Sporangila autolysis in Clostridium perfringens type A

605

Biochimica et Biophysica A cta, 338 (1974) 605--618

© ]~sevier ScientificPublishingCompany,Amsterdam--Printed in The Netherlands BBA 27319 SPORANGIAL AUTOLYSIS IN C L O S T R I D I U M P E R F R I N G E N S TYPE A

LILY K. CHEN and CHARLES L. DUNCAN Food Research Institute and Department of Bacteriology, University of Wisconsin, Madison, Wisc. 53706 (U.S.A.)

(Received August 13th, 1973)

Summary The autolytic system functioning in the release of mature spores and enterotoxin from sporangia of C l o s t r i d i u m p r e f r i n g e n s was partially characterized. After sporangial autolysis in buffer, the supernatant fluid of the suspension contained autolysin active against purified sporangial walls. The autolysin was most active at pH 8 and 37°C, in the presence of Co 2÷ (0.3 • 10 -3 M CoC12 ) and trypsin (48 pg/ml). Sodium dodecyl sulfate-treated sporangial walls further extracted with trichloroacetic acid to remove teichoic acid were a better enzyme substrate than walls treated only with sodium dodecyl sulfate. N-Acetylmuramyl-L-alanine amidase activity which released N-terminal alanine, and endolSeptidase activity which hydrolysed the D-alanyl-glycine linkage liberating N-terminal glycine and C-terminal alanine, were both functional at pH 8. It is not k n o w n if one or two enzymes are involved. Autolysin appeared in cells as early as 2 h after inoculation into sporulation medium. Two asporogenic Stage 0 mutants grown in sporulation medium also produced autolysin identical in mode of action to t h a t of the sporogenic wild type. Although the active cellular autolysin concentration subsequently decreased as cells sporulated, the walls of 8-h-old sporangia containing refractile heat-resistant spores were more susceptible to digestion by autolysin, than those of 2-, 4-, or 6-h-old cells grown in sporulation m e d i u m or of 4- or 14-h vegetative cells from growth medium. The results suggest that a progressive change may occur in the structure of the sporangial wall during spore morphogenesis, thus increasing its susceptibility to autolysis.

Introduction

The p h e n o m e n o n of autolysis has been studied with diversified groups of microorganisms. The autolysins have been f o u n d to be associated with cell walls in some cases [1--3]. Fan [1] was able to release the active autolysin f r o m Bacillus subtilis walls by incubation of the walls at a high salt concentra-

606 tion. In other cases, lytic enzymes are not tightly bound by the cell. They can be released directly into the growth medium, as indicated for a strain o f B. subtilis [4] or into the s u p ~ n a t a n t of a suspension of cells undergoing lysis in buffer, as reported for Bacillus th:tt,ringiensis var. thuringiensis [5]. A role in wall grovct~i and ;vegetative cell division has been proposed as one function of autol~s~ns ['6,7]. Similar lytic factors have been released from sporulating cells, indicating that the liberation of mature spores from sporangia is also dependent on such a system. Strange and Dark [8] reported release of two autolysins from tolueneautolysed sporulating cells of Bacillus cereus (enzyme V and enzyme S). The authors proposed!that enzyme V played a specific role in liberation of free spores, and enzyme S in spore germination. Detailed studies have been carried out by Kingan and Ensign [5] on three autolysins of B. thuringiensis var. thuringiensis, that were specifically associated with sporulation and spore liberation. It is only recently that more studies have been done on autolytic systems in the genus Clostridium. Takumi and Kawata [9] partially purified and characterized two autolytic enzymes in Clostridium botulinum, which exhibited only one pH o p t i m u m near 6.8. Activity of the enzymes was stimulated by Co 2÷ , trypsin and mercaptoethanol. Takumi and Kawata [10] also examined autolysis of 2-h vegetative cells of Clostridium perfringens Type A. Both whole cells and cell walls underwent rapid lysis, liberating NH2-terminal groups, but no reducing groups. This suggested that the autolysin attacked either the N-acetylmuramyl-L-alanine linkage or the peptide linkages or both of C. perfringens peptidoglycan. Recent research has shown that the enterotoxin of C. perfringens Type A is a sporulation-specific gene product and is released simultaneously with mature spores when sporulating cells undergo lysis [11,12]. The present study was carried o u t using autolysin and walls of sporulating cells of C. perfringens to examine the mechanism underlying the liberation of spores and the enterotoxin, and also to investigate the time of autolysin formation during spore morphogenesis.

Experimental Procedure Cultivation and harvest o f cells C. perfringens Type A, strain NCTC 8798, was used for all studies, unless stated otherwise. A 1% (v/v) inoculum was made from a cooked-meat stock culture into 5-ml volumes of Duncan--Strong sporulation medium [13], contained in screw-cap tubes. The tubes were incubated at 37°C for 16 h. The cultures were then refrigerated and saved as spore stocks. A total of 2 ml of the spore stock was heat-shocked at 75°C for 20 min, and a 1% inoculation was then made into fluid thioglycollate medium (Baltimore Biological Laboratories) for vegetative cell growth. After 12--14 h of incubation at 37 ° C, a 1% inoculum was transferred from fluid thioglycollate to 3 1 of Duncan--Strong medium, which was incubated for 8 h at 37°C. Usually, 5--6 flasks containing sporulated cells were harvested at a given time. During harvest, cells were maintained at a low temperature by the addition of crushed

607 ice to the culture flasks, and by centrifugation at 4 ° C, with a re~tive ~entrifugal force of 15 000 × g. The harvested cells containing a mixt~me o f sporulated and vegetative forms were washed once with cold distilled ~ b e v at 4°C and used in subsequent experiments.

Measurement of growth and sporulation Cell growth was followed by measuring absorbance at 650 nm with a Bausch and Lomb Spectronic 20 Colorimeter. Percent sporulation was determined by direct-counts using phase-contrast microscopy, of vegetative cells and refractile spores, immobilized on an agar block. Dry weights were determined by heating cell samples at 100°C for 24 h or longer, until constant weights were obtained.

Separation of .sporangia from vegetative cells on renografin gradients Renografin (N, N'-diacetyl-3,5-diamino-2,4,6-triiodobenzoate) was used as a supporting medium for separating sporangia and vegetative cells by isopycnic centrifugation [14]. Renografin (Squibb) solutions of various densities were prepared by mixing renografin with water in specific ratios. Step gradients were prepared by carefully layering 3 ml of each of the following densities: 1.2110, 1.2036, 1.1964, 1.1816, 1.1742 g/ml, one on top of the other in a 30-ml polyallomer tube, with the densest layer on the b o t t o m . The tubes were kept at 4°C for 15 min, then 10 ml of a cold suspension of 8-h-old sporulating and vegetative cells at an A s s 0 nm of 1.5 was layered on top of each gradient. The cells were centrifuged in a Beckman SW 25.1 rotor at 40 690 × g for 45 min, to separate the different cells forms. After centrifugation, the cells were separated into 2 bands, the sporangia banded at a density of 1.2060 g/ml and the vegetative cells at a density of 1.1782 g/ml. The 2 bands of cells were removed separately, washed with cold distilled water, and layered on fresh gradients for further separation. The procedure was repeated until a clean preparation of sporangia was obtained.

Preparation of sporangial walls The 8-h cells from Duncan--Strong medium were heated at 100°C for 10 min to inactivate any autolysins, and sporangia were obtained by isopycnic centrifugation. The sporangia were disrupted by grinding with synthetic zeolite in an ice-cooled mortar. Zeolite has been shown to be very effective for disruption o f bacterial cells, and for obtaining large fragments of cell walls [15]. To remove non-peptidoglycan wall constituents, the crude sporangial walls obtained by zeolite grinding were treated with 1% sodium dodecyl sulfate (Matheson, Coleman and Bell) for 15 h at 37°C, with shaking. They then were washed six times in cold distilled water with centrifugation at 25 000 × g, and suspended in 0.05 M potassium phosphate buffer, (1 g of wall/100 ml buffer) pH 7.5, at 37°C. Trypsin (Sigma) and ribonuclease {Worthington) were each added to a final concentration of 0.5 mg/ml, and the mixture was incubated at 37°C for 4 h, with stirring. The treated walls were washed 6 times with cold distilled water as before and were then extracted with 10% trichloroacetic acid at 60°C for 45 min. After 6 final washings to remove the trichloroacetic acid and extractable material, the sporangial walls were lyophilized and stored in a desiccator at 4°C for later use.

608 All the walls used for studies were prepared as above unless indicated otherwise. In general, walls that were treated with sodium dodecyl sulfate, trypsin and ribonuclease are subsequently referred to as sodium dodecyl sulfate-walls, and sodium dodecyl sulfate-walls extracted with 10% trichloroacetic acid are designated as trichloroacetic acid-extracted walls.

Assay for teichoic acid in trichloroacetic acid extracts After t r e a t m e n t with trichloroacetic acid as indicate above, the walls were pelleted by centrifugation at 25 000 X g, and 4 vol. of 95% ethanol were added to the supernatant to precipitate any teichoic acid present. The white precipitate was purified by redissolving in 10% trichloroacetic acid and reprecipitating with ethanol. The teichoic acids were hydrolysed with 2 M HC1 (1 mg precipit a t e / 1 0 0 pl HC1) at 100°C for 3h. The HC1 was removed in vacuo over NaOH, and the residue was assayed for inorganic phosphate by the technique of Lowry et al. [16]. The presence of phosphate equivalent to a level of 0.22 pmoles/mg dry wt of sodium dodecyl sulfate-walls, was indicative of teichoic acid extraction by 10% trichloroacetic acid. Preparation o f crude autolysate Freshly prepared 8-h sporangia were suspended in 0.05 M Tris buffer (pH 8 at 37°C). The ratio of sporangia to buffer (w/v) was 0.9, and a final concentration of 2.5% toluene was added to facilitate release of any autolysin that might be bound to the cell wall. Lysis usually began within 0.5 h, as determined by phase-contrast microscopy, but the sporangia were allowed to incubate for 3 h to ensure complete release of autolysins. Toluene was removed from the preparation by evaporation under a stream of air and the suspension then was centrifuged at 25 000 × g for 45 min. The pellet of free spores and cell debris was discarded, and the supernatant was saved for use as the crude autolysin. Enzyme assay In early experiments, 48 pg/ml of trypsin and 0.3 • 10 -3 M CoC12 were found to be stimulatory to autolysis of the sporangial walls, and were therefore used in all assays for lyric activity unless indicated otherwise. For the routine assay of lytic activity, 240 pl of crude autolysin was added to a 10 ml X 45 mm tube containing 160 pl of 0.05 M Tris buffer (pH 8) with 10-3M CoC12 incorporated (final concentration of Co 2÷ was 0.3 10 -3 M). 25 pl of 1 pg/td trypsin was also added to make a final concentration of 48 pg/ml. The pH and composition of the buffer was varied when desired. After equilibrating the mixture at 37°C for 10 min, 100 #l of a trichloroacetic acid-extracted wall suspension (2 mg dry wt/ml), also at 37°C, was added, and the preparation was immediately transferred to a 0.5-ml cuvette. The absorbance at 650 nm was monitored for 1 h using an Hitachi Perkin--Elmer spectrophotometer, equipped with an Haake constant-temperature circulator. The cuvette temperature was maintained at 37°C, and when a reading was taken, the wall suspension in the cuvette was thoroughly mixed. In control samples, the volume of crude autolysin was replaced with an equal a m o u n t of buffer. One unit of enzyme activity is defined as that a m o u n t of enzyme pro-

609 ducing an initial decrease in absorbance (650 nm) of 0.0001 in 15 min, calculated from measurements made over a 30-min period of linear decrease. The initial absorbance was adjusted to approximately 0.1, to minimize the amount of sporangial walls needed as substrate. Wall autolysis was validated b y phasecontrast microscopy.

Chemical assays Protein was determined b y the procedure of L o w r y et al. [ 1 7 ] , using crystalline bovine serum albumin (Sigma) as the standard. Reducing groups were measured by the ferricyanide reduction method of Park and Johnson [ 1 8 ] . Dinitrophenyl derivatives of free amino acids and free amino groups, N-terminal and C-terminal amino acids were identified or quantitated or both b y the methods of Ghuysen et al. [ 1 9 ] . Results

Growth and sporulation The growth of C. perfringens strain NCTC 8798 cells in Duncan--Strong medium is quite rapid. By 2.5--3 h, the increase in absrobance began to level off. Observation by phase-contrast microscopy showed the presence of forespores by 3 h, and after that, the count o f refractile spores steadily increased until a maximum percent sporulation was reached at 7--8 h, which ranged from 60--85% with different batches of cells. Free spores usually began to appear b y 10--12 h, b u t the release was relatively slow, and t o o k several hours to complete. To obtain sporangial walls and cell-free autolysin prior to spore release, sporangia containing heat-resistant, refractile spores were harvested at 8 h of incubation. Sporangial lysis Sporangia suspended in buffers of different pH at 37°C exhibited various rates o f lysis (Fig. 1). The rate of lysis was expressed as the linear decrease in absorbance (650 nm) per min X 103, as measured during a 30-min interval. At

3

~2 1 0 45

5.5

6.5

7,5 pH

85

9.5

Fig. 1. E f f e c t o f p H o n t h e r a t e o f a u t o l y s i s o f 8 - h s p o r a n g i a o f C. perfringens. S p o r a n g i a w e r e s u s p e n d e d in 0 . 0 5 M b u f f e r s o f d i f f e r e n t p H ; 4 . 5 , 5, 5.5, 6, 6 . 5 , 7, 7 . 5 , 8, 8 . 5 , 9 a n d 9 . 5 . L y s i s w a s f o l l o w e d a t 3 7 ° C f o r 1 h b y a b s o r b a n c e r e a d i n g s at 6 5 0 n m . T h e r e s u l t s aze e x p r e s s e d as t h e rate o f lysis: ~ A 6 6 0 n m / m i n X 1 0 3 . ©, a c e t i c a c i d - - s o d i u m a c e t a t e ; A K 2 H P O 4 - - N a O H ; ~ T r i s - - H C l ; • g l y c i n e - - N a O H .

610 200

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50

95

pH Fig. 2. E f f e c t o f p H o n 8-h s p o r a n g i a l a u t o l y s i n a c t i v i t y using t r i c h l o r o a c e t i c a c i d - e x t r a c t e d s p o r a n g i a l w a l l s o f C. perfringens as substrate. T h e A 6 s 0 n r a w a s f o l l o w e d at 3 7 ° C for 3 0 rain. o, a c e t i c a c i d - s o d i u m a c e t a t e ; A K 2 H P O 4 - - N a O H ; ~ T r i s ' - - H C l ; o, g l y e i n e - - N a O H .

pH 4.5 and 5, autolysis was c o m p l e t e l y inhibited. At pH 5.5 and higher, the rate o f sporangial lysis steadily increased until a plateau was reached at pH 8.5--9. At pH 9.5, autolysis was drastically reduced, and any higher alkalinity prevented lysis. The optimal t e m p e r a t u r e of sporangial autolysis was 37°C.

Optimal conditions for lytic activity using purified cell walls A comparison of the activity o f sporangial autolysin on sodium d o d e c y l sulfate-walls versus trichloroacetic acid-extracted walls was made. There was no lytic activity when sodium d o d e c y l sulfate-sporangial walls alone were used as the substrate. However, with t he addition o f Co 2÷ at a final c o n c e n t r a t i o n of 0.3 • 10 -3 M and trypsin at 48 pg/ml, activity of t he autolysin preparation was stimulated and wall lysis could be det ect ed. When trichloroacetic acid-extracted walls were used as substrate under the same conditions, a considerably higher activity was obtained, (an absorbance decrease in 60 min of 0.09 for trichloroacetic acid-extracted walls versus 0.03 for sodium d o d e c y l sulfate-walls) suggesting that the removal o f teichoic acids might have exposed more susceptible areas o f th e walls to the autolysin. Th e effect o f pH on autolysin activity was examined using trichloroacetic acid-extracted walls and sporangial autolysin, in t he presence o f Co 2÷ and trypsin. The pH--response curve shown in Fig. 2 resembles that shown in Fig. 1, where whole sporangia u n d e r w e n t autolysis in buffers of different pH. A p r o m i n e n t peak was seen at pH 8 with t he sporangial walls, hence this was established as t he optimal pH for lyric activity, and all subsequent assays were made using this pH value. As men ti one d, Co 2÷ and trypsin were stimulatory to lytic activity. Either Co ~÷ or trypsin alone could stimulate autolysin activity in 0.05 M Tris b u f f e r (pH 8), b u t if the two were added together, stimulation was comparatively greater (Fig. 3). The mechanisms w h e r e b y Co 2÷ and trypsin stimulate autolysin activity are most likely different, hence this apparent l y additive effect o f stimulation was observed. The effect of several divalent metal ions o n sporangial wall lysis at pH 8 by the autolysin preparation was also investigated. T he results are shown in Table I. Co 2÷ at a c o n c e n t r a t i o n o f 0.3 • 10 -3 M was the most stimulatory among the ions tested. However, Mg 2÷ , Mn 2÷ and Ni 2÷ were also stimulatory.

611

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Fig, 3. E f f e c t o f Co 2'~ a n d t r y p s i n o n 8-h s p o r a n g i a l a u t o l y s i n a c t i v i t y o n t r i c h l o r o a c e t i c a c i d - e x t r a c t e d w a i l s o f C. perfringens. T h e f i n a l c o n c e n t r a t i o n o f Co 2+ u s e d w a s 0 , 3 • 10 -3 M; t r y p s i n , 48 /~g/ml. I n c u b a t i o n w a s at 3 7 ° C a n d p H 8.0.

By incubating the autolysin assays at different temperatures, it was found that maximal activity was obtained at 37°C (Table II), the same as found for autolysis of whole sporangia. Kawata and Takumi [9] reported that mercaptoethanol increased the activity of the lytic system of C. botulinum T y p e A. When 0.3 • 10 -3 M mercaptoethanol was incorporated into the C. perfringens sporangial autolysin assay at pH 8, with 0.05 M Tris buffer, no effect was detected. This suggested that the enzyme system does n o t require a reducing environment for proper functioning. From the results gathered, the optimal conditions for autolysin digestion of purified sprongial walls were 37°C, pH 8, 0.05 M Tris buffer, with 0.3 10 -3 M CoC12 and 48 pg/ml trypsin added, and trichloroacetic acid-extracted sporangial walls. Time o f au tolysin production It was of interest to k n o w the time of autolysin formation during sporula-

TABLE I EFFECT OF DIVALENT METAL IONS ON LYSIS OF TRICHLOROACETIC S P O R A N G I A L W A L L S BY C R U D E A U T O L Y S I N

ACID-EXTRACTED

T h e m e t a l i o n s w e r e a d d e d as s u l f a t e s o f Mg 2+, Z n 2+ a n d M n 2+, a n d c h l o r i d e s o f Co 2+, Ca 2+ a n d Ni 2+, at f i n a l c o n c e n t r a t i o n s o f 0 . 3 " 1 0 - 3 , 0 . 3 " 1 0 - 4 a n d 0 . 3 . 1 0 - 5 M. I n c u b a t i o n w a s at 3 7 ° C in 0 . 0 5 M T r i s b u f f e r ( p H 8) a n d in t h e p r e s e n c e o f 4 8 p g / m l t r y p s i n . T h e r e s u l t s a v e r a g e d f r o m t r i p l i c a t e r u n s are prese n t e d as r e l a t i v e lysis, w i t h t h e 1 5 % d e c r e a s e i n o r i g i n a l a b s o r b a n c e at 6 5 0 n m o c c u r r i n g in t h e c o n t r o l a f t e r 30 m i n t a k e n as 1 0 0 . Metal i o n a d d e d

None (control) Co 2+ Mg 2+ Ca 2+ Mn 2+ Ni 2+ Z n 2+

R e l a t i v e lysis as c o m p a r e d t o c o n t r o l 0.3.10 -3 M

0.3-10 -4 M

0.3"10 -5 M

100 340 160 67 160 200 50

100 267 133 100 100 180 60

100 133 160 133 100 170 60

612 T A B L E II OPTIMAL TEMPERATURE FOR ACTIVITY OF CRUDE C. P E R F R I N G E N S O N P U R I F I E D S P O R A N G I A L W A L L S

AUTOLYSIN

F R O M S-h S P O R A N G I A

OF

T h e a s s a y s w e r e c o n d u c t e d w i t h 0 . 0 5 M Tris b u f f e r , p H 8, in t h e p r e s e n c e of 48 ~/g/ml t r y p s i n a n d 0.3 X 10 -3 M CoC12. L y s i s w a s f o l l o w e d at 6 5 0 n m fo." 6 0 rain.

Temperature of incubation (°C)

Units of a c t i v i t y

32

37

40

75

150

125

45 100

tion, and whether or not it was also present in vegetative cells of the same strain. Thus, exponential and stationary phase cells grown 4 h and 14 h, respectively, in fluid thioglycollate growth medium, and cells harvested at different stages of growth and sporulation in Duncan--Strong sporulation medium, were tested for the presence of autolysin. The 4-h fluid thioglycollate grown cells of NCTC 8798 lysed slowly after being suspended for 2.5 h in Tris buffer (pH 8), and lysis was complete in 4.5 h. The 14 h fluid thioglycollate cells did not autolyse in buffer, hence they were sonicated, and the extracts and the 4-h lysates were tested for lytic activity on trichloroacetic acid-extracted sporangial walls. Autolysates from cells of the same strain harvested at different stages of growth and sporulation in Duncan--Strong medium were also assayed for activity on 8-h trichloroacetic acid-extracted sporangial walls. Lytic activity could not be detected, using trichloroacetic acid-extracted sporangial walls as substrate, in 4-h fluid thioglycollate lysates or 14-h cell extracts, whereas autolysates of cells from Duncan--Strong medium had enzyme activity at all stages tested (2, 4, 6, 8 h). Surprisingly, with the increasing age of cells, there was a reduction in lytic activity, and also in specific activity and a m o u n t of autolysin/mg dry wt of cells {Table III). T A B L E III ACTIVITY OF CRUDE AUTOLYSATES OR CELL EXTRACT FROM VEGETATIVE TING CELLS ON TRICHLOROACETIC ACID-EXTRACTED SPORANGIAL WALLS

OR SPORULA-

F T G i n d i c a t e s v e g e t a t i v e ceils f r o m f l u i d t h i o g i y c o l l a t e g r o w t h m e d i u m . D-S m i x e d i n d i c a t e s m i x e d s p o r a n g i a , if p r e s e n t , and v e g e t a t i v e cells f r o m s p o r u l a t i o n m e d i u m . D-S sporangia i n d i c a t e s p u r i f i e d s p o r a n g i a s e p a r a t e d f r o m v e g e t a t i v e cells.

Autolysates from v a r i o u s cells 4-h F T G 14-h F T G 2-h D-S m i x e d 4-h D-S m i x e d 6-h D-S m i x e d 8-h D-S m i x e d 8-h D-S s p o r a n g i a

Units of activity

Specific activity

Units of activity/ m g d r y w t o f cells*

0 0 153 154 99 99 100

0 0 108 98 58 60 59

0 0 22 19 8 6 6

* Dry w t o f cells f r o m w h i c h a u t o l y s a t e w a s p r e p a r e d .

613

T A B L E IV A C T I V I T Y OF C R U D E A U T O L Y S I N F R O M S-H S P O R A N G I A ON T R I C H L O R O A C E T I C A C I D - E X T R A C T E D W A L L S P R E P A R E D F R O M V E G E T A T I V E OR S P O R U L A T I N G CELLS F T G i n d i c a t e s v e g e t a t i v e cells f r o m f l u i d t h i o g l y c o l l a t e g r o w t h m e d i u m . D-S m i x e d i n d i c a t e s m i x e d s p o r a n g i a , if p r e s e n t , a n d v e g e t a t i v e ceils f r o m s p o r u l a t i o n m e d i u m . D-S s p o r a n g i a i n d i c a t e s p u r i f i e d s p o r a n g i a s e p a r a t e d f r o m v e g e t a t i v e cells. T r i c h l o r o a c e t i c a c i d - e x t r a c t e d walls f r o m v a r i o u s cell p r e p a r a t i o n s 4-h F T G 14-h F T G 2-h D-S m i x e d 4-h D-S m i x e d 6-h D-S m i x e d 8-h D-S m i x e d 8-h D-S S p o r a n g i a

U n i t s of a c t i v i t y

Specific activities

9 10 75 90 100 130 140

-5 34 41 45 63 64

The following experiment was conducted to determine the sensitivity of walls obtained at different stages of growth and sporulation. Purified walls were prepared from 4-h and 14-h fluid thioglycollate-grown cells and 2-, 4-, 6- and 8-h Duncan--Strong cells, as previously described for sporangial walls. As the vegetative forms and sporangia of the Duncan--Stronggrown cells were not separated, the wall preparations were designated as mixed walls. Autolysin from 8-h sporangia was tested on these different types of walls, and the results are shown in Table IV. The lytic enzymes were most active against purified 8-h sporangial walls, and activity decreased in the order as followed: sporangial wall > 8-h mixed > 6-h mixed > 4-h mixed > 2-h Duncan--Strong walls > 14-h fluid thioglycollate > 4-hr fluid thioglycollate walls.

Mode of activity During the course of lysis by 8-h sporangial autolysin of purified, trichloroacetic acid-extracted sporangial walls at pH 8 and 37 ° C, 200-#1 samples were taken at regular time intervals. The residual wall fragments were pelleted by centrifugation and the supernatant was analysed for the presence of reducing groups. There was no increase in reducing constituents as lysis proceeded, indicating the absence of a functional endo-N-acetylmuramidase or endo-Nacetylglucosaminidase in the autolytic system. Similar time samples were tested for free amino groups, and as lysis continued, increasing a m o u n t s of free amino groups were produced (Fig. 4), indicating that a peptidase or amidase, or both, were present in the autolysin and were acting on the walls. Analysis of dinitrophenyl-derivatives of N-terminal amino acids by thinlayer chromatography showed that during sporangial wall lysis, there was liberation of N-terminal alanine and N-terminal glycine, and that the concentrations o f the N-terminal amino acids increased with time (Fig. 5). The data show t h a t the absorbance decreased at a faster rate than the corresponding increase in N-terminal amino acids. A 77% decrease in absorbance occurred in the first

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TIME (rain)

groups from trichloroacetic acid-extracted sporangial walls by 8-h sporangial a u t o l y s i n . 1 ml of autolysate w a s a d d e d t o 6 5 0 p l o f p H 8 b u f f e r w i t h 0 . 3 • 1 0 - 3 M C o 2+ a n d 4 8 ~zg/ml o f t r y p s i n i n c o r p o r a t e d . A f t e r 1 0 rain o f i n c u b a t i o n at 3 7 ° C , 4 5 0 / J l o f t r i c h l o r o a c e t i c acid walls ( 4 m g d r y w t / m l ) w a s a d d e d , a n d t h e d e c r e a s e in A 5 5 0 n m m e a s u r e d . A t 1 0 rain i n t e r v a l s , 2 0 0 - p l s a m p l e s were removed from the reaction mixture. The residual walls were pelleted by centrifugation a n d discarded, while the supernatants were assayed for free amino groups. F i g . 4. F o r m a t i o n

of free amino

15 min, as compared to a 40% increase in N-terminal glycine and a 38% increase in N-terminal alanine in the same period o f time. It could be that a few bonds initially cleaved in the peptidoglycan network by lytic enzymes can considerably loosen the network and result in a large drop in absorbance, at which time only a limited number of N-terminal groups would be exposed. A~ lysis of the walls continued, more peptide chains were cleaved, and hence an increased concentration of N-terminal amino acids was detected. No free amino acids could be detected. The release of N-terminal alanine appears to be indicative of N-acetylmuramyl-L-alanine amidase activity, splitting the bond between L-alanine and N-acetylmuramic acid. In the peptidoglycan structure of C. perfringens, as proposed by Leyh-Bouille et al. [ 2 0 ] , the peptide cross-bridge consists of 1 molecule of glycine linked between D-alanine of one peptide and LL-diaminopimelic acid of another peptide, hence the appearance of N-terminal glycine indicated an endopeptidase activity which cleaved the linkage between D-alanine and glycine. Determination of C-terminal amino acids yielded C-terminal alanine, which provided further evidence for cleavage of the D-alanylglycine linkage.

.20 _E o

.15

.10

F

o.5 GLYCIN|

~O

o4 ALANINE

o3 "~

, ,O

02 •

\

o.1

=

"'- ....... oo 0

5

10

15

20

25

30

T I M E (mini

F i g . 5.

Release

of

sporangia] autolysin.

N-terminal amino acids from trichloracetic acid-extracted sporangial ~ , N - t e r m i n a l a l a n i n e ; © - - c N - t e r m i n a l g l y c i n e ; ©- - o , A 6 5 0 n m .

walls

by

8-h

615 TABLE V A C T I V I T Y O F A U T O L Y S I N S F R O M A S P O R O G E N I C S T A G E 0 M U T A N T S 8-5 A N D 8-46 O N T R I C H L O R O A C E T I C A C I D - E X T R A C T E D S P O R A N G I A L W A L L S O F T H E W I L D T Y P E C. P E R F ~ t I N G E N S STRAIN NCTC 8798 Strains

Units activity

Specific a c t i v i t y

8-5 8-46 NCTC 8 7 9 8 (wild type)

125 113 140

31 41 64

Experiments were conducted to determine if the mode of action of autolysin from 2- or 10.5-h cells grown in sporulation medium was the same as that of autolysin obtained from 8-h sporulating cells. The results indicated an identical mode of action. A trypsin control experiment was run to determine whether trypsin in the autolysin assay was responsible for disrupting any peptide bonds in possible residual wail-bound protein. Trypsin (48 pg/ml) was incubated with trichloroacetic acid sporangial wail in pH 8 buffer with Co 2÷ , at 37°C, but with no autolysin present. After 45 min of incubation, the sample was analysed for N-terminai amino acids, but none was released. It was also found that autolysin alone, in the absence of trypsin, solubilized trichloroacetic acid-wail and liberated N-terminai alanine and N-terminal glycine. Therefore, it is evident that trypsin does not play a role in exposing N-terminai amino acids.

Autolysis o f asporogenic mutants o f strain NCTC 8798 Cells of two asporogenic Stage 0 mutants of NCTC 8798, strains 8-5 and 8-46 [12], were grown for 8 h in Duncan--Strong medium, harvested, washed and suspended (0.9 weight/volume) in Tris buffer (pH 8) at 37°C with 2.5% toluene. Autolysis was not detected until after 45 min of incubation, and was completed by 2 h. Autolysins from these mutants were assayed on 8-h trichloroacetic acid-extracted sporangial walls of the wild type NCTC 8798, and good activity was observed for each strain (Table V). Both 8-5 and 8-46 autolysins released N-terminal alanine and N-terminal glycine from the walls. Thus, even though the m u t a n t s have lost the ability to sporulate, they produced an autolysin similar to that of the wild type. Discussion

According to the model cell wail structure of C. perfringens proposed by Leyh-Bouille et ai. and Schleifer et al. [20,21], the tetrapeptides of the peptidoglycan consist of L-alanine--D-isoglutamine--L L-diaminopimelic acid--Dalanine, with a single glycine molecule as the cross-bridge connecting D-alanine of one chain to LL-diaminopimelic acid o f another (Fig. 6). This model differed from that suggested by Pickering [22], which had a cross-bridge of Lalanine-D-glutamic acid-diaminopimelic acid, with the glycine molecule being attached to D-glutamic acid of the tetrapeptide chain. In the current study, the presence of N-terminal glycine instead of free glycine following wail lysis supports the cell wail structure proposed by Leyh-Bouille et al. for C. perfringens.

616 Glycan Glycan L

L - A l a 4 D - G l u - NH 2

Ala~D

Glu L

NH 2

~1

~ ~ D ALa ~ GIy ~ DAP ~1, - - - G l y ~ (L)

Droposed

sptes

of

N - acetylrnu .~

Endopept

autolytlc

{L) DAP (L)

attack

rainy I - L -ala n ine

arn d a ~ e

Jdase

of the peptidoglycan polymer o f C. perfringens s h o w i n g the proposed sites o f a u t o ] y t i c a t t a c k b y the sporangial autolysin. L-Ala, L-alanine; D-GIu-NH2, D-isoglutamine; LL-DAP, LL-diaminopimelic acid; D-Ala, D-alanine; GIy, glycine. Fig. 6. Structure

Tinelli [23] reported that C. perfringens vegetative cell walls contained no autolysin; whereas Kawata and Takumi [10] observed considerable autolysis of vegetative cell walls of C. perfringens, with concomitant release of N-terminal amino groups. It was possible that in the study by Tinelli, the autolysin was not wall bound, hence no lytic activity could be detected in the walls. The contradicting reports might also have resulted from the cells being grown in different media, which may have influenced autolysin synthesis, or may have led to a slight difference in the cell wall arrangement in each case. One type of composition may be more susceptible to attack by autolysin than the other. In the present study, the fact t h a t fluid thioglycollate-grown vegetative cell walls are almost non-susceptible to dissolution by the autolysin obtained from cells grown in Duncan--Strong medium, while the Duncan--Strong walls are highly susceptible, may be a similar example. Autolysin is present in Duncan--Strong-grown cells at all stages of growth and sporulation. The autolysin concentration decreases as the cells develop into mature sporangia. However, the sporangial walls are more susceptible to the autolysin than walls of younger cells, indicating t h a t a specificity has been established in sporangial walls during spore morphogenesis. This specificity appears to result from a progressive change in the structure of the walls, but it is not known what the nature of the change is. There is the possibility of slow digestion of walls occurring during sporulation, which increases their sensitivity to subsequently added autolysin. Brown and Young [24] noted that for B. subtilis, the autolysin from one stage of growth was more active against exponential cells than post-exponential cells, and they also attributed this difference to a change in substrate, but, the nature of the change was not elucidated. Many functions have been proposed for autolysins, among these are dechaining of cells, loosening o f the cell wall net, providing receptor sites for new peptidoglycan subunits, and liberation of spores from sporangia [5--7,25 ]. It is proposed that the autolysin of C. perfringens is responsible for releasing free spores and enterotoxin. The autolysates from two asporogenic mutants of NCTC 8798 that are blocked at Stage 0 of sporulation, strains 8-5 and 8-46, can lyse sporangial walls o f the wild type, and release N-terminal alanine and N-terminal glycine as end products. This may indicate that similar lytic enzymes are present even in

617

mutant cells that cannot sporulate, b u t their cell walls are probably less susceptible to solubilization b y the enzymes, as evidenced b y an increased time required for lysis of the intact mutant cells as compared to the wild-type sporangia. Since the mutants are asporogenic, their formation of autolysin in sporulation medium that is identical in mode o f action to that o f the sporogenic wild t y p e suggests that synthesis of detectable levels o f autolysin may be dependent on the medium in which the cells are grown. It is also possible that synthesis of autolysin is one of the biochemical events in the sporulation process, b u t its formation is not d e p e n d e n t on sporulation. This may explain w h y asporogenic mutants can also produce autolysin. Kingan and Ensign [5] have indicated that the autolysins of B. thuringiensis vat. thuringiensis were sporulation-related, in that the enzymes appeared shortly before spore liberation and were absent in vegetative cells grown in the same medium. Strange and Dark [8] also reported that autolysins of B. cereus were present only in sporulating cells containing mature spores. The autolysins attacked both heat-killed vegetative cells and purified vegetative walls, b u t they were not detected in non-sporulating vegetative cells. As a contrast, the autolysin of C. perfringens is found even in 2-h-old cells grown in sporulation medium. As sporulation progresses, the concentration of autolysin decreases and the sensitivity of the walls to autolysin increases. Hence, it is implied that instead of depending on an increased level of enzyme, the nature of the sporangial walls o f C. perfringens determines wether or not autolysis will occur.

Acknowledgment This research was supported b y the College of Agricultural and Life Science, University of Wisconsin, Madison, b y research "grants 5-RO1CC00554-03, from the Center for Disease Control, 2-RO1-FD-00203-03, from the F o o d and Drug Administration, and b y contributions to the F o o d Research Institute b y member industries.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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