BAILLIE, A. et al. (1967). J . appl. Bact. 30 (2), 312-316.

Some Preliminary Observations on the Location of Esterases in Bacillus cereus

ANN BAILLIE, R. 0. THOMSON, IRENE BATTY AND P. D. WALKER Wellcome Research Laboratories, Beckenham, Kent, E ~ l ~ n d

(Received 24 November, 1966)

SUMMARY. located within the bacterial cell during different stages of the growth cycle. significance of the results is discussed.

The enzymes of Bacillus cereu8 capable of hydrolysing thiolacetic a.cicl were The

PREVIOUS INVESTIQATIONS on the esterases of bacteria have in general made use of the technique of gel electrophoresis. Analysis of the enzymes by this technique has yielded valuable information of two kinds. First, the technique of esterase analysis, in which comparison is made of the electrophoretic patterns produced by the esterases of different strains and species of bacteria has provided valuable taxonomic data. In particular the technique has been used for the identification of Bacillus thuringiensis (Norris & Burgess, 1963 ; Norris, 1964), mycobacteria (Cann & Willox, 1965; Cann, Hobbs & Shewan, 1966), streptococci (Lund, 1965) and vibrio species (Willox & Shewan, 1963). Secondly, gel electrophoresis of disintegrates of B. cereus during various stages of the growth cycle has revealed the changing pattern of enzymes during sporulation and germination (Baillie & Norris, 1963; Lund & Norris, 1963). Asingle esterase band was demonstrated in young cultures of B. cereu8 and the develop- ment of a second band was noted after 1 1 h growth. No esterase was demonstrated in mature spores.

A somewhat different approach using alternative techniques has been used by other workers ; Sierra (1963) using a manometric technique obtained indirect evidence of esterase activity in ruptured spores of B. subtilis. Roberts & Rosenkrantz (1966a) demonstrated acetyl esterase activity is spores of B. cereus. This was later extended to the demonstration of lipase and esterase activity in intact spores of B. coagulans using titrimetric and fluorometric techniques (Roberts and Rosenkrantz, 19663).

Although these methods have yielded much useful information about the enzymes, they give little indication as to the location of the enzymes in the bacterial cell. The recent advances in the field of enzyme electron histochemistry (Pearse, 1963; van Iterson, 1965) provide new tools for accurate location of enzymes in biological material. In the present paper some preliminary observations on the location during various stages of the growth cycle of the enzymes in B. cereus, which hydrolyse thiolacetic acid, are presented.

Materials and Methods Organisms and methods of culture

Two strains of B. cereus, Strain M8 (Mahmoud, 1955) and B. cereus var. terminalis were used in the present study.

312

Location oi'esterases in B. ccrcus 313

Xporulation The organisms were grown in the sporulation medium of Young (1958) to give synchronous sporulation. A young overnight culture was inoculated into the sporu- latiori medium and shaken at 30". Samples were removed for test a t appropriate periods.

Gerrninutiota A spore suspension was heat shocked by heating to 65" for 10 min, then incubated in nutrient broth containing 5 mDr L-alanine a t 30". Samplos were removed after 5 min, 1 hand 2 h .

Esternse activity All samples received the same treatment. After centrifugation the deposit was washed several times in physiological saline to free it from the growth medium and finally resuspended in the incubation medium of Barnett & Palade (1959) consisting of thiolacetic acid and lead carbonate in a cacodylic acid buffer, pH 6, and incubated a t 37". Controls were included in which either the thiolacetic acid or the lead carbonatc was excludod f?om the medium. A black colouration developed rapidly in the mixtures containing both thiolacetic acid and lead carbonate due to the formation of lead sulphide No colouration developed in the controls. After incubation for 10 min the mixtures were centrifuged, washed twice in saline arid the deposits processed for electron microscopy.

Electron naicroscopy The deposits w(:ro fixed for 18 h in the fixative of Kellenberger, Rytcr & SBchaud (1958) and embedded in Maraglass (Freeman & Spurlock, 1962). Sections were cut on an LKB ultratome (L.K.B., Anerley Rd, London S.E.20), and collected on 200 mesh Formvw coated grids. The specimens were examined in a Phillips EM200 microscope (Phillips, Eindhoven, Holland) a t 60 kV.

Results Identical results ere obtained with both strains.

Xporulation Young vegetative cells incubated in the reaction mixture showed intense deposits of lead sulphide along the cytoplasmic membrane, the membranes of the mesosomes, in the cytoplasm and in the nuclear areas (Plate 1). No deposits were seen in the control (Plate 2 ) . With the advent of sporulation additional deposits of lead sulphide were seen along the developing exosporium, the spore coat and in the cyto- plasm of the immature spore (Plates 3, 4, 5 & 7) . At this stage the cytoplasmic membrane of the vegetative cell showed fewer deposits. With the maturation of the spore, deposits of lead sulphide could no longer be found in the cytoplasm but remained on the surrounding membranes (Plates 3, 4, 5 8: 6). Again, no deposits were seen in the control preparations (Plates 8 &, 9).

D

314 Ann Baillie et al.

Germination Mature resting spores showed no deposits of lead sulphide (Plate 10). lnimediately after germination areas of lead sulphide were again evident. Some spores showed a discrete pattern on the cytoplasmic membrane and on the membrane adjacent to the spore coat (Plate l l) , whilst others showed, in addition, large deposits in the cytoplasm, on the mesosome structures of the developing cell and in the cortex (Plates 12 & 1 3 ) . Again, no deposits were seen in the control preparations (Plates 14 & 15).

Discussion Esterase activity as judged by the deposition of lead sulphide was clearly demon- strable during various stages of growth. In young vegetative cells activity is closely associated with the cytoplasmic membrane and the membranes of the mesosomes. With the onset of sporulation additional activity is associated with the developing membranes of the spore ; mature spores show no enzymic activity, but activity immediately reappears on germination and is associated with the developing cyto- plasmic membrane of the vegetative cell and the membrane adjacent to the spore coat. In considering those deposits of lead sulphide in tho cytoplasm of vegetative cells and of germinating spores, mliich do not appear to be associtttcd with morphological structures, it should be remembered that their formation is probably the result of action of soluble enzymes and therefore conclusions as to exact locations are difficult. The same criteria apply to the deposits found in the cortex during germination, although the dissolution of the c o r t a during this phase is likely to be accompanied by enzyme activity. Esterase activity in young vegetative cells is seen in the cytoplasmic membrane, a structure likely to be active in the exchange of materials between the medium and the cell, and in the membranes of the mesosomes where similar conditions prevail. During sporulation it is the developing membranes of the spore which show most activity. It is known that cells committed to sporu- Iation will do so even when washed free from the growth medium. This fact suggests a change in the chief site of biochemical activity from the membrane of the vegetative cell to the membranes of the spores developing within the sporangium. The location of esterases, as shown by this method, corresponds closely to the location of succinic dehydrogenase in vegetative cells of Eschekhia coli and B. subtilis (Sedar 85 Burde, 1965a, b ) .

The problems associated with the determination of the exact location of enzymes must always be borne in mind. In the earlier work in which formazan deposits were used as markers, the solubility of the formazan deposits in lipid granules in bacterial cytoplasm gave rise to a false impression of the location of enzymes (Weibull, 1953). Migration of deposits of lead sulphide is unlikely. Some of the larger deposits seen in the present investigation are undoubtedly due to over-incubation. It has been pointed out by Pearse (1963) that the precise location of enzymes is possible even when there is considerable loss of activity, and that it is desirable therefore to carry out further work both on whole organisms and on thin sections after fixation.

I n considering the present observations in relation to the previous results of Baillie & Norris (1963) using gel electrophoresis, it is tempting to suggest that

Location of' cs terases in B. cereus 3 15

their sccoiid band of erizyniic activity appearing after 11 11 arid persisting during sporulation, results from the increased activity we have shown associated with the developing spore membranes. We were unable to detect any esterase activity in mature spores by this method

We should like to thank Mr. J . Short for his invaluable technical assistance in the preparation of mtttrrittl for examination aiid for the electron micrographs.

References BAILLIE, ANN C XUHI~IS, J . H . (1963) . Study of onzymc: changes during aporulation irr Brwi1lu.s

cereus using starch go1 electrophoresis. J . appl. Buct. 26, 102. BARNETT, R . J . & FALADE, G. E . (1959). Enzymatic activity in the bl band. J . biophys. biochem.

Cytol. 6 , 1 6 3 . CANN, D. C., HOBBS, G. & SHEWAN, J. M. (1966) . The identification o f certain Mycobacteriu?r~

species. In Irlenbijica,/ioii Methods for Microbiologists. Part A . Ed. B. M. Gibbs & F. A. Skinner. London : Acadomic Press.

CANN, D. C. & WILLOX, MARGARET, E. (1965). Analysis o f multimolecular enzymes as an aid to the identification of certain rapidly growing mycobacteria, using starch gel electrophoresis. J . appl. Bact. 28, 165.

FREEMAN, J . A. & SPIJRLOCK, H. 0. (1962) . A new epoxy ombodmerit for electron microscopy. J . cell. Biol. 13, 437.

VAN ITERSEN, W. ( l % 5 ) . Symposium on the fine structure and replication of bacteria and their parts. 11. Hackrial oytol)laam. Bact. IZeu. 29, 299.

KELLENBERQER, E., RBTER, A . & S ~ C H A U O , J. (1958) . Electron microscope study o f DNA-coil- taining plasms. [I. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in clii'ferent physiological statea. . I . biophys. biochem. Cytol. 4 , 67 1.

LUND, BARBARA, M. (1965). A comparison by the we of gel electrophoresis of soluhlc protein compontmts anti estcrase enzymes of wjme Group 1) streptococci. f . yet&. Mierobiol. 40, 413.

LUND, BARBARA M . C NORRIS, .J. R. (1963) . Enzyme arid antigen changes during germination of bacterial spores. In I'roceediugs of the Inieributiorial Synaposium o n Physiology, Ecology and Biocheniuistry of Gc~rniiiiotioii. Grcqswald, E . Germuwy.

NAHMOUD, S. A. Z. (1955) . A study of sporeformers occuring in soil. Their germination and biochemical activity. Thesis, University of Leeds.

NORRIY, J. R. (1964) . The classification o f Bacillus th~urinqiensis. J . uppl. Buct. 27, 439. NORRIS, J. K. & BURGESS, H. D. (1963) . Esterases of crystalliferous bacteria pathogenic for insects:

epizootiological application. < J . Insect Path. 5 , 460. PEARSE, A. G. E. (1963) . Some aspects o f the localization of enzyme activity with tho electron

microscope. JZ R. microsc. SOC. 81, 107. ROBERTS, T. L. & ROSENKRANTZ, H. (1966a) . dcetyl esterase in Bacillus cereus spores. C'un. '1.

Biochem. Physiol. 44, 671. ROBERTS, T. L. & ROSENKRANTZ, H. (19666). Lipase and acetyl esterase activities in intact Bu.cillurv

coaguluns spores. Curr. J . Biochem. Physwl. 44, 677. SEDAR, A. W. & BuRISE, It. M. (1965a) . Localisation of the succinic dehydrogenesc system in

Escherichiu coli tising romhined techniques of cytochemistry and electron microscopy. J . cell Biol. 24 ,285 .

8EDA4R, A. W. & BLJROE, K. 11.2. (19656). The demonstration of the succinic dehydrogenase system in Bacillus subtilis using tetranitro-bluo tetrazolium combined with techniques of electron microscopy. J . cell Biol. 27, 53.

SIERRA, G. (1963) . Esterase in Bacillus subtilis spores and its release by ballistic disintegration, Can. ,I. Microbiol. 9, 643.

WEIBULL, C . (1953) . Observations on the stairiiiig ( .~fBnci l lus rnegaterium with triphenyltetrazolium. ,T. Buct. 66, 137.

WILLOX, MARGARET E. & SHEWAN, J . RI. (1963) . Aiinual Report, Torry Research Station, Ahercleen. YOUNG, I. ELIZABETH (1958) . Chemical and niorphological changes during sporulation in variants

of Bacillus cereus. Thesis, University of Ontario, Caiiada.

316 Ann Baillie et al.

Abbreviations used: CM, cytoplasmicmembrane ; M, mesosomes ; SC, spore coat ; E, exosporium ; N, nuclear material ; SCY, spore cytoplasm; C, cortex; COM, cortical membrane; VM, vegetative membrane.

Section of young vegetative cell of 3. cereus after incubation in the reaction medium. The deposits of lead sulphide on the cytoplasmic membrane, on membranes of the mesosomes, and in the cytoplasm indicate sites of esterase activity ( x 61,000).

Section of young vegetative cell of 3. ceTeu8 after incubation in t,he reaction medium without thiolacetic acid. There are no deposits of lead sulphide ( x 82,000).

Sections of sporulating cells of B. cereus after incubation in the reaction medium. The deposits of lead sulphide seen on the cytoplasmic membrane, the membranes surrounding the spore and in the spore and bacterial cytoplasm indicate sites of esterase activity. (Plate 3 x 61,000; Plate 4 x 76,250; Plate 5 x 50,000; Plate 6 x 82,000; Plate

Sections of sporulating cells of B. cereus after incubation in the reaction medium without thiolacetic acid. There are no deposits of lead sulphide (Plate 8 X 82,000; Plate 9 x 46,000).

Section of ungerminated spores of B. cereus after incubation in the reaction medium. There are no deposits of lead sulphide, which indicates that there is no esterase activity ( x 73,000).

PLATES 11, 12 & 13. Sections of germinating spores of B. cereus after incubation in the reaction medium. The deposits of lead sulphide in the vegetative membrane, on the membrane adjacent to the spore coat, in the cytoplasm of the developing vegetative cell and in the cortex indicate renewed esterase activity (Plate 11 x 110,000; Plate 12 x 82,000; Plate 13 x 81,000).

Sections of germinating spores of B. cereus incubated in the reaction medium without thiolacetic acid. There are no deposits of lead sulphide (Plate 14 x 110,000; Plate 15 ~ 7 6 , 2 5 0 ) .

PLATE 1.

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PLATES 3, 4, 5, 6 & 7.

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