JOURNAL OF BACTIIUOLOGY, June 1976, p. 1186-1193 Copyright X) 1976 American Society for Microbiology
Vol. 126, No. 3 Printed in USA.
Mechanism of Autolysis of Neisseria gonorrhoeae BRUCE H. HEBELER' AND FRANK E. YOUNG* Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 21 January 1976
The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (E.C. 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3Hldiaminopimelic acid). Autolytic enzymes have been found in a wide variety of gram-positive and gram-negative organisms. In many cases the mechanism of action is known. Three classes of lytic enzymes have been described (1). Glycosidases, such as endo-N-acetylglucosaminidases or endo-N-acetyl-muramidases, hydrolyze the glycan backbone of peptidoglycan. N-acetylmuramyl-L-alanine amidases hydrolyze the amide bond between N-acetyl-muramic acid and L-alanine. In addition, there are many endopeptidases that cleave the peptide moiety of the peptidoglycan. Uncontrolled, any of these enzymes can lead to eventual autolysis of the bacterial cell and the release of somatic antigens. The physiological significance of these enzymes is not well understood. Recently, it has been postulated that autolysins could facilitate cellular growth and division by: (i) providing new acceptor sites for the addition of peptidoglycan precursors; (ii) restructuring the cell wall; and (iii) aiding in cell division and separation (7). In a previous study (5) it was shown that a high rate of autolysis was observed in all strains of Neisseria gonorrhoeae examined. The data presented in this paper indicate that the major autolysin is N-acetyl-muramyl-Lalanine amidase. MATERIALS AND METHODS Organisms. Neisseria gonorrhoeae strain RD5 (colony type 4), obtained from cultures provided by F. J. Tyeryar (Naval Medical Reserve Institute, Bethesda, Md.), was used throughout this study. The maintenance of gonococcal strains, media, and growth conditions were described previously (8). Preparation of peptidoglycan. Peptidoglycan was I Present address: Department of Microbiology, University of Oregon Medical School, 3181 S. W. Sam Jackson Park Road, Portland, Ore. 97201.
isolated from 10 liters of strain RD5 cells grown in Mueller-Hinton broth supplemented per liter with: yeast extract, 10 g; glucose, 22 mM; and CaCl2, 5 mg (standard growth medium [SGMl). The harvested cells were suspended in cold 5% trichloroacetic acid for 10 min at 4 C, pelleted, and washed three times with glass-distilled water by centrifugation (8,000 x g, 10 min at 4 C). The washed trichloroacetic acid-insoluble material was suspended in boiling 4% sodium dodecyl sulfate for 3 h followed by stirring overnight at room temperature. The suspension was centrifuged in a Beckman L3-50 using a 50 Ti rotor at 20 C for 35 min at 65,000 x g. The pellet was then washed three times with glass-distilled water under similar conditions and lyophilized. Radioactive peptidoglycan was prepared from cells grown in SGM which contained 5 ACi (300 nCi/nmol) of [3H]diaminopimelic acid (DAP) (Amersham/Searle Corp., Des Plaines, Ill.). The cells were harvested at a Klett of 90 to 100, and the peptidoglycan was isolated as previously described (6). The specific activity of DAP was 0.86 nCi/nmol. Isolation of autolysin. Autolysin was liberated from envelopes by a modification of the procedure that Hartman et al. developed for Escherichia coli (4). Essentially mid-logarithmic phase cells were harvested and washed in 10 mM Tris-maleate buffer (pH 6.8) or 10 mM potassium phosphate buffer (pH 6.8). After resuspension in the same buffer the cells were ruptured in a Braun disintegrator at 4 C (three 1-min disintegrations/sample) and centrifuged (20 min at 4 C at 48,000 x g), and the pellet was washed in 10 mM Tris-maleate (pH 7.0) containing 10 mM sodium ethylenediaminetetraacetic acid. Unless indicated otherwise, the pellet was extracted at room temperature for 30 min in 10 mM Tris-maleate, pH 8.5, containing 2% Triton X-100. The extraction procedure was carried out three times. After centrifugation (30 min, 4 C, 48,000 x g) the supernatant was dialyzed at 4 C against 10 mM potassium phosphate buffer (pH 6.0) and then lyophilized. Autolysis of peptidoglycan. The assay system used to screen for enzymatic activity was adopted
1186
VOL. 126, 1976
AUTOLYSIS OF NEISSERIA GONORRHOEAE
from the procedure used by Hartman et al. (4) for E. coli. Unless otherwise indicated, 20 Al of peptidoglycan substrate labeled with [3H]DAP (containing approximately 5,000 dpm) was suspended in 150 ,ul of 10 mM Tris-maleate (pH 8.5) containing 2% Triton X-100. The enzyme solution was then added (30 ,ul), and the mixture was incubated at 37 C. After the incubation period the mixture was placed in ice, 30 ,ul of bovine serum albumin and 20 Al of cold 5% trichloroacetic acid were added, and the mixture was centrifuged at 8,000 x g for 2 min at room temperature. The supernatant was then assayed for radioactivity. Measurement of radioactivity. Radioactive samples were suspended in 5 ml of Triton X-100 scintillation fluid (15.2 g of Omnifluor, 1 liter of Triton X100, and 2.78 liters of toluene). The radioactivity was determined in a Beckman LS-230 liquid scintillation counter. Rates of autolysis of peptidoglycan were determined as a function of the first-order rate constant K = (ln Co/Ci) x min-', where Co is the initial counts per minute and Ci is the counts per minute after an incubation period. Analytical methods. Determination of 2,4-dinitrophenyl (DNP) amino acids was accomplished by thin-layer chromatography on heat-activated (60 C, 14 h) silica gel 60 thin-layer plates (EM Laboratories, Inc., New York, N.Y.) using the procedure of Ghuysen et al. (2). D- and L-alanine were measured with L-glutamate-pyruvate and D-amino acid oxidase enzymes (Sigma Chemical Co., St. Louis, Mo.) coupled to lactic acid dehydrogenase and reduced nicotinamide adenine dinucleotide according to the methods described by Ghuysen et al. (2), using reagents purchased from Sigma Chemical Co. Chromatography of L- and i.-alanine on Dowex 50 W-X (Bio-Rad Laboratories, Richmond, Calif.) was performed as described by Gomori (3). Reducing groups and protein were determined by the methods of Park and Johnson (10) and Lowry et al. (9), respectively.
1187
TABLE 1. Liberation of autolysin" DAP released (nmol)b
Total DAP
+ 0.5 M
0.7 2.5
24.2 86.5
+ 1.0 M
1.6
57.1
+ 0.5 M
2.5
86.2
+ 1.0 M
1.7
58.2
Extraction method
2% Triton X-100 2% Triton X-100 NaCl 2% Triton X-100 NaCl 2% Triton X-100 LiCl 2% Triton X-100 LiCI
a Autolysin was liberated from the cell envelope as described in Materials and Methods. Extractions were carried out in 10 mM Tris-maleate buffer (pH 8.5) for 30 min at room temperature. b Release of fragments of peptidoglycan containing DAP was determined by the standard assay method after an incubation period of 150 min at 37 C.
nCi/nmol. Table 1 shows the release of radioactive fragments from the peptidoglycan substrate after a 120-min incubation at 37 C. An optimal release of autolysin was achieved with 2% Triton X-100 containing either 0.5 M NaCl or 0.5 M LiCl. A large batch of crude enzyme was then prepared by extraction of cell envelopes utilizing 2% Triton X-100 and 0.5 M NaCl in 10 mM Tris-maleate buffer, pH 8.5. This crude enzyme was used to establish the conditions required for optimal enzymatic activity. Effect of NaCI on assay system. Whereas NaCl enhanced liberation of autolysin, there were indications that it reduced the activity of the enzyme. Therefore, 2-ml samples of enzyme extracts were dialyzed against 10 mM Tris-maRESULTS leate (pH 8.5) containing no NaCl, 0.1 M and Liberation of autolytic activity. In E. coli it 0.5 M NaCl. After dialysis the extracts were was found that the maximum solubilization of assayed for autolytic activity. The results (Tapeptidoglycan by hydrolases occurred with ble 2) show that whereas NaCl enhanced liberabuffer containing 2% Triton X-100 and 0.5 M tion of the autolysin from the cell envelopes, it NaCl (4). The conditions were initially applied inhibited enzymatic hydrolysis of the peptidoto the gonococcal system in an attempt to ex- glycan substrate. Therefore, all subsequent entract the autolysin. To determine the most ef- zyme preparations were dialyzed to remove the fective extraction procedures, a standard enve- NaCl. lope suspension was prepared from a 1-liter Release of [3H]DAP from peptidoglycan. To culture of RD5, as described in Materials and determine the optimum enzyme concentration Methods. Samples of the envelope suspension to be used in subsequent assays, varying con(0.2 ml) were mixed with 0.8 ml of 10 mM Tris- centrations of enzyme were incubated with the maleate (pH 8.5) containing 2% Triton X-100 substrate for 20 min at 37 C. Ten microliters of with or without NaCl or LiCl at various concen- enzyme solution contained 23 ,ug of protein. trations, as shown in Table 1. The samples There was a linear release of [3H]DAP from the were kept at room temperature for 30 min and peptidoglycan substrate (specific activity, 0.86 then centrifuged. The release of the autolysin nCi/nmol) with increasing enzyme concentrainto the supernatant was monitored using the tion up to 100 ,ul of enzyme (230 ,ug of protein) (Fig. 1). From these data it was decided that 50 assay system described in Materials and Methods. The specific activity of the [3H]DAP la- ,ul (115 ug of protein) of enzyme solution would beled peptidoglycan used as substrate was 0.86 be used.
1188
HEBELER AND YOUNG
J. BACTERIOL.
TABLE 2. Effect of NaCI on enzymatic activitya DAP released Total DAP possible (%) Dialyzed against (mol)5 No NaCl 2.3 78 0.1 M NaCl 1.4 49 0.5 M NaCl 0.7 23 a Autolysin was liberated from the cell envelope as described in Materials and Methods. Extractions were carred out in 10 mM Tris-maleate buffer (pH 8.5) containing 2% Triton X-100 for 30 min at room temperature. b Release of fragments of peptidoglycan containing diaminopimelic acid (DAP) was determined after an incubation period of 150 min at 37 C by the standard assay method described in Materials and Methods.
600 500 (n z O 400 0
1300 -J
0
U) 200
lized. Boiling (10 min) completely inactivated the enzyme. From these data it was determined that 0.116 nmol of DAP were released per mg of protein per min. Physiological characteristics of isolated autolysin. (i) Effect of pH on enzymatic activity. To determine if the pH optimum of the crude enzyme was similar to that of the whole cell autolysis (5), assays were run in buffers at various pH values. The rate of release of [3H]DAP from peptidoglycan as a function of pH in 10 mM sodium phosphate and 10 mM Tris-maleate buffer containing 2% Triton X-100 is shown in Fig. 3. The data present a completely different pattern than that observed in whole cell autolysis. The crude enzyme preparation was active over a broad pH range of pH 5.5 to 9.0 in Tris-maleate buffer. In sodium phosphate buffer the pH optimum ranged between 5.5 to 8.5, with a suggestion of two peaks of enzyme activity, one at 6.0 and the other at 8.0. (ii) Effect of Triton X-100 on enzymatic activity. To determine if Triton X-100 is necessary for optimal enzymatic activity the standard assay system was run in 10 mM sodium phosphate buffer (pH 6.0) containing various concentrations of Triton X-100. The rate of au-
c) 10
Ivv
0
20
40
I
I
60
80
100
120
140
160
2 35
0
ENZYME CONC. (ul)
FIG. 1. Effect of enzyme concentration on solubilization of peptidoglycan. Extraction of autolysin(s) from cell envelopes was carried out in 0.05 M Trismaleate buffer (pH 8.5) for 30 min at room temperature. The extracted enzyme(s) was dialyzed against 100 volumes of 0.05 M Tris-maleate buffer (pH 8.5) containing no NaCl or Triton X-100. Release of PH]DAP from peptidoglycan was determined using the standard assay system as described in Materials and Methods. TCA, Trichloroacetic acid.
The specific activity of this crude enzyme preparation was determined by incubating 50 gl (115 ug of protein) of enzyme solution with the radioactive substrate for various periods of time. A unit of enzyme activity is defined as the solubilization of 1 nmol of [3H]DAP per mg of protein per min at 37 C. Because previous studies have demonstrated that 95% of the wall 3H label is in DAP (6), the solubilization of [3H]DAP is a reliable estimate of the hydrolysis of peptidoglycan. The release of [3H]DAP is linear for 150 min (Fig. 2). At 150 min, 73% of the total possible DAP residues were solubi-
0
a)
X 30
E
E
Li
LU
_
25
I-
0~
± 20
0 0
0
_
_W
____a
Q-
L 0
LU
..)
H
LLI
-J 10 LUi 5 o0
25
50
75
100
125
150
180
TIME (min.)
FIG. 2. Solubilization of peptidoglycan as a function of time. Extraction of autolysin(s) from cell envelopes was carried out in 0.05 M Tris-maleate (pH 8.5) for 30 min at room temperature. The extracted enzyme(s) was dialyzed against 0.05 M Tris-maleate buffer (pH 8.5). Release of [3H]DAP from peptidoglycan was carried out using the standard assay system as described in Materials and Methods. Symbols: ( *) Peptidoglycan plus enzyme; ( 0) peptidoglycan plus inactivated enzyme (100 C, 10 min).
AUTOLYSIS OF NEISSERIA GONORRHOEAE
VOL. 126, 1976
* SODIUM PHOSPHATE, 10mM o TRIS-MALEATE, lOmM
34
30F
1189
the standard assay system. The assays were carried out using 10 mM potassium phosphate buffer (pH 6.0) prewarmed to various temperatures. The rate of hydrolysis of the peptidogly26
26 1
I
1
I
I
§I
I
0
x 22
24
Y-
-
18
I0 14~
x Y-
i0
I. 5.5
6.0
6.5
7.0
7.5
8.0
8 8.5
20 _
_
9.0
pH
FIG. 3. Effect of pH on the solubilization of peptidoglycan. Solubilization of peptidoglycan by extracted autolysin(s) was determined by the standard assay system utilizing 10 mM sodium phosphate (a) and 10 mM Tris-maleate (0) buffers containing 2% Triton X-100 at various pH values. The assay was carried out at 37 C, and the rate of autolysis was determined as described in Materials and Methods.
tolysis is affected by the amount of detergent present (Fig. 4). Maximum enzyme activity was observed at a concentration of 2% Triton X100. Note that even with these ranges of concentration of Triton X-100 there is only a 20% increase in the rate constant. (iii) Effect of ions on enzymatic activity. In whole cell autolysis (5) it was shown that there was no absolute requirement for potassium, sodium, or ammonium ions. However, it was demonstrated that potassium ions did stimulate the rate of autolysis at a concentration of 0.1 M. This stimulatory effect was previously observed with N-acetylmuramyl-L-alanine amidase in Bacillus subtilis (11). To determine if ions have the same effect on the crude enzyme, assays were run in 10 mM Tris-maleate buffer (pH 8.5) preincubated at 37 C containing varying concentrations of potassium, sodium, and magnesium ions. The results (Fig. 5) show that the rate of autolysis is markedly influenced by ions. As in whole cell autolysis, potassium ions again showed a stimulatory effect on the rate of autolysis at a concentration of 0.1 M. Higher concentrations of sodium and magnesium ions (0.1 to 0.5 M) demonstrated an inhibitory effect on autolysis. (iv) Effect of temperature on enzymatic activity. The effect of temperature on enzymatic activity was measured by determining the rate of release of [1H]DAP from peptidoglycan using
18
,.. 0
0.5
I
I
I
1.0
1.5
2.0
2.5
3.0
X-100 FIG. 4. Effect of Triton X-100 on the solubilization ofpeptidoglycan. Solubilization ofpeptidoglycan by autolysin(s) was determined by the standard assay system utilizing 10 mM sodium phosphate buffer (pH 6.0) containing varying concentrations of Triton X-100. The assay was carried out at 37 C, and the rate ofautolysis was determined as described in Materials and Methods. % TRITON
* KCI * NaCI o MgCI
32 28 24re) 0
20
x
y 16 2
8
4
103
162
60
CONC. (Molar) FIG. 5. Effect of ions on the solubilization ofpeptidoglycan. Solubilization of peptidoglycan by extracted autolysin(s) was determined by the standard assay system utilizing 10 mM Tris-maleate (pH 8.5) containing 2% Triton X-100 and varying concentrations ofKCI (-), NaCl (A), and MgCl (U). The assay was carried out at37 C, and the rate of solubilization was then determined as described in Materials and
Methods.
1190
HEBELER AND YOUNG
J. BACTERIOL.
can substrate was dependent on temperature, TABLE 3. Extraction of autolytic activity from cell envelopes" with the maximum rate occurring at 40 C (Fig. 6). At 60 C no enzymatic activity was observed. Total Recovery These results are similar to those obtained for Fraction protein of protein Sp act (mg) (%) autolysis of whole cells. Attempted purification of autolysins. In an Braun suspension 4080 100.0 0.03 attempt to further purify the crude enzyme Braun supernatant 2190 53.7 NDb 104 2.5 ND preparation, 10-liter cultures of N. gonorrhoeae Pellet wash 264 6.5 0.21 were grown in SGM and harvested at late- 1st NaCl extraction 83 2.0 0.27 logarithmic phase (Klett = 100). The autoly- 2nd NaCl extraction 69 1.7 0.08 sin(s) was liberated following the procedure de- 3rd NaCl extraction a Enzymatic activity was measured by the standscribed in Material and Methods. At all steps in the isolation procedure, samples were taken ard assay procedure as described in Materials and and assayed for amount of protein and for enzy- Methods. All NaCl extractions were performed in 10 matic activity. Table 3 shows the total protein, mM potassium phosphate buffer (pH 6.0) containing the recovery of protein, the presence of enzy- 2% Triton X-100 for 30 min at room temperature. matic activity, and the specific activity for each Specific activity is defined as the nanomoles of diaacid in fragments of peptidoglycan solstep in the initial isolation of the autolysin. The minopimelic by 1 mg of protein per min. first NaCl extraction was performed on the ubilized b ND, Not detected. same day as growth. The second and third extraction occurred 24 h later. The salt extractions were dialyzed against 10 mM potassium from the release of ['3HIDAP from the peptidophosphate buffer for 24 h in the cold. The spe- glycan substrate by each fraction as a function cific activity shown in Table 3 was determined of time. Since the first and second NaCl extractions had the same specific activity, the samples were combined and then subjected to further purification which included the following 30 procedures: (i) ammonium sulfate fractionation; (ii) column chromatography on diethyl20 _ aminoethyl-cellulose, Cellex-CM, Cellex-P, Cellex-BD, and sephadex; and (iii) differential extraction. The results obtained with the first two methods were variable. In some experi10 ments complete loss of enzymatic activity ocPo 8 curred. An attempt was then made to obtain a x 6 crude enzyme preparation of a higher specific y activity by differential extraction of the enve4 lope suspension. Another 10-liter batch of cells was grown in SGM and harvested at a Klett of 75. An envelope suspension was then prepared as described in Materials and Methods. This 2 envelope suspension was first extracted in 10 mM potassium phosphate buffer, pH 6.0, containing 0.5 M NaCl for 30 min at room temperature. This step was then followed by extraction 34 31 32 30 33 35 36 37 in the same buffer containing 1% Triton X-100 IT under the same conditions. On the following FIG. 6. Effect of temperature on the solubilization day (14 h later) the envelope suspension was of peptidoglycan. Solubilization of peptidoglycan by again extracted twice more in the same buffer extracted autolysin(s) was determined by the stand- containing 2% Triton X-100. All extracts were ard assay system utilizing 10 mM potassium phos- dialyzed overnight in cold 50 mM potassium phate buffer (pH 6.0) containing 2% Triton X-100. phosphate buffer, pH 6.0, before being assayed The buffer samples containing enzyme were preincu- for autolytic activity. The autolysin obtained by bated at the various temperatures for 15 min before this method did not have a significantly higher the addition of the radioactive labeled substrate. The rate of solubilization was then determined, using the specific activity. Because of the variabilities encountered in the attempt to purify the autolyassay method described in Materials and Methods. The reciprocal of absolute temperature is plotted on sin, it was decided to focus attention on the the abscissa and the rate constant is plotted on the mechanism of action of the autolysin. ordinate. Determination of mechanism of action. The I
4
VOL. 126, 1976
1191
AUTOLYSIS OF NEISSERIA GONORRHOEAE
fourth extract from the previous experiment was used in a further attempt to determine the mechanism of action of the autolysin(s). The experiment involved the following parameters: (i) solubilization of [3HIDAP from peptidoglycan; (ii) release of reducing groups; (iii) release of N-terminal amino acids; and (iv) identification of the N-terminal amino acids. The assays consisted of: (i) 2 ml of crude enzyme solution (6.2 mg of protein); (ii) 2 ml of inactivated enzyme solution (100 C, 10 min) containing 4 mg of peptidoglycan; and (iii) 2 ml of active enzyme solution containing 4 mg of peptidoglycan. One hundred microliters of [3H]DAP-labeled peptidoglycan (200,000 counts/min) was added to the last two samples. After the addition of 10 ,umol of sodium azide, all tubes were incubated at 37 C. Samples, taken at various time periods, were boiled (100 C, 5 min), centrifuged (10,000 x g, 5 min), and filtered (Millipore Corp., Boston, Mass., HA 0.45 anm). The supernatants were collected and subsequently assayed for the solubilization of [3H]DAP and release of N-terminal amino acids. Reducing group determinations were performed on unfiltered samples using the control of enzyme alone as a blank. [3H]DAP (59.7%) was solubilized after a 22-h incubation period (Fig. 7A). During this same incubation period, a 3.2% increase in reducing group equivalents was observed (Fig. 7B). This corresponded to a reduction in average chain length from 105 to 32 disaccharide units. The assay for free N-terminal amino acids demonstrated an increase of 48.1% after a 22-h incubation period. Subsequent analysis of the DNPderivatives by thin-layer chromatography on Silica Gel G developed sequentially in: (i) Nbutanol-1% ammonia (vol/vol), 1:1, upper phase; and (ii) chloroform-methanol-acetic acid, (vol/vol/vol), 85:14:1, showed the presence of N-terminal alanine which increased as a function of time. A 28.8% increase due to Nterminal alanine was observed at the end of the 22-h incubation period (Fig. 7C). Also observed on the thin-layer plates were variable concentrations of DNP-NH3 and free DNP. These DNP by-products accounted for the differences observed in the amount of free N-terminal groups and the amount of N-terminal alanine. The presence of N-terminal D- or L-alanine was measured by the procedure of Ghuysen et al. (2). Samples were taken from the supernatants obtained after the 22-h incubation period and split into two equal aliquots. Total D-alanine and L-alanine was determined in one aliquot after hydrolysis (4 N HCI, 105 C, 8 h) and subsequent lyophilization to remove the HCI. The other aliquot was subjected to treatment with DNP prior to hydrolysis. After removal of DNP
70 60
1
1
1
l
1
1
l
1
-
A. 3H-DAP-
40 30 -/20 _ 10
w C.)
w 'I z
0 4 3 2
.
-
.
K
.
.
I
I
II
I
I
I
B. REDUCING GROUPS
6
r
I
I
12
I
I
I
16
TIME (hours) FIG. 7. Release of products during hydrolysis of peptidoglycan. Purified peptidoglycan was mixed with extracted autolysin(s) and incubated at 37 C in 10 mM potassium phosphate (pH 6.0). The increase of fragments of peptidoglycan containing [3H]DAP (A) and N-terminal-alanine (C) in the supernatant fraction was determined. Reducing groups (B) in the particulate fraction were estimated as described in Materials and Methods. Closed symbols represent peptidoglycan plus enzyme. Open symbols represent peptidoglycan plus inactivated enzyme (100 C, 10 min).
amino acids by ether extraction, the sample was passed through a Dowex 50 colunm to remove any by-products formed during the DNP reaction. The alanine was eluted from the column with 1.5 N HCl and lyophilized. The amount of r-alanine and L-alanine was then determined. The ratio of total L-alanine to 1alanine was 0.97, whereas the ratio of L-alanine to i>alanine after removal of N-terminal DNPamino acids was 0.36 (Table 4). These experiments establish that N-terminal L-alanine is released during autolysis and that the major autolysin of N. gonorrhoeae RD5 type 4 is Nacetylmuramyl-L-alanine amidase (E.C. 3.5.1.28).
DISCUSSION The cell envelope of gram-negative bacteria is composed of two morphologically distinct layers: the inner cytoplasmic membrane and an outer membrane. In N. gonorrhoeae the autolysin appears to be tightly bound to this envelope complex. However, treatment of the complex for a short duration with non-ionic detergent, Triton X-100, and NaCl effectively extracted
1192
HEBELER AND YOUNG
J. BACTERIOL.
TABLE 4. Analysis of autblysate for N-terminal alanine' Treatment before hydrolysis
. L-alanine (nmol)
o-alanine (nmol)
Ratio of
L-ala-
nine/>alanine
None DNFB
141 ± 2 146 ± 4 0.97 47 ± 5 132 ± 2 0.33 "Autolysates were boiled, centrifuged, filtered, and separated into two aliquots. One aliquot was left untreated before hydrolysis in 4 N HCI at 105 C for 8 h. The other aliquot was treated with dinitrofluorobenzene (DNFB) before hydrolysis. The DNPamino acids were removed by ether extraction and the DNP by-products were removed by column chromatography on Dowex 50. Determination of Dand L-alanine were as described in Materials and Methods, and performed in duplicate.
the autolysin. An optimal liberation of autolysin was achieved with 2% Triton X-100 containing 0.5 M NaCl. Triton X-100 or NaCl alone did not effectively liberate the autolysin from the envelope. Similar findings were also reported for E. coli by Hartman et al. (4). Since effective solubilization is dependent on a combination of Triton X-100 and sodium chloride, it was suggested that the autolysin may be fixed in the envelope by both hydrophobic and ionic bonds. While NaCl proved to be effective in the extraction of the autolysin, it was also shown that it reduced the activity of the enzyme. Therefore, all extracts required dialysis to remove the NaCl before attempting further analyses. After removal of NaCl from the extracts it was shown that the release of fragments of peptidoglycan labeled with I3HIDAP was dependent on enzyme concentration as well as time of incubation. In a previous study (5) the physiological conditions for optimal autolysis of whole cells were established. To determine whether the enzyme(s) extracted from the cell envelope is in fact responsible for whole cell autolysis, a comparison of the conditions necessary for autolysis of whole cells and hydrolysis of purified peptidoglycan was made. It was found that the pH optimum of the autolysin in whole cells is broad, with a maximum at pH 9.0. In the cellfree system the pH optimum was again broad, but with no sharp maximal rate. There was, however, a suggestion of two optima in potassium phosphate buffer, one at 6.0 and the other at 8.0. The pH optimum of lysozyme and most ,B-N-acetylhexosamidases is below pH 7.0. The pH optimum of B. subtilis 168 N-acetylmuramyl-L-alanine amidase, one of the more extensively studied autolysins, is pH 9.5 (11). The rates of autolysis in both systems were similar
and were dependent on temperature, with the maximum rate occurring at 40 C. The decline in the rate of autolysis above 40 C is attributed to the inactivation of the autolysin. Treatment at 60 C for 15 min completely inactivates the
autolysin. Potassium ions influenced the rate of autolysis of both whole cells and cell-free systems at a concentration of 0.1 M. This effect was also reported for the amidase of B. subtilis 168 (11). High concentrations of sodium ions (0.1 M to 0.5 M) decreased the rate of autolysis as did magnesium ions. Magnesium ions were also shown to decrease enzymatic activity in E. coli (4). Due to similar conditions necessary for maximal rate of autolysis of whole cell and cell-free systems, it is postulated that the extracted enzyme(s) are responsible for whole cell lysis. Difficulties were encountered in attempts to purify the autolysin(s). One of the major problems was the presence of Triton X-100 which is necessary for the extraction of the autolysin. Triton X-100 is not effectively removed by dialysis, ultrafiltration, or Cellex-BD column chromatography. Concentration of enzyme extracts yielded a viscous material which was not suitable for column chromatography. Column chromatography of unconcentrated extracts led to variable results. If the column was eluted with buffer containing 2% Triton X-100 the collected samples could not be concentrated for further analysis. If the eluting buffer did not contain Triton X-100, the Triton X-100 containing samples formed micelles and passed through the column in the void volume. Often column chromatography resulted in an irreversible loss of enzymatic activity. Mixing of the fractions also failed to restore activity. These difficulties must be overcome before further attempts are made to purify the autolysin(s). The mechanism of action of bacteriolytic enzymes can be established by analysis of the lytic products of peptidoglycan. Hexosamidases cause an increase in reducing groups of either N-acetylglucosamine or N-acetylmuramic acid depending on whether the enzyme is an Nacetylglucosamidase or an N-acetylmuramidase. These reducing groups can be detected by borohydride reduction or by the Park-Johnson assay (10). N-acetylmuramyl-L-alanine amidase liberates N-terminal L-alanine and an equal molar amount of N-acetylmuramyl carboxyl groups. Unfortunately, there is no easy assay to test for the appearance of N-acetylmuramyl carboxyl groups. However, an increase in N-terminal L-alanine without an increase in C-tenninal amino acid during autolysis or a decrease of L-alanine to D-alanine ratios after incubation of the autolysate with dinitrofluoro-
VOL. 126, 1976
AUTOLYSIS OF NEISSERIA GONORRHOEAE
benzene is indicative of an N-acetylmuramylL-alanine amidase. By the use of these techniques it was determined that the major autolysin of N. gonorrhoeae RD5 type 4 is an N-acetylmuramyl-L-alanine amidase. During the autolysis of envelopes there is a slight increase in reducing groups. Although this could be due to the presence of a hexaminidase, we currently favor the interpretation that the increment in reducing groups is related to "unmasking" of the terminus of glycan chains during autolysis. Therefore, we conclude that the N-acetylmuramyl-L-alanine amidase appears to be responsible for the rapid turnover observed in the peptidoglycan of N. gonorrhoeae (6). ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant 1 R01 AI-11709 from the National Institute of Allergy and Infectious Diseases. B.H.H. was a Public Health Service predoctoral trainee funded by grant 5 T01GM-00592 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Ghuysen, J.-M. 1968. Use of bacteriolytic enzymes in determination of wall structure and their role in cell
1193
metabolism. Bacteriol. Rev. 32:425-464. 2. Ghuysen, J.-M., D. J. Tipper, and J. L. Strominger. 1966. Enzymes that degrade bacterial cell walls, p. 685-699. In E. F. Neufeld and V. Ginsburg (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 3. Gomori, G. 1955. Preparation of buffers for use in enzyme studies, p. 138-146. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. I. Academic Press Inc., New York. 4. Hartman, R., S. B. Bock-Hennig, and U. Schwarz. 1974. Murein hydrolases in the envelope of Escherichia coli. Properties in situ and solubilization from the envelope. Eur. J. Biochem. 41:203-208. 5. Hebeler, B. H., and F. E. Young. 1975. Autolysis of Neisseria gonorrhoeae. J. Bacteriol. 122:385-392. 6. Hebeler, B. H., and F. E. Young. 1976. Chemical composition and turnover rate of peptidoglycan of Neisseria gonorrhoeae. J. Bacteriol 126:1180-1185. 7. Higgins, M. L., and G. D. Shockman. 1971. Procaryotic cell division with respect to walls and membranes. Crit. Rev. Microbiol. 1:29-72. 8. La Scolea, L. J., Jr., and F. E. Young. 1974. Development of a defined minimal medium for the growth of Neisseria gonorrhoeae. Appl. Microbiol. 28:70-76. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 10. Park, J. T., and M. J. Johnson. 1949. A submicrodetermination of glucose. J. Biol. Chem. 181:149-151. 11. Young, F. E. 1966. Autolytic enzyme associated with cell walls of Bacillus subtilis. J. Biol. Chem. 241:3462-3467.
Vol. 126, No. 3 Printed in USA.
Mechanism of Autolysis of Neisseria gonorrhoeae BRUCE H. HEBELER' AND FRANK E. YOUNG* Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 21 January 1976
The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (E.C. 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3Hldiaminopimelic acid). Autolytic enzymes have been found in a wide variety of gram-positive and gram-negative organisms. In many cases the mechanism of action is known. Three classes of lytic enzymes have been described (1). Glycosidases, such as endo-N-acetylglucosaminidases or endo-N-acetyl-muramidases, hydrolyze the glycan backbone of peptidoglycan. N-acetylmuramyl-L-alanine amidases hydrolyze the amide bond between N-acetyl-muramic acid and L-alanine. In addition, there are many endopeptidases that cleave the peptide moiety of the peptidoglycan. Uncontrolled, any of these enzymes can lead to eventual autolysis of the bacterial cell and the release of somatic antigens. The physiological significance of these enzymes is not well understood. Recently, it has been postulated that autolysins could facilitate cellular growth and division by: (i) providing new acceptor sites for the addition of peptidoglycan precursors; (ii) restructuring the cell wall; and (iii) aiding in cell division and separation (7). In a previous study (5) it was shown that a high rate of autolysis was observed in all strains of Neisseria gonorrhoeae examined. The data presented in this paper indicate that the major autolysin is N-acetyl-muramyl-Lalanine amidase. MATERIALS AND METHODS Organisms. Neisseria gonorrhoeae strain RD5 (colony type 4), obtained from cultures provided by F. J. Tyeryar (Naval Medical Reserve Institute, Bethesda, Md.), was used throughout this study. The maintenance of gonococcal strains, media, and growth conditions were described previously (8). Preparation of peptidoglycan. Peptidoglycan was I Present address: Department of Microbiology, University of Oregon Medical School, 3181 S. W. Sam Jackson Park Road, Portland, Ore. 97201.
isolated from 10 liters of strain RD5 cells grown in Mueller-Hinton broth supplemented per liter with: yeast extract, 10 g; glucose, 22 mM; and CaCl2, 5 mg (standard growth medium [SGMl). The harvested cells were suspended in cold 5% trichloroacetic acid for 10 min at 4 C, pelleted, and washed three times with glass-distilled water by centrifugation (8,000 x g, 10 min at 4 C). The washed trichloroacetic acid-insoluble material was suspended in boiling 4% sodium dodecyl sulfate for 3 h followed by stirring overnight at room temperature. The suspension was centrifuged in a Beckman L3-50 using a 50 Ti rotor at 20 C for 35 min at 65,000 x g. The pellet was then washed three times with glass-distilled water under similar conditions and lyophilized. Radioactive peptidoglycan was prepared from cells grown in SGM which contained 5 ACi (300 nCi/nmol) of [3H]diaminopimelic acid (DAP) (Amersham/Searle Corp., Des Plaines, Ill.). The cells were harvested at a Klett of 90 to 100, and the peptidoglycan was isolated as previously described (6). The specific activity of DAP was 0.86 nCi/nmol. Isolation of autolysin. Autolysin was liberated from envelopes by a modification of the procedure that Hartman et al. developed for Escherichia coli (4). Essentially mid-logarithmic phase cells were harvested and washed in 10 mM Tris-maleate buffer (pH 6.8) or 10 mM potassium phosphate buffer (pH 6.8). After resuspension in the same buffer the cells were ruptured in a Braun disintegrator at 4 C (three 1-min disintegrations/sample) and centrifuged (20 min at 4 C at 48,000 x g), and the pellet was washed in 10 mM Tris-maleate (pH 7.0) containing 10 mM sodium ethylenediaminetetraacetic acid. Unless indicated otherwise, the pellet was extracted at room temperature for 30 min in 10 mM Tris-maleate, pH 8.5, containing 2% Triton X-100. The extraction procedure was carried out three times. After centrifugation (30 min, 4 C, 48,000 x g) the supernatant was dialyzed at 4 C against 10 mM potassium phosphate buffer (pH 6.0) and then lyophilized. Autolysis of peptidoglycan. The assay system used to screen for enzymatic activity was adopted
1186
VOL. 126, 1976
AUTOLYSIS OF NEISSERIA GONORRHOEAE
from the procedure used by Hartman et al. (4) for E. coli. Unless otherwise indicated, 20 Al of peptidoglycan substrate labeled with [3H]DAP (containing approximately 5,000 dpm) was suspended in 150 ,ul of 10 mM Tris-maleate (pH 8.5) containing 2% Triton X-100. The enzyme solution was then added (30 ,ul), and the mixture was incubated at 37 C. After the incubation period the mixture was placed in ice, 30 ,ul of bovine serum albumin and 20 Al of cold 5% trichloroacetic acid were added, and the mixture was centrifuged at 8,000 x g for 2 min at room temperature. The supernatant was then assayed for radioactivity. Measurement of radioactivity. Radioactive samples were suspended in 5 ml of Triton X-100 scintillation fluid (15.2 g of Omnifluor, 1 liter of Triton X100, and 2.78 liters of toluene). The radioactivity was determined in a Beckman LS-230 liquid scintillation counter. Rates of autolysis of peptidoglycan were determined as a function of the first-order rate constant K = (ln Co/Ci) x min-', where Co is the initial counts per minute and Ci is the counts per minute after an incubation period. Analytical methods. Determination of 2,4-dinitrophenyl (DNP) amino acids was accomplished by thin-layer chromatography on heat-activated (60 C, 14 h) silica gel 60 thin-layer plates (EM Laboratories, Inc., New York, N.Y.) using the procedure of Ghuysen et al. (2). D- and L-alanine were measured with L-glutamate-pyruvate and D-amino acid oxidase enzymes (Sigma Chemical Co., St. Louis, Mo.) coupled to lactic acid dehydrogenase and reduced nicotinamide adenine dinucleotide according to the methods described by Ghuysen et al. (2), using reagents purchased from Sigma Chemical Co. Chromatography of L- and i.-alanine on Dowex 50 W-X (Bio-Rad Laboratories, Richmond, Calif.) was performed as described by Gomori (3). Reducing groups and protein were determined by the methods of Park and Johnson (10) and Lowry et al. (9), respectively.
1187
TABLE 1. Liberation of autolysin" DAP released (nmol)b
Total DAP
+ 0.5 M
0.7 2.5
24.2 86.5
+ 1.0 M
1.6
57.1
+ 0.5 M
2.5
86.2
+ 1.0 M
1.7
58.2
Extraction method
2% Triton X-100 2% Triton X-100 NaCl 2% Triton X-100 NaCl 2% Triton X-100 LiCl 2% Triton X-100 LiCI
a Autolysin was liberated from the cell envelope as described in Materials and Methods. Extractions were carried out in 10 mM Tris-maleate buffer (pH 8.5) for 30 min at room temperature. b Release of fragments of peptidoglycan containing DAP was determined by the standard assay method after an incubation period of 150 min at 37 C.
nCi/nmol. Table 1 shows the release of radioactive fragments from the peptidoglycan substrate after a 120-min incubation at 37 C. An optimal release of autolysin was achieved with 2% Triton X-100 containing either 0.5 M NaCl or 0.5 M LiCl. A large batch of crude enzyme was then prepared by extraction of cell envelopes utilizing 2% Triton X-100 and 0.5 M NaCl in 10 mM Tris-maleate buffer, pH 8.5. This crude enzyme was used to establish the conditions required for optimal enzymatic activity. Effect of NaCI on assay system. Whereas NaCl enhanced liberation of autolysin, there were indications that it reduced the activity of the enzyme. Therefore, 2-ml samples of enzyme extracts were dialyzed against 10 mM Tris-maRESULTS leate (pH 8.5) containing no NaCl, 0.1 M and Liberation of autolytic activity. In E. coli it 0.5 M NaCl. After dialysis the extracts were was found that the maximum solubilization of assayed for autolytic activity. The results (Tapeptidoglycan by hydrolases occurred with ble 2) show that whereas NaCl enhanced liberabuffer containing 2% Triton X-100 and 0.5 M tion of the autolysin from the cell envelopes, it NaCl (4). The conditions were initially applied inhibited enzymatic hydrolysis of the peptidoto the gonococcal system in an attempt to ex- glycan substrate. Therefore, all subsequent entract the autolysin. To determine the most ef- zyme preparations were dialyzed to remove the fective extraction procedures, a standard enve- NaCl. lope suspension was prepared from a 1-liter Release of [3H]DAP from peptidoglycan. To culture of RD5, as described in Materials and determine the optimum enzyme concentration Methods. Samples of the envelope suspension to be used in subsequent assays, varying con(0.2 ml) were mixed with 0.8 ml of 10 mM Tris- centrations of enzyme were incubated with the maleate (pH 8.5) containing 2% Triton X-100 substrate for 20 min at 37 C. Ten microliters of with or without NaCl or LiCl at various concen- enzyme solution contained 23 ,ug of protein. trations, as shown in Table 1. The samples There was a linear release of [3H]DAP from the were kept at room temperature for 30 min and peptidoglycan substrate (specific activity, 0.86 then centrifuged. The release of the autolysin nCi/nmol) with increasing enzyme concentrainto the supernatant was monitored using the tion up to 100 ,ul of enzyme (230 ,ug of protein) (Fig. 1). From these data it was decided that 50 assay system described in Materials and Methods. The specific activity of the [3H]DAP la- ,ul (115 ug of protein) of enzyme solution would beled peptidoglycan used as substrate was 0.86 be used.
1188
HEBELER AND YOUNG
J. BACTERIOL.
TABLE 2. Effect of NaCI on enzymatic activitya DAP released Total DAP possible (%) Dialyzed against (mol)5 No NaCl 2.3 78 0.1 M NaCl 1.4 49 0.5 M NaCl 0.7 23 a Autolysin was liberated from the cell envelope as described in Materials and Methods. Extractions were carred out in 10 mM Tris-maleate buffer (pH 8.5) containing 2% Triton X-100 for 30 min at room temperature. b Release of fragments of peptidoglycan containing diaminopimelic acid (DAP) was determined after an incubation period of 150 min at 37 C by the standard assay method described in Materials and Methods.
600 500 (n z O 400 0
1300 -J
0
U) 200
lized. Boiling (10 min) completely inactivated the enzyme. From these data it was determined that 0.116 nmol of DAP were released per mg of protein per min. Physiological characteristics of isolated autolysin. (i) Effect of pH on enzymatic activity. To determine if the pH optimum of the crude enzyme was similar to that of the whole cell autolysis (5), assays were run in buffers at various pH values. The rate of release of [3H]DAP from peptidoglycan as a function of pH in 10 mM sodium phosphate and 10 mM Tris-maleate buffer containing 2% Triton X-100 is shown in Fig. 3. The data present a completely different pattern than that observed in whole cell autolysis. The crude enzyme preparation was active over a broad pH range of pH 5.5 to 9.0 in Tris-maleate buffer. In sodium phosphate buffer the pH optimum ranged between 5.5 to 8.5, with a suggestion of two peaks of enzyme activity, one at 6.0 and the other at 8.0. (ii) Effect of Triton X-100 on enzymatic activity. To determine if Triton X-100 is necessary for optimal enzymatic activity the standard assay system was run in 10 mM sodium phosphate buffer (pH 6.0) containing various concentrations of Triton X-100. The rate of au-
c) 10
Ivv
0
20
40
I
I
60
80
100
120
140
160
2 35
0
ENZYME CONC. (ul)
FIG. 1. Effect of enzyme concentration on solubilization of peptidoglycan. Extraction of autolysin(s) from cell envelopes was carried out in 0.05 M Trismaleate buffer (pH 8.5) for 30 min at room temperature. The extracted enzyme(s) was dialyzed against 100 volumes of 0.05 M Tris-maleate buffer (pH 8.5) containing no NaCl or Triton X-100. Release of PH]DAP from peptidoglycan was determined using the standard assay system as described in Materials and Methods. TCA, Trichloroacetic acid.
The specific activity of this crude enzyme preparation was determined by incubating 50 gl (115 ug of protein) of enzyme solution with the radioactive substrate for various periods of time. A unit of enzyme activity is defined as the solubilization of 1 nmol of [3H]DAP per mg of protein per min at 37 C. Because previous studies have demonstrated that 95% of the wall 3H label is in DAP (6), the solubilization of [3H]DAP is a reliable estimate of the hydrolysis of peptidoglycan. The release of [3H]DAP is linear for 150 min (Fig. 2). At 150 min, 73% of the total possible DAP residues were solubi-
0
a)
X 30
E
E
Li
LU
_
25
I-
0~
± 20
0 0
0
_
_W
____a
Q-
L 0
LU
..)
H
LLI
-J 10 LUi 5 o0
25
50
75
100
125
150
180
TIME (min.)
FIG. 2. Solubilization of peptidoglycan as a function of time. Extraction of autolysin(s) from cell envelopes was carried out in 0.05 M Tris-maleate (pH 8.5) for 30 min at room temperature. The extracted enzyme(s) was dialyzed against 0.05 M Tris-maleate buffer (pH 8.5). Release of [3H]DAP from peptidoglycan was carried out using the standard assay system as described in Materials and Methods. Symbols: ( *) Peptidoglycan plus enzyme; ( 0) peptidoglycan plus inactivated enzyme (100 C, 10 min).
AUTOLYSIS OF NEISSERIA GONORRHOEAE
VOL. 126, 1976
* SODIUM PHOSPHATE, 10mM o TRIS-MALEATE, lOmM
34
30F
1189
the standard assay system. The assays were carried out using 10 mM potassium phosphate buffer (pH 6.0) prewarmed to various temperatures. The rate of hydrolysis of the peptidogly26
26 1
I
1
I
I
§I
I
0
x 22
24
Y-
-
18
I0 14~
x Y-
i0
I. 5.5
6.0
6.5
7.0
7.5
8.0
8 8.5
20 _
_
9.0
pH
FIG. 3. Effect of pH on the solubilization of peptidoglycan. Solubilization of peptidoglycan by extracted autolysin(s) was determined by the standard assay system utilizing 10 mM sodium phosphate (a) and 10 mM Tris-maleate (0) buffers containing 2% Triton X-100 at various pH values. The assay was carried out at 37 C, and the rate of autolysis was determined as described in Materials and Methods.
tolysis is affected by the amount of detergent present (Fig. 4). Maximum enzyme activity was observed at a concentration of 2% Triton X100. Note that even with these ranges of concentration of Triton X-100 there is only a 20% increase in the rate constant. (iii) Effect of ions on enzymatic activity. In whole cell autolysis (5) it was shown that there was no absolute requirement for potassium, sodium, or ammonium ions. However, it was demonstrated that potassium ions did stimulate the rate of autolysis at a concentration of 0.1 M. This stimulatory effect was previously observed with N-acetylmuramyl-L-alanine amidase in Bacillus subtilis (11). To determine if ions have the same effect on the crude enzyme, assays were run in 10 mM Tris-maleate buffer (pH 8.5) preincubated at 37 C containing varying concentrations of potassium, sodium, and magnesium ions. The results (Fig. 5) show that the rate of autolysis is markedly influenced by ions. As in whole cell autolysis, potassium ions again showed a stimulatory effect on the rate of autolysis at a concentration of 0.1 M. Higher concentrations of sodium and magnesium ions (0.1 to 0.5 M) demonstrated an inhibitory effect on autolysis. (iv) Effect of temperature on enzymatic activity. The effect of temperature on enzymatic activity was measured by determining the rate of release of [1H]DAP from peptidoglycan using
18
,.. 0
0.5
I
I
I
1.0
1.5
2.0
2.5
3.0
X-100 FIG. 4. Effect of Triton X-100 on the solubilization ofpeptidoglycan. Solubilization ofpeptidoglycan by autolysin(s) was determined by the standard assay system utilizing 10 mM sodium phosphate buffer (pH 6.0) containing varying concentrations of Triton X-100. The assay was carried out at 37 C, and the rate ofautolysis was determined as described in Materials and Methods. % TRITON
* KCI * NaCI o MgCI
32 28 24re) 0
20
x
y 16 2
8
4
103
162
60
CONC. (Molar) FIG. 5. Effect of ions on the solubilization ofpeptidoglycan. Solubilization of peptidoglycan by extracted autolysin(s) was determined by the standard assay system utilizing 10 mM Tris-maleate (pH 8.5) containing 2% Triton X-100 and varying concentrations ofKCI (-), NaCl (A), and MgCl (U). The assay was carried out at37 C, and the rate of solubilization was then determined as described in Materials and
Methods.
1190
HEBELER AND YOUNG
J. BACTERIOL.
can substrate was dependent on temperature, TABLE 3. Extraction of autolytic activity from cell envelopes" with the maximum rate occurring at 40 C (Fig. 6). At 60 C no enzymatic activity was observed. Total Recovery These results are similar to those obtained for Fraction protein of protein Sp act (mg) (%) autolysis of whole cells. Attempted purification of autolysins. In an Braun suspension 4080 100.0 0.03 attempt to further purify the crude enzyme Braun supernatant 2190 53.7 NDb 104 2.5 ND preparation, 10-liter cultures of N. gonorrhoeae Pellet wash 264 6.5 0.21 were grown in SGM and harvested at late- 1st NaCl extraction 83 2.0 0.27 logarithmic phase (Klett = 100). The autoly- 2nd NaCl extraction 69 1.7 0.08 sin(s) was liberated following the procedure de- 3rd NaCl extraction a Enzymatic activity was measured by the standscribed in Material and Methods. At all steps in the isolation procedure, samples were taken ard assay procedure as described in Materials and and assayed for amount of protein and for enzy- Methods. All NaCl extractions were performed in 10 matic activity. Table 3 shows the total protein, mM potassium phosphate buffer (pH 6.0) containing the recovery of protein, the presence of enzy- 2% Triton X-100 for 30 min at room temperature. matic activity, and the specific activity for each Specific activity is defined as the nanomoles of diaacid in fragments of peptidoglycan solstep in the initial isolation of the autolysin. The minopimelic by 1 mg of protein per min. first NaCl extraction was performed on the ubilized b ND, Not detected. same day as growth. The second and third extraction occurred 24 h later. The salt extractions were dialyzed against 10 mM potassium from the release of ['3HIDAP from the peptidophosphate buffer for 24 h in the cold. The spe- glycan substrate by each fraction as a function cific activity shown in Table 3 was determined of time. Since the first and second NaCl extractions had the same specific activity, the samples were combined and then subjected to further purification which included the following 30 procedures: (i) ammonium sulfate fractionation; (ii) column chromatography on diethyl20 _ aminoethyl-cellulose, Cellex-CM, Cellex-P, Cellex-BD, and sephadex; and (iii) differential extraction. The results obtained with the first two methods were variable. In some experi10 ments complete loss of enzymatic activity ocPo 8 curred. An attempt was then made to obtain a x 6 crude enzyme preparation of a higher specific y activity by differential extraction of the enve4 lope suspension. Another 10-liter batch of cells was grown in SGM and harvested at a Klett of 75. An envelope suspension was then prepared as described in Materials and Methods. This 2 envelope suspension was first extracted in 10 mM potassium phosphate buffer, pH 6.0, containing 0.5 M NaCl for 30 min at room temperature. This step was then followed by extraction 34 31 32 30 33 35 36 37 in the same buffer containing 1% Triton X-100 IT under the same conditions. On the following FIG. 6. Effect of temperature on the solubilization day (14 h later) the envelope suspension was of peptidoglycan. Solubilization of peptidoglycan by again extracted twice more in the same buffer extracted autolysin(s) was determined by the stand- containing 2% Triton X-100. All extracts were ard assay system utilizing 10 mM potassium phos- dialyzed overnight in cold 50 mM potassium phate buffer (pH 6.0) containing 2% Triton X-100. phosphate buffer, pH 6.0, before being assayed The buffer samples containing enzyme were preincu- for autolytic activity. The autolysin obtained by bated at the various temperatures for 15 min before this method did not have a significantly higher the addition of the radioactive labeled substrate. The rate of solubilization was then determined, using the specific activity. Because of the variabilities encountered in the attempt to purify the autolyassay method described in Materials and Methods. The reciprocal of absolute temperature is plotted on sin, it was decided to focus attention on the the abscissa and the rate constant is plotted on the mechanism of action of the autolysin. ordinate. Determination of mechanism of action. The I
4
VOL. 126, 1976
1191
AUTOLYSIS OF NEISSERIA GONORRHOEAE
fourth extract from the previous experiment was used in a further attempt to determine the mechanism of action of the autolysin(s). The experiment involved the following parameters: (i) solubilization of [3HIDAP from peptidoglycan; (ii) release of reducing groups; (iii) release of N-terminal amino acids; and (iv) identification of the N-terminal amino acids. The assays consisted of: (i) 2 ml of crude enzyme solution (6.2 mg of protein); (ii) 2 ml of inactivated enzyme solution (100 C, 10 min) containing 4 mg of peptidoglycan; and (iii) 2 ml of active enzyme solution containing 4 mg of peptidoglycan. One hundred microliters of [3H]DAP-labeled peptidoglycan (200,000 counts/min) was added to the last two samples. After the addition of 10 ,umol of sodium azide, all tubes were incubated at 37 C. Samples, taken at various time periods, were boiled (100 C, 5 min), centrifuged (10,000 x g, 5 min), and filtered (Millipore Corp., Boston, Mass., HA 0.45 anm). The supernatants were collected and subsequently assayed for the solubilization of [3H]DAP and release of N-terminal amino acids. Reducing group determinations were performed on unfiltered samples using the control of enzyme alone as a blank. [3H]DAP (59.7%) was solubilized after a 22-h incubation period (Fig. 7A). During this same incubation period, a 3.2% increase in reducing group equivalents was observed (Fig. 7B). This corresponded to a reduction in average chain length from 105 to 32 disaccharide units. The assay for free N-terminal amino acids demonstrated an increase of 48.1% after a 22-h incubation period. Subsequent analysis of the DNPderivatives by thin-layer chromatography on Silica Gel G developed sequentially in: (i) Nbutanol-1% ammonia (vol/vol), 1:1, upper phase; and (ii) chloroform-methanol-acetic acid, (vol/vol/vol), 85:14:1, showed the presence of N-terminal alanine which increased as a function of time. A 28.8% increase due to Nterminal alanine was observed at the end of the 22-h incubation period (Fig. 7C). Also observed on the thin-layer plates were variable concentrations of DNP-NH3 and free DNP. These DNP by-products accounted for the differences observed in the amount of free N-terminal groups and the amount of N-terminal alanine. The presence of N-terminal D- or L-alanine was measured by the procedure of Ghuysen et al. (2). Samples were taken from the supernatants obtained after the 22-h incubation period and split into two equal aliquots. Total D-alanine and L-alanine was determined in one aliquot after hydrolysis (4 N HCI, 105 C, 8 h) and subsequent lyophilization to remove the HCI. The other aliquot was subjected to treatment with DNP prior to hydrolysis. After removal of DNP
70 60
1
1
1
l
1
1
l
1
-
A. 3H-DAP-
40 30 -/20 _ 10
w C.)
w 'I z
0 4 3 2
.
-
.
K
.
.
I
I
II
I
I
I
B. REDUCING GROUPS
6
r
I
I
12
I
I
I
16
TIME (hours) FIG. 7. Release of products during hydrolysis of peptidoglycan. Purified peptidoglycan was mixed with extracted autolysin(s) and incubated at 37 C in 10 mM potassium phosphate (pH 6.0). The increase of fragments of peptidoglycan containing [3H]DAP (A) and N-terminal-alanine (C) in the supernatant fraction was determined. Reducing groups (B) in the particulate fraction were estimated as described in Materials and Methods. Closed symbols represent peptidoglycan plus enzyme. Open symbols represent peptidoglycan plus inactivated enzyme (100 C, 10 min).
amino acids by ether extraction, the sample was passed through a Dowex 50 colunm to remove any by-products formed during the DNP reaction. The alanine was eluted from the column with 1.5 N HCl and lyophilized. The amount of r-alanine and L-alanine was then determined. The ratio of total L-alanine to 1alanine was 0.97, whereas the ratio of L-alanine to i>alanine after removal of N-terminal DNPamino acids was 0.36 (Table 4). These experiments establish that N-terminal L-alanine is released during autolysis and that the major autolysin of N. gonorrhoeae RD5 type 4 is Nacetylmuramyl-L-alanine amidase (E.C. 3.5.1.28).
DISCUSSION The cell envelope of gram-negative bacteria is composed of two morphologically distinct layers: the inner cytoplasmic membrane and an outer membrane. In N. gonorrhoeae the autolysin appears to be tightly bound to this envelope complex. However, treatment of the complex for a short duration with non-ionic detergent, Triton X-100, and NaCl effectively extracted
1192
HEBELER AND YOUNG
J. BACTERIOL.
TABLE 4. Analysis of autblysate for N-terminal alanine' Treatment before hydrolysis
. L-alanine (nmol)
o-alanine (nmol)
Ratio of
L-ala-
nine/>alanine
None DNFB
141 ± 2 146 ± 4 0.97 47 ± 5 132 ± 2 0.33 "Autolysates were boiled, centrifuged, filtered, and separated into two aliquots. One aliquot was left untreated before hydrolysis in 4 N HCI at 105 C for 8 h. The other aliquot was treated with dinitrofluorobenzene (DNFB) before hydrolysis. The DNPamino acids were removed by ether extraction and the DNP by-products were removed by column chromatography on Dowex 50. Determination of Dand L-alanine were as described in Materials and Methods, and performed in duplicate.
the autolysin. An optimal liberation of autolysin was achieved with 2% Triton X-100 containing 0.5 M NaCl. Triton X-100 or NaCl alone did not effectively liberate the autolysin from the envelope. Similar findings were also reported for E. coli by Hartman et al. (4). Since effective solubilization is dependent on a combination of Triton X-100 and sodium chloride, it was suggested that the autolysin may be fixed in the envelope by both hydrophobic and ionic bonds. While NaCl proved to be effective in the extraction of the autolysin, it was also shown that it reduced the activity of the enzyme. Therefore, all extracts required dialysis to remove the NaCl before attempting further analyses. After removal of NaCl from the extracts it was shown that the release of fragments of peptidoglycan labeled with I3HIDAP was dependent on enzyme concentration as well as time of incubation. In a previous study (5) the physiological conditions for optimal autolysis of whole cells were established. To determine whether the enzyme(s) extracted from the cell envelope is in fact responsible for whole cell autolysis, a comparison of the conditions necessary for autolysis of whole cells and hydrolysis of purified peptidoglycan was made. It was found that the pH optimum of the autolysin in whole cells is broad, with a maximum at pH 9.0. In the cellfree system the pH optimum was again broad, but with no sharp maximal rate. There was, however, a suggestion of two optima in potassium phosphate buffer, one at 6.0 and the other at 8.0. The pH optimum of lysozyme and most ,B-N-acetylhexosamidases is below pH 7.0. The pH optimum of B. subtilis 168 N-acetylmuramyl-L-alanine amidase, one of the more extensively studied autolysins, is pH 9.5 (11). The rates of autolysis in both systems were similar
and were dependent on temperature, with the maximum rate occurring at 40 C. The decline in the rate of autolysis above 40 C is attributed to the inactivation of the autolysin. Treatment at 60 C for 15 min completely inactivates the
autolysin. Potassium ions influenced the rate of autolysis of both whole cells and cell-free systems at a concentration of 0.1 M. This effect was also reported for the amidase of B. subtilis 168 (11). High concentrations of sodium ions (0.1 M to 0.5 M) decreased the rate of autolysis as did magnesium ions. Magnesium ions were also shown to decrease enzymatic activity in E. coli (4). Due to similar conditions necessary for maximal rate of autolysis of whole cell and cell-free systems, it is postulated that the extracted enzyme(s) are responsible for whole cell lysis. Difficulties were encountered in attempts to purify the autolysin(s). One of the major problems was the presence of Triton X-100 which is necessary for the extraction of the autolysin. Triton X-100 is not effectively removed by dialysis, ultrafiltration, or Cellex-BD column chromatography. Concentration of enzyme extracts yielded a viscous material which was not suitable for column chromatography. Column chromatography of unconcentrated extracts led to variable results. If the column was eluted with buffer containing 2% Triton X-100 the collected samples could not be concentrated for further analysis. If the eluting buffer did not contain Triton X-100, the Triton X-100 containing samples formed micelles and passed through the column in the void volume. Often column chromatography resulted in an irreversible loss of enzymatic activity. Mixing of the fractions also failed to restore activity. These difficulties must be overcome before further attempts are made to purify the autolysin(s). The mechanism of action of bacteriolytic enzymes can be established by analysis of the lytic products of peptidoglycan. Hexosamidases cause an increase in reducing groups of either N-acetylglucosamine or N-acetylmuramic acid depending on whether the enzyme is an Nacetylglucosamidase or an N-acetylmuramidase. These reducing groups can be detected by borohydride reduction or by the Park-Johnson assay (10). N-acetylmuramyl-L-alanine amidase liberates N-terminal L-alanine and an equal molar amount of N-acetylmuramyl carboxyl groups. Unfortunately, there is no easy assay to test for the appearance of N-acetylmuramyl carboxyl groups. However, an increase in N-terminal L-alanine without an increase in C-tenninal amino acid during autolysis or a decrease of L-alanine to D-alanine ratios after incubation of the autolysate with dinitrofluoro-
VOL. 126, 1976
AUTOLYSIS OF NEISSERIA GONORRHOEAE
benzene is indicative of an N-acetylmuramylL-alanine amidase. By the use of these techniques it was determined that the major autolysin of N. gonorrhoeae RD5 type 4 is an N-acetylmuramyl-L-alanine amidase. During the autolysis of envelopes there is a slight increase in reducing groups. Although this could be due to the presence of a hexaminidase, we currently favor the interpretation that the increment in reducing groups is related to "unmasking" of the terminus of glycan chains during autolysis. Therefore, we conclude that the N-acetylmuramyl-L-alanine amidase appears to be responsible for the rapid turnover observed in the peptidoglycan of N. gonorrhoeae (6). ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant 1 R01 AI-11709 from the National Institute of Allergy and Infectious Diseases. B.H.H. was a Public Health Service predoctoral trainee funded by grant 5 T01GM-00592 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Ghuysen, J.-M. 1968. Use of bacteriolytic enzymes in determination of wall structure and their role in cell
1193
metabolism. Bacteriol. Rev. 32:425-464. 2. Ghuysen, J.-M., D. J. Tipper, and J. L. Strominger. 1966. Enzymes that degrade bacterial cell walls, p. 685-699. In E. F. Neufeld and V. Ginsburg (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 3. Gomori, G. 1955. Preparation of buffers for use in enzyme studies, p. 138-146. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. I. Academic Press Inc., New York. 4. Hartman, R., S. B. Bock-Hennig, and U. Schwarz. 1974. Murein hydrolases in the envelope of Escherichia coli. Properties in situ and solubilization from the envelope. Eur. J. Biochem. 41:203-208. 5. Hebeler, B. H., and F. E. Young. 1975. Autolysis of Neisseria gonorrhoeae. J. Bacteriol. 122:385-392. 6. Hebeler, B. H., and F. E. Young. 1976. Chemical composition and turnover rate of peptidoglycan of Neisseria gonorrhoeae. J. Bacteriol 126:1180-1185. 7. Higgins, M. L., and G. D. Shockman. 1971. Procaryotic cell division with respect to walls and membranes. Crit. Rev. Microbiol. 1:29-72. 8. La Scolea, L. J., Jr., and F. E. Young. 1974. Development of a defined minimal medium for the growth of Neisseria gonorrhoeae. Appl. Microbiol. 28:70-76. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 10. Park, J. T., and M. J. Johnson. 1949. A submicrodetermination of glucose. J. Biol. Chem. 181:149-151. 11. Young, F. E. 1966. Autolytic enzyme associated with cell walls of Bacillus subtilis. J. Biol. Chem. 241:3462-3467.
