Vol. 18, No. 1
INFECTION AND IMMUNITY, Oct. 1977, p. 210-219 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Cell Envelope of Neisseria gonorrhoeae: Relationship Between Autolysis in Buffer and the Hydrolysis of Peptidoglycan WARNER S. WEGENER,t BRUCE H. HEBELER, AND STEPHEN A. MORSE* Department ofMicrobiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201 Received for publication 2 May 1977
Neisseria gonorrhoeae readily underwent autolysis when suspended in N-2'hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at alkaline pH values. Autolysis was inhibited by the addition of Mg2" or other divalent cations. Autolysis was also suppressed at acid pH (pH 6.0). Suspension of cells in buffer was accompanied by the hydrolysis of peptidoglycan. The rate of peptidoglycan hydrolysis in HEPES buffer was maximal at pH 8.5 and was similar in the presence or absence of Mg2+. Therefore, divalent cation stabilization against autolysis is not mediated by inhibition of peptidoglycan hydrolysis. Peptidoglycan hydrolysis occurred in HEPES buffer (pH 6.0), but at a rate that was 50% of the maximum. Incubation of cells with chloramphenicol or rifampin before suspension in HEPES buffer (pH 8.5) partially prevented autolysis; under these conditions, peptidoglycan hydrolysis still occurred, but at a reduced rate. Old and new peptidoglycans were hydrolyzed at similar rates. Peptidoglycan hydrolysis results in solubilization of both the peptide and glycan moieties. When grown in complex media containing glucose, the peptidoglycan (PG) of virulent and avirulent gonococci constitutes 1 to 2% of the dry weight of the cell and contains muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid (DAP) in a ratio of 1:1:2:1:1 (9). A lipoprotein, covalently attached to the PG, has not been demonstrated in Neisseria gonorrhoeae (9, 22). The PG of this organism turns over at a high rate during exponential growth (approximately 50% per generation) (9). Hebeler and Young (10) reported that the enzyme principally responsible for this rapid turnover was N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), which cleaves the amide linkage between N-acetylmuramic acid and L-alanine. This enzyme, when extracted by salt and detergent from cell walls and assayed in vitro with [3H]DAP-labeled PG as the substrate, exhibited a broad pH optimum and was insensitive to Mg2'. No hexaminidase (endo N-acetylglucosaminidase or endo N-acetylmuramidase), which catalyzes the hydrolysis of the glycan backbone of the PG, has been reported in N. gonorrhoeae. Gonococci resuspended in buffer at an alkaline pH undergo rapid autolysis (8). This autolysis can be prevented by the addition of divalent cations or by osmotic stabilization (3, 4); howt Present address: Department of Microbiology, Indiana University School of Medicine, Indianapolis, IN 46202.
ever, these conditions do not prevent a concomitant loss of viability. Gonococci also lose viability when grown in media containing limiting concentrations of glucose (15). The loss of viability coincides with glucose depletion and is accompanied by cell lysis. The regulatory mechanisms that trigger these processes in the gonococcus are not known. Since the autolysis of a number of gram-positive bacteria can be correlated with PG hydrolysis, we have undertaken a study of the conditions that influence PG hydrolysis in gonococci suspended in buffer. MATERIALS AND METHODS Organism. N. gonorrhoeae strain JW-31 was used in these studies. This strain was originally isolated from a cervical culture of a symptomatic female. A type 4 (T-4) colony type of strain JW-31 was selected after in vitro passage on GC agar (Difco) plates. The maintenance and diagnostic criteria for identification of N. gonorrhoeae were described previously (15). Medium and growth conditions. The basal medium (LGCB) contained the following, per liter of distilled water: proteose peptone no. 3 (Difco), 15 g; K2HPO4, 4 g; KH2PO4, 1 g; NaCl, 5 g; and soluble starch, 1 g. The final pH of the medium was 7.2. A growth factor supplement similar to Iso-VitaleX (BBL), but lacking glucose, and NaHCO3 (42 mg/liter) were added after autoclaving. Where indicated, glucose (0.5%), sodium lactate (0.5%), or sodium pyruvate (0.5%) was aseptically added as the energy source. Frozen stock cultures were prepared according to 210
VOL. 18, 1977
CELL ENVELOPE OF N. GONORRHOEAE
211
the method of La Scolea and Young (11) and used by sequential 5-ml washings of the inside of the tube for inocula as previously described (7). All liquid cul- with 8% hot sodium dodecyl sulfate and water to give tures were incubated at 37°C in a gyratory shaker a final volume of 20 ml of 4% sodium dodecyl sulfate. (New Brunswick Scientific Co., New Brunswick, N.J.). After incubation for 4 h at 100°C, the insoluble PG Turbidity was measured by Klett-Summerson colo- was pelleted by centrifugation at 90,000 x g for 40 min rimetry, filter no. 54 (540 nm). (type 30 rotor) in a Spinco model L ultracentrifuge. Chemicals and radioisotopes. All reagents were The clear gel-like pellets were washed twice with 20 purchased from Sigma Chemical Co., St. Louis, Mo. ml of water and then suspended in 1 ml of water. The following isotopes were obtained from New Eng- Samples (0.2 ml in triplicate) were placed in vials land Nuclear Corp., Boston, Mass.: [6-3H]glucose (spe- containing 15 ml of Aquasol (New England Nuclear cific activity, 33.9 Ci/mmol); [1-'4C]glucose (specific Corp., Boston, Mass.), and radioactivity was deteractivity, 9.6 mCi/mmol); [6-3H]glucosamine (specific mined in a liquid scintillation spectrometer (Beckman activity, 15 Ci/mmol); and [1-_4C]glucosamine (specific Instruments, Inc., Fullerton, Calif.). With these samactivity, 9.1 mCi/mmol). [DL + meso]-2,6-Diamino-[G- ples, the counting efficiency was 50% for 14C and 33% 3H]pimelic acid (specific activity, 699 mCi/mmol) was for 3H, with a 30% overlap of "4C into the 3H channel. purchased from Amersham/Searle Corp., Arlington In all cases, the overlap of 3H into the 14C channel Heights, Ill. was less than 0.01%. Measurement of autolysis. Log-phase cells of N. Incorporation of labeled substrates. To detergonorrhoeae JW-31 (100 to 150 Klett units) were mine the percentage of radioactivity incorporated into centrifuged (4,100 x g for 5 min), and the cell pellet cell material, 0.5- to 1.0-ml samples of cells from culwas resuspended at 100 to 120 Klett units in 50 mM N- tures growing in medium containing a labeled sub2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid strate were pipetted into an equal volume of 20% (HEPES) buffer, adjusted to the desired pH with (wt/vol) cold trichloroacetic acid and incubated at NaOH. In some experiments, the cell pellet was resus- 4°C for 30 to 60 min. The suspensions were filtered pended at the appropriate pH in buffers prepared onto Whatman GF/C filter disks, and the filters were with either N-tris(hydroxymethyl)methyl-2-amino- sequentially washed with 15 ml of cold 10% trichloromethane-sulfonic acid or tris(hydroxymethyl)amino- acetic acid and 15 ml of cold 95% ethanol. The filters methane. MgCl2 was added at a final concentration were placed in scintillation vials and incubated for 1 of 10 mM or as indicated. Other additions were made to 2 h with NCS solubilizer (Amersham/Searle). Raas indicated. Autolysis was measured as the rate of dioactivity was determined after addition of 10 ml of decrease in turbidity at 540 nm. The first-order rate scintillation fluid (16). constant k was calculated as k = logio (CO/C1) x 2.303 Electron microscopy. Cells of N. gonorrhoeae x min-', where Co and C1 are turbidities at to and ti. JW-31 were removed from 18-h GC agar (Difco) plates Preparation of labeled PG. N. gonorrhoeae was and prefixed with 3% glutaraldehyde in 0.125 M phosgrown in LGCB containing pyruvate to mid-log phase phate buffer (pH 7.4) for 2 h. Cells were pelleted by (90 to 100 Klett units), at which time [6-3H]glucose centrifugation and embedded in 2% purified agar (0.5 to 2.0 ,Ci/ml of medium) .was added and the (Difco), washed in phosphate buffer overnight, and culture was incubated for 20 to 30 min. Unlabeled postfixed in 1% osmium tetroxide for 2 h. Sections glucose (0.2%) was added, and the culture was incu- were cut from Epon 812-embedded samples on a Porbated for an additional 10 min to facilitate incorpora- ter-Blum MT2 ultramicrotome and stained with uration of any residual [6-3H]glucose in the intracellular nyl acetate and lead citrate. Samples were examined pool. Cells were then harvested by centrifugation in a Phillips EM 300 electron microscope at 60 kV. (4,100 x g for 5 min at 20°C), and the pellet was RESULTS resuspended in appropriate buffer. Under these conditions, the newly synthesized PG is labeled with 3H. Effect of pH, divalent cations, and growth To obtain old 3H-labeled PG, the cell pellet was resus- conditions on the rate of autolysis. Gonopended in four to five times its original volume in cocci are fragile organisms that exhibit a rapid LGCB medium containing glucose and incubated for decrease in viability when transferred to nonan additional two generations (average generation growth conditions. Suspension of cells in various time, 75 min). Where indicated, PG from log-phase cells grown in LGCB media with pyruvate was labeled buffers results in cell lysis (autolysis). The rate with [6-3H]glucosamine, [1-'4C]glucosamine, [1-'4C] of autolysis of N. gonorrhoeae JW-31 (T4) in 50 mM HEPES buffer is shown in Fig. 1. The glucose, or DL-(meso)-[G-3H]DAP. Measurement of PG hydrolysis. At 15-min inter- rates of autolysis were lowest at an acid pH (pH vals, 10-ml samples of 3H- or "C-labeled cell suspen- 6.0) and in the presence of divalent cations sions were pipetted into centrifuge tubes containing 1 (Mg2e) and was highest at an alkaline pH (pH ml of unlabeled carrier cells of strain JW-31 (10 to 20 8.5) or in the presence of ethylenediaminetetramg [dry weight] of cells) at 4°C. The tubes were acetic acid (EDTA). Although an acid pH and immediately centrifuged (3,400 x g for 3 min), and the the presence of divalent cations suppressed ausupernatants were decanted. PG was isolated from tolysis, they did not prevent a loss of viability the cell pellets by the procedure of Hebeler and Young In was (3). Autolysis temperature dependent. (9) as follows. The pellets were suspended in 5 ml of distilled water and placed in screw-capped glass tubes HEPES buffer (pH 8.5), it was markedly recontaining 5 ml of 4% sodium dodecyl sulfate at 100°C. duced at 4°C and did not occur when cells had Residual cell material was quantitatively recovered been heated at 80°C for 10 min.
212
_j 70 z
60
0
37C,HEPES
pH60.0
v 37C,HEPES pH 8.5- Mg+
4C,HEPESpH8.5
Z
C..)50
40 0
INFECT. IMMUN.
WEGENER, HEBELER, AND MORSE
* 37C,HEPES pH 6.0+EDTA A
37C,HEPESpH 8.5
10
20
30
40
50
60
TIME (min.)
FIG. 1. Inhibition of autolysis of N. gonorrhoeae JW-31 by Mg" and pH. Log-phase cells were harvested and suspended at 37 or 4°C in 50 mM HEPES buffer at pH 8.5 or 6.0 in the presence or absence of 10 mM MgCl2 or 5 mM EDTA. Autolysis was measured as described in the text.
Some buffers appeared to be more effective than others in promoting autolysis (Table 1). However, in all cases, the autolytic rate was highest at an alkaline pH and was reduced in the presence of Mg2". The optimal pH for autolysis appeared to be 8.0 to 8.5. In addition to Mg2+, other divalent cations (Ca2 Mn2 and Ba2+) were effective in preventing autolysis of cell suspensions (Table 2). The addition of monovalent cations (Na+, K+, Li+) had little effect, whereas trivalent cations (Fe3+, Al3+) showed intermediate activity. Autolysis was not prevented by the sulfhydryl inhibitors N-ethylmaleimide or iodoacetate, which are able to penetrate the gonococcal cell wall, as indicated by their inhibition of glucose incorporation and growth (data not shown). However, autolysis was totally inhibited by the addition of 1 mM H2+. Hg2~ It was of interest to determine whether the chemical composition of the envelope or the synthesis or activity of an autolytic enzyme could be altered by variations in growth conditions. Accordingly, the energy source, pH, growth temperature, and the concentration of Mg2e in the medium were varied, and rates of cellular autolysis were determined in HEPES buffer at pH 8.5 and 6.0 with and without Mg2+ and EDTA. There were no significant differences in either the patterns or the rates of autolysis between cells initially grown: (i) at 37°C in LGCB media with glucose, pyruvate, or lactate as the energy source; (ii) at 37°C in LGCBglucose media buffered with HEPES over a pH range of 6.0 to 8.0; (iii) in LGCB-glucose media incubated at 37, 31, or 28°C; and (iv) at 37°C in LGCB-glucose media containing a large excess (5 to 20 mM) of MgCl2. ,
,
Incorporation of radioactivity into PG. The PG of N. gonorrhoeae exhibits a high rate of turnover during exponential growth (9). Therefore, we developed a procedure by which to radioactively label PG of strain JW-31 at a high specific activity during a short incubation period. Unless noted otherwise, PG was labeled during a 30- to 40-min period by the addition of [6-3H]glucose to log-phase cultures in LGCB media containing pyruvate as the energy source. Since glucose is metabolized primarily by the Entner-Doudoroff pathway in N. gonorrhoeae (16), the label in position 6 is readily incorporated into cell material. Figure 2 compares the kinetics of incorporation of [6-3H]glucose and [6-3H]glucosamine into trichloroacetic acid-insoluble cell material during growth in LGCB-pyruvate medium. In the TABLE 1. Effect ofpH and buffer on rate of autolysis of N. gonorrhoeae JW-31 Rate of autolysis (k x 10-3)a
pH
HEPES
TES
Tris-hydro- Tris-machloride
leate
INFECTION AND IMMUNITY, Oct. 1977, p. 210-219 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Cell Envelope of Neisseria gonorrhoeae: Relationship Between Autolysis in Buffer and the Hydrolysis of Peptidoglycan WARNER S. WEGENER,t BRUCE H. HEBELER, AND STEPHEN A. MORSE* Department ofMicrobiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201 Received for publication 2 May 1977
Neisseria gonorrhoeae readily underwent autolysis when suspended in N-2'hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at alkaline pH values. Autolysis was inhibited by the addition of Mg2" or other divalent cations. Autolysis was also suppressed at acid pH (pH 6.0). Suspension of cells in buffer was accompanied by the hydrolysis of peptidoglycan. The rate of peptidoglycan hydrolysis in HEPES buffer was maximal at pH 8.5 and was similar in the presence or absence of Mg2+. Therefore, divalent cation stabilization against autolysis is not mediated by inhibition of peptidoglycan hydrolysis. Peptidoglycan hydrolysis occurred in HEPES buffer (pH 6.0), but at a rate that was 50% of the maximum. Incubation of cells with chloramphenicol or rifampin before suspension in HEPES buffer (pH 8.5) partially prevented autolysis; under these conditions, peptidoglycan hydrolysis still occurred, but at a reduced rate. Old and new peptidoglycans were hydrolyzed at similar rates. Peptidoglycan hydrolysis results in solubilization of both the peptide and glycan moieties. When grown in complex media containing glucose, the peptidoglycan (PG) of virulent and avirulent gonococci constitutes 1 to 2% of the dry weight of the cell and contains muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid (DAP) in a ratio of 1:1:2:1:1 (9). A lipoprotein, covalently attached to the PG, has not been demonstrated in Neisseria gonorrhoeae (9, 22). The PG of this organism turns over at a high rate during exponential growth (approximately 50% per generation) (9). Hebeler and Young (10) reported that the enzyme principally responsible for this rapid turnover was N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), which cleaves the amide linkage between N-acetylmuramic acid and L-alanine. This enzyme, when extracted by salt and detergent from cell walls and assayed in vitro with [3H]DAP-labeled PG as the substrate, exhibited a broad pH optimum and was insensitive to Mg2'. No hexaminidase (endo N-acetylglucosaminidase or endo N-acetylmuramidase), which catalyzes the hydrolysis of the glycan backbone of the PG, has been reported in N. gonorrhoeae. Gonococci resuspended in buffer at an alkaline pH undergo rapid autolysis (8). This autolysis can be prevented by the addition of divalent cations or by osmotic stabilization (3, 4); howt Present address: Department of Microbiology, Indiana University School of Medicine, Indianapolis, IN 46202.
ever, these conditions do not prevent a concomitant loss of viability. Gonococci also lose viability when grown in media containing limiting concentrations of glucose (15). The loss of viability coincides with glucose depletion and is accompanied by cell lysis. The regulatory mechanisms that trigger these processes in the gonococcus are not known. Since the autolysis of a number of gram-positive bacteria can be correlated with PG hydrolysis, we have undertaken a study of the conditions that influence PG hydrolysis in gonococci suspended in buffer. MATERIALS AND METHODS Organism. N. gonorrhoeae strain JW-31 was used in these studies. This strain was originally isolated from a cervical culture of a symptomatic female. A type 4 (T-4) colony type of strain JW-31 was selected after in vitro passage on GC agar (Difco) plates. The maintenance and diagnostic criteria for identification of N. gonorrhoeae were described previously (15). Medium and growth conditions. The basal medium (LGCB) contained the following, per liter of distilled water: proteose peptone no. 3 (Difco), 15 g; K2HPO4, 4 g; KH2PO4, 1 g; NaCl, 5 g; and soluble starch, 1 g. The final pH of the medium was 7.2. A growth factor supplement similar to Iso-VitaleX (BBL), but lacking glucose, and NaHCO3 (42 mg/liter) were added after autoclaving. Where indicated, glucose (0.5%), sodium lactate (0.5%), or sodium pyruvate (0.5%) was aseptically added as the energy source. Frozen stock cultures were prepared according to 210
VOL. 18, 1977
CELL ENVELOPE OF N. GONORRHOEAE
211
the method of La Scolea and Young (11) and used by sequential 5-ml washings of the inside of the tube for inocula as previously described (7). All liquid cul- with 8% hot sodium dodecyl sulfate and water to give tures were incubated at 37°C in a gyratory shaker a final volume of 20 ml of 4% sodium dodecyl sulfate. (New Brunswick Scientific Co., New Brunswick, N.J.). After incubation for 4 h at 100°C, the insoluble PG Turbidity was measured by Klett-Summerson colo- was pelleted by centrifugation at 90,000 x g for 40 min rimetry, filter no. 54 (540 nm). (type 30 rotor) in a Spinco model L ultracentrifuge. Chemicals and radioisotopes. All reagents were The clear gel-like pellets were washed twice with 20 purchased from Sigma Chemical Co., St. Louis, Mo. ml of water and then suspended in 1 ml of water. The following isotopes were obtained from New Eng- Samples (0.2 ml in triplicate) were placed in vials land Nuclear Corp., Boston, Mass.: [6-3H]glucose (spe- containing 15 ml of Aquasol (New England Nuclear cific activity, 33.9 Ci/mmol); [1-'4C]glucose (specific Corp., Boston, Mass.), and radioactivity was deteractivity, 9.6 mCi/mmol); [6-3H]glucosamine (specific mined in a liquid scintillation spectrometer (Beckman activity, 15 Ci/mmol); and [1-_4C]glucosamine (specific Instruments, Inc., Fullerton, Calif.). With these samactivity, 9.1 mCi/mmol). [DL + meso]-2,6-Diamino-[G- ples, the counting efficiency was 50% for 14C and 33% 3H]pimelic acid (specific activity, 699 mCi/mmol) was for 3H, with a 30% overlap of "4C into the 3H channel. purchased from Amersham/Searle Corp., Arlington In all cases, the overlap of 3H into the 14C channel Heights, Ill. was less than 0.01%. Measurement of autolysis. Log-phase cells of N. Incorporation of labeled substrates. To detergonorrhoeae JW-31 (100 to 150 Klett units) were mine the percentage of radioactivity incorporated into centrifuged (4,100 x g for 5 min), and the cell pellet cell material, 0.5- to 1.0-ml samples of cells from culwas resuspended at 100 to 120 Klett units in 50 mM N- tures growing in medium containing a labeled sub2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid strate were pipetted into an equal volume of 20% (HEPES) buffer, adjusted to the desired pH with (wt/vol) cold trichloroacetic acid and incubated at NaOH. In some experiments, the cell pellet was resus- 4°C for 30 to 60 min. The suspensions were filtered pended at the appropriate pH in buffers prepared onto Whatman GF/C filter disks, and the filters were with either N-tris(hydroxymethyl)methyl-2-amino- sequentially washed with 15 ml of cold 10% trichloromethane-sulfonic acid or tris(hydroxymethyl)amino- acetic acid and 15 ml of cold 95% ethanol. The filters methane. MgCl2 was added at a final concentration were placed in scintillation vials and incubated for 1 of 10 mM or as indicated. Other additions were made to 2 h with NCS solubilizer (Amersham/Searle). Raas indicated. Autolysis was measured as the rate of dioactivity was determined after addition of 10 ml of decrease in turbidity at 540 nm. The first-order rate scintillation fluid (16). constant k was calculated as k = logio (CO/C1) x 2.303 Electron microscopy. Cells of N. gonorrhoeae x min-', where Co and C1 are turbidities at to and ti. JW-31 were removed from 18-h GC agar (Difco) plates Preparation of labeled PG. N. gonorrhoeae was and prefixed with 3% glutaraldehyde in 0.125 M phosgrown in LGCB containing pyruvate to mid-log phase phate buffer (pH 7.4) for 2 h. Cells were pelleted by (90 to 100 Klett units), at which time [6-3H]glucose centrifugation and embedded in 2% purified agar (0.5 to 2.0 ,Ci/ml of medium) .was added and the (Difco), washed in phosphate buffer overnight, and culture was incubated for 20 to 30 min. Unlabeled postfixed in 1% osmium tetroxide for 2 h. Sections glucose (0.2%) was added, and the culture was incu- were cut from Epon 812-embedded samples on a Porbated for an additional 10 min to facilitate incorpora- ter-Blum MT2 ultramicrotome and stained with uration of any residual [6-3H]glucose in the intracellular nyl acetate and lead citrate. Samples were examined pool. Cells were then harvested by centrifugation in a Phillips EM 300 electron microscope at 60 kV. (4,100 x g for 5 min at 20°C), and the pellet was RESULTS resuspended in appropriate buffer. Under these conditions, the newly synthesized PG is labeled with 3H. Effect of pH, divalent cations, and growth To obtain old 3H-labeled PG, the cell pellet was resus- conditions on the rate of autolysis. Gonopended in four to five times its original volume in cocci are fragile organisms that exhibit a rapid LGCB medium containing glucose and incubated for decrease in viability when transferred to nonan additional two generations (average generation growth conditions. Suspension of cells in various time, 75 min). Where indicated, PG from log-phase cells grown in LGCB media with pyruvate was labeled buffers results in cell lysis (autolysis). The rate with [6-3H]glucosamine, [1-'4C]glucosamine, [1-'4C] of autolysis of N. gonorrhoeae JW-31 (T4) in 50 mM HEPES buffer is shown in Fig. 1. The glucose, or DL-(meso)-[G-3H]DAP. Measurement of PG hydrolysis. At 15-min inter- rates of autolysis were lowest at an acid pH (pH vals, 10-ml samples of 3H- or "C-labeled cell suspen- 6.0) and in the presence of divalent cations sions were pipetted into centrifuge tubes containing 1 (Mg2e) and was highest at an alkaline pH (pH ml of unlabeled carrier cells of strain JW-31 (10 to 20 8.5) or in the presence of ethylenediaminetetramg [dry weight] of cells) at 4°C. The tubes were acetic acid (EDTA). Although an acid pH and immediately centrifuged (3,400 x g for 3 min), and the the presence of divalent cations suppressed ausupernatants were decanted. PG was isolated from tolysis, they did not prevent a loss of viability the cell pellets by the procedure of Hebeler and Young In was (3). Autolysis temperature dependent. (9) as follows. The pellets were suspended in 5 ml of distilled water and placed in screw-capped glass tubes HEPES buffer (pH 8.5), it was markedly recontaining 5 ml of 4% sodium dodecyl sulfate at 100°C. duced at 4°C and did not occur when cells had Residual cell material was quantitatively recovered been heated at 80°C for 10 min.
212
_j 70 z
60
0
37C,HEPES
pH60.0
v 37C,HEPES pH 8.5- Mg+
4C,HEPESpH8.5
Z
C..)50
40 0
INFECT. IMMUN.
WEGENER, HEBELER, AND MORSE
* 37C,HEPES pH 6.0+EDTA A
37C,HEPESpH 8.5
10
20
30
40
50
60
TIME (min.)
FIG. 1. Inhibition of autolysis of N. gonorrhoeae JW-31 by Mg" and pH. Log-phase cells were harvested and suspended at 37 or 4°C in 50 mM HEPES buffer at pH 8.5 or 6.0 in the presence or absence of 10 mM MgCl2 or 5 mM EDTA. Autolysis was measured as described in the text.
Some buffers appeared to be more effective than others in promoting autolysis (Table 1). However, in all cases, the autolytic rate was highest at an alkaline pH and was reduced in the presence of Mg2". The optimal pH for autolysis appeared to be 8.0 to 8.5. In addition to Mg2+, other divalent cations (Ca2 Mn2 and Ba2+) were effective in preventing autolysis of cell suspensions (Table 2). The addition of monovalent cations (Na+, K+, Li+) had little effect, whereas trivalent cations (Fe3+, Al3+) showed intermediate activity. Autolysis was not prevented by the sulfhydryl inhibitors N-ethylmaleimide or iodoacetate, which are able to penetrate the gonococcal cell wall, as indicated by their inhibition of glucose incorporation and growth (data not shown). However, autolysis was totally inhibited by the addition of 1 mM H2+. Hg2~ It was of interest to determine whether the chemical composition of the envelope or the synthesis or activity of an autolytic enzyme could be altered by variations in growth conditions. Accordingly, the energy source, pH, growth temperature, and the concentration of Mg2e in the medium were varied, and rates of cellular autolysis were determined in HEPES buffer at pH 8.5 and 6.0 with and without Mg2+ and EDTA. There were no significant differences in either the patterns or the rates of autolysis between cells initially grown: (i) at 37°C in LGCB media with glucose, pyruvate, or lactate as the energy source; (ii) at 37°C in LGCBglucose media buffered with HEPES over a pH range of 6.0 to 8.0; (iii) in LGCB-glucose media incubated at 37, 31, or 28°C; and (iv) at 37°C in LGCB-glucose media containing a large excess (5 to 20 mM) of MgCl2. ,
,
Incorporation of radioactivity into PG. The PG of N. gonorrhoeae exhibits a high rate of turnover during exponential growth (9). Therefore, we developed a procedure by which to radioactively label PG of strain JW-31 at a high specific activity during a short incubation period. Unless noted otherwise, PG was labeled during a 30- to 40-min period by the addition of [6-3H]glucose to log-phase cultures in LGCB media containing pyruvate as the energy source. Since glucose is metabolized primarily by the Entner-Doudoroff pathway in N. gonorrhoeae (16), the label in position 6 is readily incorporated into cell material. Figure 2 compares the kinetics of incorporation of [6-3H]glucose and [6-3H]glucosamine into trichloroacetic acid-insoluble cell material during growth in LGCB-pyruvate medium. In the TABLE 1. Effect ofpH and buffer on rate of autolysis of N. gonorrhoeae JW-31 Rate of autolysis (k x 10-3)a
pH
HEPES
TES
Tris-hydro- Tris-machloride
leate
