Acid hydrolysis of the spores of B. cereus; a correlation of chemical and cytological findings

346

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 1 4 (1954)

ACID H Y D R O L Y S I S O F T H E S P O R E S O F B. CEREUS," A C O R R E L A T I O N O F C H E M I C A L AND C Y T O L O G I C A L F I N D I N G S * by P. C. F I T Z - J A M E S * * , C. F. R O B I N O W

AND G. H. B E R G O L D

Departments of Biochemistry and Bacteriology, University o/ Western Ontario, London, Canada and Laboratory of Insect Pathology, Sault Ste. Marie (Canada)

Acid hydrolysis, which forms part of the standard procedure for demonstrating the nuclear material of bacteria, produces an odd effect on resting spores. Whereas the Feulgen-positive material (chromatin) of a vegetative cell remains within the cell during acid hydrolysis, that of the resting spore is extruded beyond the cell outline, appearing as an eccentric plate, or side body. (STILLE1;DELAPORTE~;and ROBINOW3). It is now known that, during this hydrolysis, the nuclear structure of the resting spore is markedly rearranged (RoBINOW4). In earlier studies of this reaction, RomNow 5 observed that if potassium permanganate were added to the acid (hydrochloric or nitric) and the hydrolysis carried out at room temperature, the eccentric basophilic structure of the spore could be selectively removed by extraction with dilute buffers at a p H greater than 6.8. This mild alkaline washing did not remove the side body from spores hydrolyzed without permanganate. As the side body contains the Feulgen-positive substance of the spore, the fact that it could be selectively dissolved suggested a simple means of separating nuclear from cytoplasmic material. Experiments were therefore designed to study: a. The effect on the phosphorus fractions of resting spores of hydrolysis with acid mixtures, with and without permanganate. b. The further effect of mild alkali extraction on the phosphorus fractions of spores which had been hydrolyzed with and without the addition of permanganate. The effects of hydrolysis on other materials in the spore were also observed. A preliminary report of this work has already been presented ( F I T Z - J A M E S ~ ) . MATERIALS AND METHODS Spores of B. cereus were used e x c l u s i v e l y . These were h a r v e s t e d a f t e r t e n d a y s g r o w t h on t h e a g a r m e d i u m of t I o w l E AND CRUIKSHANK 7, s u p p l e m e n t e d w i t h i % c a s a m i n o acids. Spores w e re w a s h e d e i g h t to t w e l v e t i m e s w i t h d i s t i l l e d w a t e r a t r o o m t e m p e r a t u r e . The w a s h i n g w a s e x t e n d e d o v e r one to t w o days, a n d a f t e r each c e n t r i f u g a t i o n (6o00 t o 9ooo g) t h e u p p e r l a y e r of t h e p e l l e t w a s s c r a p e d to r e m o v e v e g e t a t i v e debris. A spore s u s p e n s i o n free of v e g e t a t i v e ceils w a s t h u s o b t a i n e d . One or t w o m l of t h e w a s h e d spore s u s p e n s i o n , c o n t a i n i n g f r o m 3 ° t o 50 m g of spores, w a s p i p e t t e d to a series of 15 m l c e n t r i f u g e t u b e s . T e n to t w e l v e ml of a f r e s h l y p r e p a r e d s o l u t i o n of N/3 n i t r i c a c i d c o n t a i n i n g o.i % p o t a s s i u m p e r m a n g a n a t e ( " K " solution) w a s a d d e d t o one g r o u p of spores w h i c h w a s t h e n a l l o w e d to s t a n d for 20 m i n u t e s a t r o o m t e m p e r a t u r e . H y d r o c h l o r i c acid, to a final c o n c e n t r a t i o n of I N, w a s a d d e d to a n o t h e r g r o u p a n d t h e t u b e s h e a t e d a t 6o ° C for i o * S u p p o r t e d b y a g r a n t f r o m t h e N a t i o n a l R e s e a r c h Council of C a n a d a . ** H o l d e r of a G r a d u a t e M e d i c a l R e s e a r c h F e l l o w s h i p , N a t i o n a l R e s e a r c h C ounc i l of C a n a d a .

Re#rences p. 355.

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AClI~ HYDROLYSIS OF SPORES

347

minutes. The samples were centrifuged and decanted. Following hydrolysis, the residues were washed w i t h distilled water, and the clear h y d r o l y s a t e s and their washes pooled. The p h o s p h o r u s c o n t e n t and, where possible, the ultraviolet a b s o r p t i o n spectra of the h y d r o l y s a t e s were measured. Those samples of hydrolyzed spores to be f u r t h e r extracted by mild alkali were suspended in 0. 5 o.,/osodium acetate (ROBINOW5) for 5 to 20 m i n u t e s at roonl t e m p e r a t u r e , centrifuged at 2000 r.p.m. and, depending on the degree of extraction desired, either washed with water or re-extracted with sodium acetate. Tile cytological effect of these t r e a t m e n t s was followed b y coverslip s m e a r s stained with crystal violet and m o u n t e d in w a t e r from which p h o t o m i c r o g r a p h s were made. The p i t of the various spore suspensions was m e a s u r e d w i t h a glass-electrode p H meter. Duplicate samples of the control spores, as well as the residues following hydrolysis and mild alkali t r e a t m e n t were subjected to the p h o s p h o r u s fractionation procedures of SCHNEIDER8 and of SCHMIDT AND THANNHAUSER9. Since the D N A of hydrolyzed spores was not precipitated from the alkali e x t r a c t of SCHMIDT AND THANNHAUSER on acidification, this m e t h o d of fractionation was of limited use. The individual nucleic acid values in treated spores are therefore based on the MEJBAUM10 orcinot reaction for R N A and the DISCHE 11 d i p h e n y l a m i n e reaction for DNA. The ribonucleic acid s t a n d a r d was a yeast R N A from Schwartz and C o m p a n y ; the desoxyribonucteic acid s t a n d a r d a sample of highly polymerized D N A p r e p a r e d from calf t h y m u s b y the MIRSKY AND [~OLLISTER m e t h o d 12 and obtained from Dr. G. C. BUTLER, University of Toronto. At low concentrations of D N A (as in spores free of side body) the a b s o r p t i o n s p e c t r u m of the colour complex developed in the Dische test was e x a m i n e d in a Becknlan (Model DU) s p e c t r o p h o t o m e t e r , at wave lengths fronl 55 ° to 675 m#. This helped to decide w h e t h e r the t r u e colour complex or some interfering s u b s t a n c e was responsible for the optical d e n s i t y at 600 m/~. Total nucleic acid in trichloracetic acid (TCA) e x t r a c t s was estimated b y the ultraviolet absorption procedure described b y LOGAN, MANNELL AND ROSSITER 13. Total P was determined on all samples b y the KING1~ method, except t h a t " m e t o l " was used instead of KING'S reducing agent (C-OMORI15). I n o r g a n i c p h o s p h o r u s was m e a s u r e d b y the m e t h o d of ]~RNSTER, ZETTERSTR6M AND LINDBERG1~. All values are expressed as mg p h o s p h o r u s per ioo g of original spores dried at 80 ° C and stored i n v a c u o over p h o s p h o r u s pentoxide to a c o n s t a n t weight. Optical e q u i p m e n t consisted of a Zeiss a c h r o m a t i c condenser, N.A. i .4, a c h r o m a t i c oil i m m e r s i o n objective 90 x , N.A. 1.3, in conjunction with a c o m p e n s a t i n g eyepiece, 15 × . The light source was a t u n g s t e n r i b b o n lamp and the e q u i p m e n t was arranged to give Koehler illumination. The p h o t o g r a p h s were t a k e n using a Baird Associates interference filter having a t r a n s m i s s i o n peak at 539 m#. Tile p h o t o g r a p h s , with a magnification of 18oo, were enlarged twice before m o u n t i n g . I n one series of studies, the effect of the hydrolysis and acetate t r e a t m e n t s was followed b y electron microscopy. Small drops of dilute spore suspension were dried on collodion-covered electron m i c r o s c o p y grids, and s h a d o w e d w i t h u r a n i u m (8 rag, distance 12 cm) at an angle of a b o u t i to 4 The pictures were t a k e n w i t h an RCA electron microscope, t y p e E M U 2 D.

RESULTS

Hydrolysis with acid permanganate solution Eight experiments were performed to study the effect on spore phosphorus fractions of acid permanganate hydrolysis ("K" treatment) and subsequent mild alkali extraction. The results of a typical experiment are presented. Within a few minutes of suspending the spores in the K solution, the permanganate colour was markedly reduced. The spores settled out as a brownish floc, leaving a pale pink supernatant. Fig. I illustrates the crystal-violet-stained appearance of spores treated in bulk with the K solution. The refractile, non-staining, resting spores, one of which still persists in the upper left of Fig. I, are differentiated into two basophilic parts, a deep blue side body and a purple central zone separated from the spore surface by a non-staining layer. The central basophilic zone is connected to the side body by a short neck. The electron micrograph of this change is shown in Figs. 6A and 6B. The exosporium still envelopes the hydrolyzed spore. Table I records the chemical changes during K treatment. Some 60 % of the RNA-P was lost from the protein-bound fraction, while the residue P (Schneider fractionation) was reduced to 40 to 50 % of its original whole spore level. Studies on this TCA-residue P of B. cereus spores are included in a publication now in preparation. A large part of it Re#rences p. 355.

348

P . c . FITZ-JAMES, C. F. ROBINOW, G. H. BERGOLD

VOL. 14 (I954)

was f o u n d to be an alkali-soluble a n d acid-labile substance possessing the p r o p e r t i e s of a p o l y p h o s p h a t e . A small, acid-stable p a r t of the TCA-residue P of B . cereus spores was b o t h acid a n d alkali insoluble (equal to the D N A - f r e e residue P of the SCHMII~I aND THa~'NI~,~USER p r o c e d u r e Table I I I ) a n d is associated with the spore coats. TABLE I THE P H O S P H O R U S D I S T R I B U T I O N OF A C I D - P E R M A N G A N A T E H Y D R O L Y Z E D ( K T R E A T E D ) SPORES, W A T E R AND SODIUM ACETATE VCASHED

Phosphorus (P) as mg/Ioo g spores K treated spores Unfree*ted

.";odzum acetate washed ~,ater a,ashed side body revealed

Cyhdogical effects

K Extract ist Acetate wash 2nd Acetate wash Acid-soluble P Lipid-soluble P

Total P Total P Total I)

On~e side body partly removed

T*c'ice side body completelyremoved

90

90 291

8 3

i0o io

29 io

90 291 ioo 14 8

499 372 90

270 145 84

189 ioo 37

112 72 o

148

63

58

26

-

-

TCA Extract Total P RNA P (orcinol) DNA P (diphenylamine)

( S C H N E I D E R 8)

TCA Residue

Total P

F r o m Table I it can be seen t h a t the loss of R N A a n d residue P from the b o u n d s forms is largely b a l a n c e d b y an increase in acid-soluble P a n d b y t h e p h o s p h o r u s in t h e K e x t r a c t . Labile p h o s p h o r u s studies showed t h a t p a r t of t h e d r o p in TCA-residue P in the K - t r e a t e d , w a t e r - w a s h e d spores of Table I m a y be a c c o u n t e d for b y an increased acid-labile P in the TCA e x t r a c t s (when a p p e a r s as KING-GOMORI phosphorus). The D N A - P , b a s e d on DISCHE'S t e s t for desoxyribose, was never r e d u c e d b y m o r e t h a n IO % after K t r e a t m e n t a n d was u s u a l l y w i t h i n 2 % of t h e u n t r e a t e d spores. S u r p r i s i n g l y high e s t i m a t e s for t o t a l nucleic acid P were e n c o u n t e r e d on a p p l y i n g t h e u l t r a v i o l e t a b s o r p t i o n p r o c e d u r e to the TCA (SCHNEIDER 8) e x t r a c t s of u n t r e a t e d spores. The u l t r a v i o l e t a b s o r p t i o n , expressed as nucleic acid phosphorus, could be as m u c h as twice the true p h o s p h o r u s c o n t e n t of the e x t r a c t . A n e x a m p l e of this interference is shown in Table I I . A s e p a r a t e s t u d y r e v e a l e d t h a t a n o n - p h o s p h o r u s s u b s t a n c e with a p e c u l i a r u l t r a v i o l e t a b s o r p t i o n s p e c t r u m was e x t r a c t e d b y hot TCA from whole resting spores a n d c o m p l e t e l y m a s k e d t h e p u r i n e - p y r i m i d i n e s p e c t r u m . This interfering substance could be r e a d i l y r e m o v e d or e x t r a c t e d into cold TCA if t h e spores were first d i s r u p t e d , h e a t killed, g e r m i n a t e d or (as i n d i c a t e d in Table II) acid h y d r o l y s e d . A s u b s t a n c e w i t h a similar a b s o r p t i o n s p e c t r u m to t h a t e n c o u n t e r e d here has been r e p o r t e d b y POWELL AND STRANGE17 in the spores of a n u m b e r of bacteria. POWELLxs has identified this m a t e r i a l as t h e calcium salt of dipicolinic acid. T h e s p e c t r u m of this m a t e r i a l is also e v i d e n t in u l t r a v i o l e t studies of spore suspensions (MoROWlTZ19). ReJerences p. 355.

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ACID

HYDROLYSIS

OF

SPORES

349

Fig. I. Resting spores hydrolyzed in bulk for 20 minutes with permanganate-nitricsolution (K treated) washed twice with water and stained with crystal violet. All but one spore in the upper left corner have differentiated into a structure with a central core and side body. TABLE II THE BOUND PHOSPHORUS OF ACID-PERMANGANATE HYDROLYZED (I~-TREATED) SPORES, WATER AND SODIUM ACETATE WASHED (SHOWING THE EFFECT OF THE TREATMENTS ON THE ESTIMATION OF NUCLEIC ACID P BY ULTRAVIOLET ABSORPTION)

mg P/lOO g spores K treated spores Untreated spores

TCA Extract (SCHNEIDER8) Total P u-v absorption as nucleotide P RNA-P (orcinol) DNA-P (diphenylamine) TCA Residue-P

Water washed

Sodium acetatewashed

476

38o

250

lO2O 4o0 9o

144 26o 95

76 14o 46

15o

75

45

After hydrolysis ( " K " or HCI t r e a t m e n t ) the ultraviolet s p e c t r u m of the TCA extracts was p r e d o m i n a n t l y t h a t of purines a n d pyrimidines. B u t the ultraviolet absorption expressed as nucleic acid P (Table II) gave values below the total P of the extracts or the sum of the nucleic acid P based on the sugar tests. These discrepancies m a y be readily exp]ained b y the degraded state of the nucleic acid residues left b y the hydrolysis, as well as b y the shift of labile residue P m e n t i o n e d above.

Mild-alkali washing o/acid-permanganate hydrolyzed spores W h e n the K - t r e a t e d spores were suspended in 0.5% sodium acetate solution sufficient in q u a n t i t y (5-1o ml) to elevate the p H of the suspension above 6.5 (the level

Re/erences p. 355. 26

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VOL. 1 4

(1954)

after K treatment and washing was p H 2.o) the side bodies could be removed. Fig. 2 shows the effect on K-treated spores of a 5 minute rinse in sodium acetate. The phosphorus distribution of these spores is recorded in Table I "Sodium acetate washed once". Some 56% of the DNA (DlSCHE11) of the spores has been removed, along with a further 12 % of the original spore RNA. Fig. 3 shows that two rinses in sodium acetate completely dissolved the side bodies, leaving a spore residue containing a basophilic core. The ultraviolet absorption spectrum of the acetate extracts indicated the presence of a nucleoprotein. The phosphorus distribution of the K-treated spores after two washes with sodium acetate is also shown in Table I. Besides removing the entire spore DNA, the two

Fig. 2. K - t r e a t e d spores after a s h o r t rinse (5 m i n u t e s ) in o. 5 % s o d i u m acetate. T h e side bodies are p a r t l y dissolved (stained w i t h c r y s t a l violet). Fig. 3. K - t r e a t e d spores after two rinses w i t h 0. 5 % s o d i u m acetate. T h e side bodies are c o m p l e t e l y dissolved l e a v i n g a basophilic c e n t r a l core (stained with c r y s t a l violet).

acetate extractions removed a further 19% of the spore RNA-P and a portion of the TCA-residue phosphorus. This left behind, then, a structure devoid of DNA but still containing some 20% of the original RNA and a small amount of residue P. Further RNA could be removed by extending the acetate extraction beyond the time necessary for the complete removal of the side body. This was associated with some shrinkage of the basophilic central cores shown in Fig. 3. Occasionally, the acetate washing was of insufficient alkalinity to raise the p H above 6.5. In these cases, although the side body did not appear to be altered in basophilia or size, some 5 ° % of the DNA was extracted. Little or no drop in the acid-labile TCAresidue P occurred with such partial treatment. On being rewashed with fresh acetate, however, the spores lost their side bodies together with the remaining DNA and the remaining alkMi-soluble part of the residue P. These chemical and cytological effects of K treatment and acetate extraction on Re/erences p. 355.

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the bound phosphorus fractions of B. cereus spores are summarized graphically in Fig. 5. The acid-labile part of the non-nucleic-acid P (non NA-P) includes the acidlabile P present both in the TCA extracts and the TCA residue. The acid-stable P is acid and alkali-insoluble material associated with the spore coats. The changes recorded in Fig. 5 are the averages of several studies.

Fig. 4. K - t r e a t e d spores after e x t r a c t i o n of nucleic acids w i t h 5 % TCA (9 o° C, 15 min), w a t e r w a s h e d a n d s t a i n e d w i t h c r y s t a l violet. T h e side b o d y a n d c e n t r a l core, b o t h slightly c o n t r a c t e d , are still stainable. WHOLE

SPORES

ACID PERM~NOANATE HYDROL/SED SPORES wafer washed sodium acetate washed

~. o.35;!

a: ~D

~ 030 O25 020

g o.f5

~c

o.1o 0.05

Fig. 5. A s u m m a r y of t h e effect of nitric a c i d - p e r m a n g a n a t e h y d r o l y s i s (K t r e a t m e n t ) a n d s u b s e q u e n t w a s h i n g w i t h 0. 5 o~ s o d i u m a c e t a t e on t h e b o u n d p h o s p h o r u s of B. cereus spores.

To investigate further the dissolution process of the side body, some electron microscopic studies were made. Fig. 6 ,"C" to "G" shows the changes in electron density as the side body is dissolved. These include a mushroom-like swelling and the gradual disappearance of the side body. The stump, or neck, on the central core is indicated in Fig. 6G.

Hydrolysis with hydrochloric acid The effect of the familiar Feulgen hydrolysis on the phosphorus fractions of resting spores and the further effect of 0.5 % sodium acetate extraction on Feulgen hydrolyzed Referesces p. 355.

352

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Fig. 6. Electron m i c r o g r a p h s showing tile effect of acidp e r m a n g a n a t e hydrolysis (K t r e a t m e n t ) and s u b s e q u e n t extraction with 0. 5 % s o d i u m acetate on the resting spores of B . c e r e u s (magnification as indicated in G). A. u n t r e a t e d resting spores. B. K-treated resting spores (from the same lot as [Zig. r). C to G. K-treated spores extracted with sodimn acetate showing the dissolving side body. The a r r o w s in G p o i n t to the necks left on tile central core.

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TABLE III T I l E P H O S P H O R U S D I S T R I B U T I O N OF F E U L G E N H Y D R O L Y Z E D SPORES,

WATER AXD SODIUM ACETATEWASHED (pH 7.8) Phosphorus as mg/Ioo g spores Unlreated spores

Total P in HC1 Total P in Acetate washes

HCI hydrolysed spores Water washed

Acetate washed (2 ×)

567 --

565 ii

39 12

15 3°

3°

645 non-nucleotide interference 56o 77

141

135

59 57 72

56 57 66

143

I io

112

--Method of SCHNEIDER8

Fractionation o] residues

Acid-soluble P Lipid P TCA Extracts Total P u.v. absorption as P RNA-P (orcinol) DNA-P (diphenylamine) Residue P Method

IO

o f S C H M I D T AND T H A N N H A U S E R 9

Supernatant o / " S and T "

Total P Inorganic P (phosphoprotein)

653 44 609

217 44 173

207 44 163

TCA Extract of D N A precipitate Total P Diphenylamine-colour P

80 78

3 o

2 o

"S and T " Residue P

3°

27

26

R N A - P + Excess P*

* Excess P, largely present as 7' acid-labile P. Studies describing this material are being published separately. "S and T", by the method of SCHMIDTAND THANNHAUSER.

spores (side bodies not removed) were compared with the above p e r m a n g a n a t e - n i t r i c studies. Cytologically, the spores hydrolyzed in bulk with HC1 were all differentiated a n d similar in appearance to the K - t r e a t e d spores (Fig. I). The chemical effects of Feulgen hydrolysis on the phosphorus fractions of B . cereus spores are shown in Table I I I . The HC1 t r e a t m e n t had altered the R N A so t h a t 90% of it was no longer protein-bound. Some 25 % of the TCA-residue P h a d also been removed from its n o r m a l position. The D N A - P , however, although non-recoverable as a p o l y n u cleotide in the SCHMIDT-THANNHAUSER fractionation, was n o t significantly altered when estimated b y the d i p h e n y l a m i n e reaction (method of SCHNEIDERS), Unexpectedly, the genera] appearance of the crystal-violet-stained K - t r e a t e d a n d HC1 hydrolyzed spores was n o t m a r k e d l y altered when the nucleic acids were r e m o v e d b y hot 5 % TCA. Fig. 4 shows a group of K (or HC1) t r e a t e d spores extracted with hot TCA, washed a n d s t a i n e d with crystal violet. A large part of the side b o d y remains, b u t it now stains a reddish colour similar to the central core. Stains more specific t h a n crystal References p. 35.5.

27

354

p . c . FITZ-JAMES, C. F. ROBINOW, C. H. BERGOLD

VOL. 14 (I954)

violet for DNA (SO,-azure A and SO2-thionine, after DELAMATER2°),although effective in staining the side body before were not taken up after hot TCA extraction by these hydrolyzed preparations. The acid dye Ponceau de xylidene R R (o.I % in o.I N HC1 for I hour) only faintly stained the side bodies of TCA extracted, K-treated spores but stained deep red those of TCA extracted Feulgen hydrolyzed spores. DISCUSSION Acid hydrolysis of resting spores was accompanied b y extraction and degradation of phosphorus compounds. On studying the effect of acid hydrolysis (N HC1, 60 ° C) on vegetative cells, VENDRELY AND LIPARD¥ ~ found that, in addition to completely extracting the RNA, some purines were sprit from the DNA. The results recorded here indicate that similar changes in the nucleic acids of spores occur on acid hydrolysis. Thus, there is no reason to suppose that spore nucleic acids are more resistant to hydrolysis than those of vegetative cells. Two different hydrolysis systems were studied. One of these, a N/3 nitric acid solution with added potassium permanganate (o.I %) and carried out at room temperature, produces a basophilic side body which can be selectively removed b y adjusting the p H to neutrality. The other, the usual Feulgen hydrolysis (N HC1 at 60 ° C), produces a side body resistant to mild alka]i extraction. These differences were found to be related to the presence or absence of permanganate in the acids used (RoBINOWS). Although the hot HCI hydrolysis did remove more RNA and less of the residual labile phosphorus than the room temperature nitric-permanganate hydrolysis, there is no evidence that these chemical effects are related to the difference in solubility of the side bodies (and DNA remnant) in mild alkali solutions. It would be more reasonable to assume that the protein of the side body, possibly the spore histone, had been more markedly damaged b y the permanganate hydrolysis. This assumption is supported by the observation that side bodies of permanganate hydrolyzed spores do not stain with an acid dye, while those of HCl-treated spores do stain. When the p H was raised to above 6.5 the complete removal of the side body was accompanied by a total extraction of desoxyribose. This suggests that the "peripheral nucleus" (RomNow 5) contains all the degraded DNA. The electron micrographic appearance of the side body, the ultraviolet absorption spectrum of the acetate extract and the stained appearance of the TCA residue of K-treated spores all indicate that this side body also contains protein. The extraction b y acetate of an acid-labile residue P coincidental with the removal of the side body suggests this substance as another possible component of spore chromatin. Recent studies applying the techniques of thin sectioning (ROBINOW4) and disruption in fixatives (FITZ-JA~tES 22) have revealed the chromatin of the resting spore as a band or layer of variable thickness surrounding an RNA-containing central m~ss. When these sectioned or disrupted spores are acid hydrolyzed, the chromatin, like that of the vegetative cell, is not apparently shifted. Thus, the extrusion of the spore chromatin as a side body during hydrolysis depends, at least, upon the presence of an intact spore coat. SUMMARY Combined chemical (phosphorus) and cytological studies on spores hydrolyzed at room temperature with a nitric acid-permanganate solution followed by dilute alkali washing (pH 7.8) have supported the suggestion (ROBINOWS; BISSET AND HALE, I95I ) that the "peripheral body" of Re[erences p. 355.

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ACID HYDROLYSIS OF SPORES

355

hydrolyzed spores is nuclear in origin. The selective dissolution of the basophilic side body of such hydrolyzed spores was accompanied by the removal of all the spore DNA (as measured by the DISCHE diphenylamine reaction) along with 15 to 20% of the RNA. Combined cytological and chemical evidence indicate t h a t protein and possibly a labile phosphorus material also constitute spore chromatin. Both the DNA remnant and the peripheral nucleus of Feulgen hydrolyzed spores remained with the spore on washing at p H 7.8. Both methods of hydrolysis degrade and extract phosphorus compounds, but to different degrees. RI~SUMt~ Des dtudes chimiques (phosphore) et cytologiques simultan~es sur des spores hydrolys~es la temperature ordinaire par une solution acide nitrique-permanganate puis lav~es par un alcali dilud (pH 7.8) corroborent l'hypoth~se (RoBINOWS; BISSET ST HALE, 1951) selon laquelle le "corps p~riph~rique" des spores hydrolys~es serait d'origine nucl6aire. La dissolution sdlective du corps basophile de spores ainsi hydrolys~es s'accompagne de l'~limination de la totalit~ du DNA des spores (ddtermin~ par la r~action de DISCHE ~t la diph6nylamine) et de 15 ~ 2o % du RNA. Des indications cytologiques et chimiques associ6es montrent qu'une prot~ine et peut-Stre 6galement un produit phosphor~ labile constituent la chromatine des spores. Le reste du DNA et le noyau p~riph~rique de spores hydrolys~es au Feulgen restent dans la spore apr~s lavage ~ p H 7.8. Les deux m6thodes d'hydrolyse d6gradent et extraient des corps phosphor~s mais ~ des degrds diff6rents. ZUSAMMENFASSUNG Gleichzeitige chemische und cytologische Studien an Sporen yon B. cereus, die bei Raumtemperatur mit einer L6sung yon Salpeters~ure-Permanganat hydrolysiert und anschliessend mit verdfinnter alkalischer L6sung (pH 7.8) gewaschen wurden, unterstiitzen die Annahme (RoBINOW~; BlSSET OND HALE, I95I), dass der "periphere K6rper" der hydrolysierten Sporen vom Kern herstammt. Gleichzeitig mit der selektiven Aufl6sung der basophilen Seitenk6rper solch hydrolysierter Sporen verschwindet alles DNA der Sporen (gemessen durch die DlSCHE Diphenylamin Reaktion) zugleich mit 15-2o %' der RNA. Ferner finden sich chemische und cytologische Beweise dafiir, dass auch das Chromatin der Sporen aus einem Protein und m0glicherweise einer labilen Phosphorverbindung aufgebaut ist. Nach Feulgen hydrolysierte Sporen enthalten nach Waschen bei pH 7.8 sowohl einen Rest an DNA wie auch den peripheren Kern. Beide Hydrolysierungsmethoden erniedrigen und extrahieren Phosphorverbindungen, jedoch in verschiedenem Ausmass. REFERENCES 1 B. STILLE, Arch. Mikrobiol., 8 (1937) 125. 2 B . DELAPORTE, Thesis, Paris, 1939. 3 C. F. ROBINOW, Proc. Roy. Soc. B, 13o (1942) 299. 4 C. F. ROBINOW, J. Bact., 66 (1953) 3oo. 5 C. F. ROBINOW, J. Gen. Microbiol., 5 (1951) 439. 6 p. C. FITZ-JAMES, Exp. Cell Res., Suppl. 2 (1951) 343. 7 j . W. HOWlE AND J. CRUIKSHANK,J. Path. Bact., 2 (194 o) 235. s W. C. SCHNEIDER, J. Biol. Chem., 161 (1945) 293. 9 G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., i 6 i (1945) 2 9 3 . 10 W. MEJBAUM, Hoppe-Seyl. Z., 258 (1939) 117. n Z. DlSCHE, Mihrochemie, 8 (193 o) 4. 12 A. E. MIRSKY AND A. W. POLLISTER, Proc. Nat. Acad. Sci., 344 (1942) 281. 13 j . E. LOGAN, W. A. MANNELL AND R. J. ROSSlTER, Biochem. J., 51 (z952) 47 o. 1~ E. J. KING, Biochem. J., 26 (1932) 292. 15 G. GOMORI, J. Lab. Clin. Med., 27 (1941) 955. 16 L. ERNSTER, R. ZETTERSTROM AND O. LINDBERGH, Acta Chem. Scand., 4 (195 °) 942. 17 j . F. POWELL AND R. E. STRANGE, Biochem. J., 54 (1953) 205. 18 j . F . POWELL, Biochem. J., 54 (1953) 2 1 o . 19 H. J. MOROWlTZ, Arch. Biochem. Biophys., 47 (1953) 325 • 20 F. D. D1~LAMATER, Stain Technol., 26 (1951) 199. 81 R. VENDRELY AND J. LIPARDY, Compt. rend., 223 (1946) 342. 22 p . C. FITZ-JAMES, J. Bact., 66 (1953) 312. R e c e i v e d F e b r u a r y 9 t h , 195 4