminal amino groups and reducing groups. Free amino groups were determined by the reaction with 2,4-dinitrofluorobenzene (DNFB) as described by Ghuysen,
Autolysis of streptococci
89
Tipper & Strominger (1966).The liberation of reducing groups was assayed by the sensitive method of Park & Johnson (1949).Samples of walls were incubated for autolysis for various periods of time and then centrifuged at 20000 g for 15 min. The supernatants were stored frozen until assayed for free amino groups and reducing groups. The dinitrophenylated amino acids were also estimated by thin-layer chromatography on silica-gel plates (Merck, Darmstadt, Germany) according to Ghuysen et al. (I 966). Chemicals. DNase I, RNase A type I-A, DNFB and DNFB-amino acids were all obtained from Sigma. RESULTS
Autolysis of whole cells Whole cells of S.mitis ATCC903 were not studied as their autolysis has already been reported (Linder, 19743).With the other strains, samples were harvested from the exponential and stationary growth phases, washed twice and suspended in Na,HPO,/NaH,PO, buffer to give an extinction of about 12,equivalent to approximately 4 mg dry wt ml-l. The effect of pH and salt concentration was studied. The results (Figs I and 2) show that all strains autolysed in phosphate buffer, the extent of lysis being affected by pH and salt concentration. The pH optimum for autolysis of these six strains, as for the previouslystudied S. mitis ATCC903 (Linder, 1974b),was above pH 7. One strain, S.sanguis 804, also had a high autolytic activity at pH 4-5(Fig. I a). Streptococcus mitis ATCCI5909 autolysed to a high degree over a broad range of pH values above 5-5 (Fig. re). Exp. cells exhibited higher autolytic activity than stat. cells except for two strains, S. mutans IB and S.mutans KPSK2 (Figs ~ d c), , which showed maximal lysis in stat. cells. The pH curves for exp. cells and stat. cells were almost parallel in the other strains, with differences in lysis of 5 to 20 yo. Salt concentration had a more varied affect on lysis than pH. Some strains (Figs 26, e) showed maximal lysis in 0.05 to 0.2M buffers, while S. mitis ATCCI5912 and exp. cells of S.mutans IB showed increasing autolysis with increasing salt concentration (Figs 2f, d ) . As can be seen from Figs I and 2, S.mitis ATCCI5912 and S.mitis ATCCI5909 exhibited the highest autolytic activity, being at maximum more than 80 and 60 % respectively. Whole cells (both exp. and stat.) were also tested for autolysis at 45 "C and J O "C at various pH values and at various salt concentrations, as in the experiments at 37 "C. Optimal autolysis at 45 "C occurred at the same values of pH and salt molarity as in the experiments at 37 "C.Maximum autolysis was o to 5 yoless at 45 "Cthan at 37 "Cfor both exp. and stat. cells. Autolysis also occurred at 10"Cbut at a reduced rate: the maximum autolytic activity was 40 to 50% of that at 37 "C. Autolysis of walls To study autolysis of walk, three strains of s. mitis, ATCCI59 I 2, ATCCI 5909 and ATCC903, were chosen because of their high autolytic activity as demonstrated here and previously (Linder, rg74b). Walls were incubated for 20 h in Na,HPO,/NaH,PO, buffer under the same conditions of pH, salt molarity and temperature as for whole cells. The pH optimum for strain A T C C I ~was ~ IpH ~ 7.0,and for strains ATCCI5909 and ATCC903, pH 7.5 (Fig. 3a). Salt concentration affected lysis of the walls differently in the three strains (Fig. 3b). Walls ~I~ maximum lysis at high salt concentration (1.0M), while those of strain A T C C I ~exhibited of strains A T C C ~ Oand ~ ATCCI5909 showed two maxima, one at 0.01M and the other at 0.5 to 1.0M. In experiments performed at 45 "Cand at 10 "C(Figs 4a,b, c), wall lysis was reduced by
90
M-L.S U N D A N D L. L I N D E R
I
I
I
I
60
.3" 40 h
.-
v) v)
3 20
? 7.0
5.0
9.0
5.0
7.0 PH
9.0
5.0
7.0
9.0
Fig. I . Autolysis of whole cells of six strains of viridans streptococci in 0-2M - S O ~ ~ phosphate U ~ buffer of different pH values. (a) Streptococcus sanguis 804; (b) S. sanguis OPCI; (c)S. mutans K P S K ~ ;(d) S. mu tans^^; (e)S. mitis A T C C I ~ ~(f) O ~S.; mitis ~ ~ ~ ~ 1 5 Closed 9 1 2 .symbols, exp. cells; open symbols, stat. cells.
yo
10 to 20 at 45 "C and by 30 to 40% at 10"C compared with lysis at 37 "C.In strain ATCCg03, lysis at 10"C was reduced by about 80 % and remained at a practically constant
level over the pH range studied.
Mode of action of the autolytic enzyme system of S. mitis A T C C I ~ ~ I ~ Figures I to 3 show clearly that, of all the strains tested, S. mitis A T C C I ~had ~ Ithe ~ highest autolytic activity for both whole cells and walls. Autolysis of this strain was characterized by a decrease in extinction and by release of soluble N-terminal amino groups. From an initial extinction of the wall suspensions of about 21 (corresponding to g mg dry wt ml-l), autolysis started without lag (Fig. 5) and was accompanied by a progressive release of soluble amino groups that was proportional to the decrease in extinction. No release of reducing groups was detected. Dinitrophenylated amino acids were identified by comparison with known standards of DNFB-derivatives of amino acids on thin-layer plates of silica gel (Ghuysen et al., 1966). Walls were autolysed at 37 "Cfor I, 3 and 20 h in 0.2 M-Na,HPO,/NaH,PO, buffer, pH 7-5. Two controls were used : a zero-time autolysis supernatant, and the supernatant of autolysinfree walls incubated in the same buffer as the test samples for 20 h at 37 "C.Dinitro-
9=
Autolysis of streptococci
70
50
30 0.2
1.0
0.6
0.2
0-2
1.0
0.6
0.6
Phosphate concn (M) Fig. 2. Autolysis of whole cells of six strains of viridans streptococci in sodium phosphate buffer pH 7.5 of different molarities. Strains and symbols as in Fig. I . 80
60
-
I
I
(6)
(a)
I
I
I
+
-
-
v
40
-
7.0 9.0 0.2 0.6 1.0 PH Phosphate concn (M) Fig. 3. Autolysis of walls of three S. mitis strains in sodium phosphate buffer at different values, (b) molarities. m, A T C C ~ ;O ~ ,ATCCI5909 ; A, ATCCI 5912, 5.0
1.0
M-L. S U N D A N D L. L I N D E R
92
80
60
40
20 7.0 9.0 5.0 PH Fig. 4. Effect of temperature on autolysis of walls from three S. mitis strains in 0-2M-sodium ; ATCCI5909; (c) ~~~(215912. A, 10"c: phosphate buffer of different pH values. (a) A T c C ~ O ~(6) OY37 "C; a 4 5 "C.
7.0
5.0
9.0
6-
I
I
I
1
-
0
.d
3 .r
I
I
I
60 Incubation time (min)
7 :14
.I I20
Fig. 5. Kinetics of autolysis of S. mitis A T C C I ~ walls, ~ I ~ incubated in 0.2 M-sodium phosphate buffer pH 7-5at 37 "C.Extinction ( 0 )was measured at 550 nm, and free:amino groups(.) were determined with I-fluor0-2~4-dinitrobenzene. Alanine was used as the standard.
Table
I.
Amino acids released into the soluble fraction during autolysis of walls 01 S. mitis ATCCI5912 Walls (initial concentration, g mg dry wt ml-l) were incubated for 5 h at 37 "C.
Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline GIycine
Concn (,urn01ml-l)
Amino acid Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
0.868 trace ND
0.297 0.379 0.658 0.8 I 2
0.305
0.684 ND,
Not detected.
Concn (pmol mi-l)
1.134 trace 0.726 0.135 0.476 0.700 0.185 0.285
Autolysis of streptococci 93 phenylated amino groups of alanine, glutamic acid, lysine, valine, serine, glycine and leucine occurred in all three test supernatants (1,3 and 20 h). All spots which were visible in the 20 h tests were also seen at I and 3 h, although they were fainter. No dinitrophenylated products were obtained from the two controls. The release of free amino acids during autolysis of walls at 37 "C for 5 h was investigated using an amino-acid analyser. Autolysin-free walls were incubated in parallel as a control. Between 14 and 16 amino acids were released into the soluble fraction during autolysis (Table I), alanine, lysine and glutamic acid being released in greatest quantity. .
DISCUSSION
It was suggested in a previous paper that autolysis in S. mitis is a physiologically useful In this strain (ATCC~O~), lysis in sodium mechanism for release of enzymes (Linder, 1974~~). phosphate buffer is regulated by the salt concentration. A striking characteristic of autolysis of this strain is that a constant proportion of the cells is resistant to autolysis regardless of whether they are harvested in the exponential growth phase or in the stationary (autolytic) phase. The potentiating effect of high salt concentration on lysis in S.mitis ATCCI5912 and S.mutans IB is similar to that in S.mitis ATCC903 but has not been reported with other bacteria. However, in S.faecalis ~ ~ ~ ~ 9autolysis 7 9 0 ,of exp. cells is most rapid at phosphate buffer concentrations of 0.01M and 0.3 M (Shockman et al., 1961).The present investigation has shown that all strains of viridans streptococci tested autolyse in phosphate buffer and that cells harvested in the stationary phase possess almost the same autolytic activity as exponential cells. This contrasts with the autolytic behaviour of S.faecalis ATCC9790 (Shockman et al., 1961)and strains of Lactobacillus (Coyette & Ghuysen, 1970;Neujahr & Logardt, 1973). Many of the amino acids released during autolysis of the walls of S.mitis ATCCI5912 were probably not part of the peptidoglycan portion of the wall and may represent a polypeptide or protein. The observation that autolysin-free walls did not release free amino groups or amino acids indicates that the peptide or protein may have been a wall protein rather than an intracellular or membrane contamination of the wall preparation. It appears that the autolytic system of S.mitis A T C C I ~contains ~ I ~ endopeptidases which hydrolyse bonds both within the tetrapeptide of the peptidoglycan and within the wall protein. Bacteriolytic enzymes splitting bonds within the tetrapeptide are extremely rare; only two reports on such enzymes have appeared (Neujahr & Logardt, 1973;Welker, 1971).These enzymes are of considerable physiological importance and of experimental and taxonomic interest. We have attempted to isolate the autolytic enzymes of S.mitis ATCCI5912 but without success. Soluble enzyme activity could not be recovered from the supernatant fluid after wall lysis, perhaps because the autolytic enzymes are firmly bound to the walls, as in S. faecalis (Shockman, Pooley & Thompson, 1967),and are released only at unusually high concentrations of salt (Pooley, Porres-Juan & Shockman, 1970). It is also possible that the enzymes are rapidly inactivated after release from the wall. Thus Coyette & Shockman (1973)found that only 20 % of the original wall lytic activity could be recovered in the supernatant after lysis of walls of Lactobacillus acidophilus for 16h at 37 "C. This investigation was supported by grants from the Faculty of Odontology, Karolinska Institute, from Patentmedelsfonden for odontologisk profylaxforskning and from The Swedish Dental Society. We wish to thank Dr G. Samuelsson, Uppsala, for performing the amino-acid analyses.
94
M-L. S U N D A N D L. LINDER REFERENCES
CARLSSON, J. (1965). Zooglea-forming streptococci resembling Streptococcus sanguis isolated from dental plaque in man. Odontologisk Revy 16, 348-358. CARLSSON, J. (1968). A numerical taxonomic study of human oral streptococci. Odontologisk Revy 19, 137-160. COYETTE, J. & GHUYSEN, J-M. (1970). Wall autolysin of Lactobacillus acidophilus strain 63 AM Gasser. Biochemistry, New York 9, 2952-2955. COYETTE, J. & SHOCKMAN, G. D. (1973). Some properties of the autolytic N-acetylmuramidase of Lactobacillus acidophilus. Journal of Bacteriology 114, 34-41. DAVIS,R. H. & SALTON, M. R. J. (1975). Some properties of a ~-alaninecarboxypeptidase in envelope fractions of Neisseria gonorrhoeae. Infection and Immunity 12, I 065-1 078. EDEBO, L. (1960). A new press for the disruption of micro-organismsand other cells. Journal of Biochemical and Microbiological Technology and Engineering 2,453-479. FAN,D. P. (1970~).Cell wall binding properties of the Bacillus subtilis autolysin(s).Journal of Bacteriology 103,488-493. FAN,D. P. (197ob). Autolysin(s) of Bacillus subtilis as dechaining enzyme. Journal of Bacteriology 103, 494-499. GHUYSEN, J-M. & SHOCKMAN, G. D. (1973). Biosynthesis of peptidoglycan. In Bacterial Membranes and Walls, pp. 37-130. Edited by L. Leive. New York: Marcel Dekker. GHUYSEN, J-M., TIPPER, D. J. & STROMINGER, J. L. (1966). Enzymes that degrade bacterial cell walls. In Methods in Enzymology, vol. 8, pp. 685-699. Edited by S. P. Colowick and N. 0. Kaplan. New York : Academic Press. JOSEPH, R. & SHOCKMAN, G. D. (1974). Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis 9790 : release of cell wall, autolysin and formation of stable autoplasts. Journal of Bacteriology 118, 735-746. KRASSE, B. (1966). Human streptococci and experimental caries in hamsters. Archives of Oral Biology 119429-436. LINDER, L. ( 1 9 7 4 ~ ~Formation ). and release of hyaluronidase and aminopeptidase in growing cultures of Streptococcus mitis A T C C ~ O Acta ~ . pathologica et microbiologica scandinavica B 82, 608-6 14. LINDER, L. (I974 b). Extraction of cell-bound hyaluronidase and aminopeptidase from Streptococcus mitis A T C C ~ Acta O ~ . pathologica et microbiologica scandinavica B 82, 593-601. LINDER, L. (I 974 c). Formation and release of hyaluronidase and aminopeptidase in non-growing cells of Streptococcus mitis ATCC903. Acta pathologica et microbiologica scandinavica B 82, 61 5-624. LINDER,L., HOLME, T. & FROSTELL, G. (1974). Hyaluronidase and aminopeptidase activity in cultures of Streptococcus mitis ATCC903. Acta pathologica et microbiologica scandinavica B 82, 52 1-526. MOHAN,R. R., KRONISH, D. P., PIANOTTI, R. S., EPSTEIN, R. L. & SCHWARTZ, B. S. (1965). Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. Journal of Bacteriology 90, 1355-1 364. NEUJAHR, H. Y. & LOGARDT, I. M. (1973). Autolytic enzyme system from Lactobacillus fermentii. Biochemistry, New York 12, 2578-2583. PARK,J. T. & JOHNSON,M. J. (1949). A method for determination of reducing power. Journal of Biological Chemistry 181, 149-151. POOLEY, H. M. & SHOCKMAN, G. D. (1969). Relationship between the latent form and the active form of the autolytic enzyme of Streptococcus faecalis. Journal of Bacteriology 100,617-624. POOLEY, H. M., PORRES-JUAN, J. M. & SHOCKMAN, G. D. (1970). Dissociation of an autolytic enzyme-cell wall complex by treatment with unusually high concentrations of salt. Biochemical and Biophysical Research Communications 38, I 134-1 140. POOLEY, H. M., SHOCKMAN, G. D., HIGGINS,M. L. & PORRES-JUAN, J. M. (1972). Some properties of two autolytic-defectivemutants of Streptococcus faecalis ~ ~ C C 9 7 9Journal 0. of Bacteriology 109, 423-43 I . ROGERS, H. J. (1970). Bacterial growth and the cell envelope. Bacteriological Reviews 34, 194-214. SALTON, M. R. J. & FREER,J. H. (1965). Composition of the membranes isolated from several Grampositive bacteria. Biochimica et biophysica acta 107, 530-538. SHOCKMAN, G. D., CONOVER, M. J., KOLB,J. J., PHILLIPS, P. M., RILEY, L. S. & TOENNIES, G. (1961). Lysis of Streptococcus faecalis. Journal of Bacteriology 81, 36-43. SHOCKMAN, G. D., POOLEY, H. M. & THOMSON, J. S. (1967) Autolytic enzyme system of Streptococcus faecalis. 111. Localisation of the autolysin at the sites of cell wall synthesis. Journal of Bacteriology 94, 1525-1530. WELKER, N. E. (1971). Structure of the cell wall of Bacillus stearothermophilus:mode of action of a thermophilic bacteriophage lytic enzyme. Journal of Bacteriology 107, 697-703.
