BAILLIE, ANN & WALKER, P. D. (1968). J . apppl. Bact. 31, 114-1 19.

Enzymes of Thermophilic Aerobic Sporeforming Bacteria

ANN BAELIE* AND P. D. WALKER Wellcome Research Laboratories, Langley Court, Beckenhum, Kent, England

(Received 7 September 1967)

SUMMARY. Esterase patterns of 217 strains of thermophilic aerobic sporeforming bacteria were examined by starch gel electrophoresis. Classification of these strains by this method corresponded closely with the results obtained by biochemical tests. Protein patterns were also examined by acrylamide gel electrophoresis. I n order to study the heat resistance and other properties of the esterases, an attempt was made to purify them using high voltage carrier-free electrophoresis.

ANALYSIS OF the starch gel electrophoretic patterns produced by the esterases of different strains and species of bacteria has provided information valuable to the taxonomist. This technique has been used in the classification of Bacillus thuringiensis (Norris & Burgess, 1963; Norris, 1964), Vibrio spp. (Willox & Shewan, 1963), myco- bacteria (Cann & Willox, 1965; Cann, Hobbs & Shewan, 1966), streptococci (Lund, 1965) and corynebacteria (Robinson, 1966). The 230 strains of B. stearothermophilus, classified by Walker & Wolf (1968, in preparation) by normal biochemical tests, have been subjected to 'esterase analysis' to determine whether the method could be applied to this group. In addition, the protein patterns have been studied by acrylamide gel electrophoresis, with a view to obtaining additional taxonomic data.

Apart from looking a t the esterase pattern by starch gel electrophoresis, it was decided that a study of heat resistance, substrate specificity and other properties of the enzymes would yield useful information. Prior to these studies an attempt was made to partially purify the enzymes. This was done by preparative high voltage carrier-free electrophoresis. The results described here are of a preliminary nature and i t is hoped to extend them a t a later date.

Materials and Methods Organisms

The strains of thermophilic aerobic sporeformers included in this survey are those described by Walker & Wolf (1968, in preparation). Of the 230 strains described by these workers, 13 were not available for examination-one from group la , 3 from group lb , 1 from group Id, 1 from group 2, 2 from group 3a, 3 from group 3b and 2 from group 3c.

Cultures Strains were grown in Roux bottles either on Oxoid Nutrient Agar or in Oxoid Nutrient Broth. Cultures were incubated a t 55" for 6-8 h. Cells were harvested and washed 3 times in saline prior to disintegration. *Present address: Department of Microbiology, University of Aberdeen, Scotland.

[I 141

Enzymes of thermophiles " 5

Disintegration Washed cells were resuspended in a minimal volume of saline and subjected to ultrasonic vibration using the M.S.E. Ultrasonic Disintegrator (M.S.E. Ltd., Spencer Street, London, S.W.1). The cell disintegrates were clarified by centrifugation and stored at -20".

Low voltage electrophoresis Disintegrates were examined by electrophoresis both in starch and acrylamide gels. Starch gel electrophoresis and the detection of esterase activity was carried out &s

described by Baillie & Norris (1963), and acrylamide gel electrophoresis and the location of proteins according to the method of Baillie (1967).

Preparative high voltage electrophoresis Disintegrated cell suspensions were separated in the Elphor VAP 2 (Northern Media Supply Ltd., Blanket Row, Kingston upon Hull). This instrument allows continuous separation of components of a protein solution such as a cell-free disintegrate of bacteria or of particulate matter by the use of high voltage applied to a free buffer a m . The separation chamber is arranged vertically (Fig. 1) and synchronization of the

Anode

Buffer feed into top of chamber

------ L i 4 4 1 C -

Cathode

Separated samples run off from 50 collection tubes

Fig. 1. Diagram of separating chamber of Elphor VAP2. I, sample input points; T, temperature probe; S, separating sample.

buffer feed pump and the buffer run off pump ensures that the buffer film travels through the chamber a t a uniform speed. A small dosing pump enables the material to be fed into the chamber at one of 8 possible points, and the temperature of the chamber is controlled by means of a small resistance thermometer in the buffer film. Cooling is effected by means of 30 Peltier batteries situated behind the separating chamber. The latter consists of two sheets of plate glass, measuring 48 x 48 cm and held 0.5 mm apart with the electrodes applied vertically at either side of the chamber. The sample

I 16 Ann Baillie and P. D. Walker

under study is fed in near the top of the chamber and is separated under the high voltage as it is drawn through in the buffer film; it is run off a t 50 points along the bottom of the chamber into collecting tubes held in a cooled trough. The instrument may be operated continuously for several days and can separate 2-5 g dry wt/day. For the separation of esterases, disintegrated material was fed into the chamber a t a rate of 1.5 ml/h with a buffer flow rate of 230 ml/h. Oxoid Barbitone Acetate Buffer (pH 8.6, p = 0.1) was used as electrode rinse buffer and diluted 114 for use in the separating chamber. Protein distribution among the separated samples was estimated by measuring absorption a t 280 mp using the Unicam SP500 spectrophotometer (Unicam Instruments Ltd., York Street, Cambridge). Esterase activity was estimated by diluting the samples with equal volumes of Tris-maleic buffer, pH 6.4, prior to adding 0.5 ml of the stain used for esterases in starch gel. The colour produced was read in a Hilger Absorptiometer B10.1 (Hilger & Watts, 98 St. Pancras Way, London, N.W.l) against a no. 52 filter.

Separated samples found to contain esterase activity were concentrated either by ultrafiltration through a collodion membrane (Membranfiltergesellschaft, Gottingen) or, for larger volumes, by freeze drying.

The voltage was 2kv and the current 200 mA.

Results Esterase analysis

The esterase patterns found in the 217 cultures examined are shown in Fig. 2. Biochemically these strains fall into three groups: group 1, which forms gas anaerobi- cally from nitrate and has a slight action on starch; group 2, which is inactive bio- chemically; group 3, which produce wide zones of hydrolysis in starch agar plates

-

la Ib, Ib, I c Id le 2 3a 3b 3c Fig. 2. Diagrammatic representation of the esterase patterns obtained by starch gel

electrophoresis of extracts of the 217 strains of thermophilio aerobic sporefonners.

(see Walker & Wolf, 1968, in preparation, from which further details of biochemical patterns can be obtained). Group 1 was further divisible into 5 subgroups according to their esterase patterns. Subgroup l a (38 cultures) all had identical esterases.

Enzymes of thermophiles 1'7

Although the 67 cultures of subgroup l b had similar biochemical properties, esterase analysis split them further (lb, and lb2). Seventeen cultures fell into khe former. Of the 10 cultures forming subgroup lc, 9 had a characteristic esterase pattern, the tenth (T112) falling into subgroup lb,. This strain differed from the other 9 by pro- ducing a wide zone of hydrolysis on starch. The 4 cultures comprising subgroup Id were more heterogeneous-2 (T92 and T119) had an esterase pattern identical with that of subgroup 3a, one (T116) fell into subgroup lb,, and one (T127) into lb,. Subgroup le, comprising 3 cultures failing to produce gas anaerobically from nitrate, all shared a characteristic esterase pattern.

The 38 strains in group 2 were very inert biochemically-a fact reflected in the difficulty in detecting any esterase activity in disintegrates. Concentration of the extracts by ultrafiltration through collodion membrane was necessary before esterases could be demonstrated on starch gel electrophoresis. All 38 strains had an identical esterase pattern.

The 46 cultures comprising subgroup 3a shared a common esterase pattern. The two strains of B. calidolactis (Reading) had an est erase pattern (subgroup 3b) which differed from that of B. calidohctis (Galesloot). These 3 strains had the same esterase pattern as the 4 strains of B. thermoliquefaciens (Gales1oot)-subgroup 3c.

h n

- + 0 Insert

Fraction

Fig. 3. Proteins and esterases of group la (T31) separated by preparative high voltage electrophoresis. Shaded area, esterase activity.

I 18 Ann Baillie and P. D. Walker

Heat resistance of esterases The heat resistances of the enzymes were studied by heating extracts a t 60,80 and 100" for 10 min immediately prior to electrophoresis. In general there was no visible effect a t 60", a slight decrease in activity a t 80" and complete destruction a t 100". Results with a typical heat resistance are shown in Plate 1.

Proteins Extracts of all strains were examined for protein components by acrylamide gel electrophoresis. The protein patterns of the different strains fell into the same 3 principal groups as the esterase patterns, but subdivisions within groups 1 and 3 were less evident. Typical gels stained to detect protein are shown in Plates 2 and 3.

High voltage electrophoresis-separation of esterases Extracts of two strains were fractionated by preparative high voltage electrophoresis. The group l a strain (T31) showed 4 peaks of esterase activity (Fig. 3) as did the lb , strain (T62), (Fig. 4). Some separation of the proteins of the extracts was also obtained.

!

h II

I r

Insert Fraction

Fig. 4. Proteins and esterases of group lb, (T62) separated by preparative high voltage electrophoresis. Shaded area, esterase activity.

PLATE I . Photograph of a starch gel stained to detect, esterasc activity. All inserts were estracts of a group l a strain (T31). Inserts (left t o right) wcre extract unheated, extract heated at 60°, 80' arid 100O for 10 min, rmpectively.

Bact. f.p. 118.

PLATE 2. Photograph of an acrylamide gel stained to detect protein. Inserts (left to right) were extracts of strains in groups la (T15), lb, (T47), Ih, (T72), Ic (T101) and Id (T92).

PLATE 3. Photograph of an acrylamide gel stained to detect protein. Inserts (left to right) were extracts of strains in groups le (T107), 2 (T143), 3a (T188), 3b (T208) and 3c (T229).

Enzymes of thermophiles 119

Discussion The results of esterase and protein analysis of the 217 strains of thermophilic aerobic sporeformers examined placed them into essentially the same groups as did more usual biochemical tests used by Walker 6 Wolf (1968, in preparation). The classical strains of B. stearothemnophilus (group 3a) which produce wide zones of hydrolysis on starch agar plates and form nitrite from nitrate without the production of gas all shared the same esterase pattern. The two strains of B. calidolactis from Reading had an esterase pattern similar, to but not identical with, the 3 from Galesloot (Holland). No difference could be detected between the latter strains and those of B. thermolipuefaciens obtained from the same source. The biochemically inert group of isolates from milk and soil (group 2) fell into a single esterase group. Among the gas producing strains (group 1) esterase grouping again followed biochemical grouping with a few exceptions. Subgroup Id proved heterogeneous, 2 strains having the esterase pattern of the classical B. stearothermophilus (subgroup 3a) and 2 falling into subgroup lb.

Esterase analysis by starch gel electrophoresis proved more sensitive than exami- nation of proteins by acrylamide gel electrophoresis.

In addition to using esterase patterns as a taxonomic tool, it would be desirable to have more information on the properties of these enzymes. In order to study them in more detail, purification is necessary. The separations obtained by preparative high voltage electrophoresis are promising, but the results are preliminary in nature. Attempts to concentrate the separated enzymes either by ultrafiltration or by freeze drying have resulted so far in the loss of most of the esterase activity. It is hoped, however, that further work along these lines will yield purified enzyme preparations.

We are grateful to Miss R. Harding, Miss L. Reeve and Miss J. Winton for technical assistance.

References BAILLIE, A" (1967). Antigens of Bacillus cerew: a comparison of a parent strain, an asporogenic

variant and cell fractions. J . appl. Bact. 30, 230. BAILLIE, A" & NORRIS, J. R. (1963). Studies of enzyme changes during sporulation in Bacillw,

cereu8 using starch gel electrophoresis. J . appl. Bact. 26, 102. CANN, D. C., HOBBS, G. & SHEWAN, J. M. (1966). The identification of certain Mycobacterium

species. In Identi$cation Methods for Microbiologists (Part A). Eds F. A. Skinner & B. M. Gibbs. London: Academic Press.

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

LUND, B. M. (1966). A comparison by the use of gel electrophoresis of soluble protein components and esteraae enzymes of some Group D Streptococci. J . gem. Microbwl. 40, 413.

NORRIS, J. R. (1964). The classification of Bacillus thuringiensis. J . appl. Bact. 27, 439. NORRIS, J. R. & BURGESS, H. D. (1963). Esterases of crystalliferous bacteria pathogenic for

ROBINSON, K. (1966). An examination of Cotynebacterium spp. by gel electrophoreeis. J . appl

WILLOX, M. E. & SHEWAN, J. M. (1963). Annual Report, Torry Research Station, Aberdeen.

insects; epizootiological application. J . Insect Path. 5 , 460.

Bact. 29, 179.

AJMAL, M. (1968). J . appl. Bact. 31, 120-123

Growth and Toxin Production of Clostridium botulinum type E

M. AJMAL* Food Hygiene Laboratory, Central Public Health Laboratory,

Colindale, London, England

(Received 1 June 1967)

SUMMARY. Growth and toxin production of' Clostridium botulinum type E was studied at 37, 30, 22 and 4". Incubation at 30" yielded maximum numbers of viable cells but the toxicity was not different from cultures grown a t 22", thus indicating that the toxin synthesis/cell was greater a t the latter temperature. Growth as well as toxin production was less pronounced at 37". The ability to grow at 4" was influenced by the medium and the type of inoculum used. The role of the enzymic activity of the culture during and after the exponential phase of growth is emphasized in relation to the final toxicity of type E cultures in terms of MLD/ml.

ALTHOUGH IT IS generally agreed that an incubation temperature between 22 and 30" is best suited for the detection and isolation of Clostridium botulinum type E (Kushnir, Brun & Paikina, 1937 ; Hazen, 1937 ; Slocum, 1964), precise information regarding its growth and toxigenicity in relation to temperature is inadequate. Ohye & Scott (1957) reported that with type E strains the maximum rate of growth occurred a t 35". The toxin titres were lower at 30 and 37" than at 25" and on occasions cultures grown a t 37" were nontoxic. Pedersen (1955) and Abrahamsson, Gullmar & Molin (1966) concluded that type E strains do, in fact, form toxin a t 37" but the toxin is inactivated during incubation for a few days at that temperature.

The present investigation was undertaken to elucidate the effect of temperature and period of incubation on the growth as well as toxin production by the organism.

Materials and Methods Strain FDA 70 (received from Dr. G. Slocum, Washington) of C1. botulinum type E was used throughout the experiment. The spores wcre prepared in the TPG medium of Schmidt, Nank & Lechowich (1962). Studies were made a t 37, 30, 22 and 4".

The cultures were grown in cooked meat medium (CMM) comprising Hartley's digest broth with veal meat particles, pH 6.8-7.0. Washed spores (100 spores/ml) were used as the inoculum. Growth was assessed by the number of viable cells forming colonies, by the Miles & Misra (1938) technique. Fresh duplicate samples were taken at each sampling time and the mean counts ofthe two samples were recorded.

For toxin production 1% of glucose was added to CMM just before use. After incubation the culture was centrifuged at 3000 rev/min for 30 min and serial dilutions of the supernatant fluid were made in gelatin-phosphate buffer (pH 6.2); 0.4 ml of each dilution was inoculated intraperitoneally into each of two white mice of weight 18-20 g. The highest dilution which killed both the inoculated mice in 48 h was *Present address: Royal Veterinary College, London, N.W. 1, England.

CZ. botulinurn: Growth and toxin production 121

regarded as the titre of the toxin and was expressed as mouse minimum lethal doses (MLD/ml). Trypsin activation of toxin was done as described by Duff, Wright & Yarinsky (1956).

Results There was no significant difference between the turbidities of the cultures grown at the 3 temperatures. The maximum numbers of viable cells occurred in cultures grown a t 30" (Fig. 1). The comparable toxicity results are presented graphically in Fig. 2 and in Table 1 the toxicities of the supernatant fluid and of the washed cellular deposit of the 1 day old culture are given. At all three temperatures, i.e. 37, 30 and 22", the pH of the cultures fell from 6.8 to 6.1 after 1 day and to c. 5.5 after two days, after which it remained almost constant (5 .5) .

7

Period (days) Fig. I Fig. 2

Fig. 1. Growth of CZ. botulinum type E, FDA70, at differont temperatures. Cultures were grown in cooked meat medium. Triangles, 37"; open circles, 30"; closed circles, 22'.

Toxin production by CZ. botulinum type E FDA70 at different temperatures. Cultures were grown in cooked meat medium containing 1 yo of glucose. Triangles, 37"; squares, 30"; circles, 22". Open symbols, untreated supernatant fluid; closed symbols, trypsinized supernatant fluid.

Pig. 2.

At 4", growth occurred in 5 weeks in CMM from a mixed inoculum of vegetative cells and spores, whereas there was no growth in 8 weeks when it was inoculated with spores only. With the addition of 1 yo of glucose to CMM, however, toxic outgrowth occurred from a spore inoculum within 4 weeks ; the toxicities of the crude supernatant fluids did not exceed 25 MLD/ml.

Discussion The results indicate that growth of Cl. botulinum type E occurs better a t 30" than a t 37 or 22". I n a separate study (unpublished) on other type E strains, similar results

I 2 2 M. Ajmal

were obtained; it was also found that about half as many spores germinated a t 37" as a t 30". It is reasonable to assume that the total number of cells produced in cultures grown at 37" would be less than a t 30". Turbidity measurements might give erroneous results because of the unequal rate of lysis and/or sporulation at the two temperatures.

It may be noted that after 1 day incubation, the supernatant fluid from the culture a t 37" was more toxic than that of the culture a t 30"; the cellular deposit of the former, on the other hand, was less toxic than that of the latter. After 2 days, the toxicities of the supernatant fluids of the cultures grown a t 30 and 37" were almost equal whereas that a t 22" was much less toxic. It follows that the toxicity of young cultures grown a t different temperatures would depend on whether only the supernatant fluid or the whole culture was tested.

TABLE 1 Toxicity of 1-duy old cultures of C1. botulinum type E strain FDA70

Toxicity (MLD/ml) of samples from cultures grown

at Material tested

37" 30"

50 10

trypsinized 2.0 x lo4 5.0 x los

50 5.0 X loa

Auid from culture

Washed cells (untreated

trypsinized 5.0 X lo4 2.0 X lo6

suspended in acetate buffer pH 6.0

Cultures were grown in cooked meat medium containing 1 % glucose.

Contrary to the observations of previous workers (Pedersen, 1955 ; Abrahamsson et ul., 1966) there was no evidence of any substantial change in the toxicity of the fluids from cultures incubated a t 37" for 2-14 days. Although the growth of the organism was better at 30" than a t 22", the yields of toxin at the two temperatures were almost equal. Thus, it appears that the total number of cells present in the culture as well as the amount of toxin synthesized/cell are important, and the two factors might be independent of each other. As suggested by Ohye t Scott (1957) it is reasonable to expect that at 37" some of the metabolic activities of the organism may not be fully expressed because this is above optimum temperature.

The toxicity of crude supernatant fluids from cultures submitted to prolonged incubation at 30 and 22" gradually increased. It is unlikely that this increase was due to the synthesis of more crude toxin because the cultures as indicated by the viable cell count reached the decline phase after 5 days (Fig. 1). This is further supported by the evidence that there was no substantial difference between the toxicities of culture fluids trypsinized after 5 and 14 days incubation. Moreover, with longer incubation periods the ratio between the toxicities of trypsinized and crude fluids decreased. It appears that some degree of activation of the toxin precursor occurs

CZ. botulinum: Growth and toxin production '23

during the further incubation period at 30 and 22", this activity being absent or less pronounced in the cultures incubated a t 37".

Dolman (1953, 1957) referred to the unusually potent toxin sometimes produced by a mixed culture comprised of the toxigenic (Tox) and proteolytic (TP) variant of C1. botulinum type E strains. Ajmal & Hobbs (1967) confirmed Dolman's findings and also demonstrated Ghat various colony forms in type E cultures might vary in their toxigenicity; thus, the amount of toxin produced by a given type E culture would be a property of the culture as a whole and may not be judged by testing an individual colony. Although the total numbers of cells remain important, the enzymic activity of the culture both during and after the exponential phase of growth appears to be quite significant in determining the final toxicity of the culture in terms of the MLD/ml.

I express my gratitude to the Nuffield Foundation for the Postgraduate Scholar- ship in Food Science which enabled me to undertake this research programme. I wish to thank Professor D. G . Evans and Dr. Betty C. Hobbs for their guidance, personal interest and constant encouragement.

References ABRAHAMSSON., K, GULLMAR, A. & MOLIN, N. (1966). The effect of temperature on toxin formation

and toxin stability of Clostridium botulinum type E in different environments. Can. J . Microbwl. 12, 385.

The incidence and significance of colonial variation in Cloatridium botulinum type E. In Botulism 1966, p. 459. Eds T. A. Roberts and M. Ingram. London: ChaDman & Hall.

AJMAL, M. & HOBBS, B. C. (1967).

DOLMAN, C. E. (f963). Cloatridium botulinum type E. Atti. vi Cong. Intermz. Microbiol. Roma 4, 130.

DOLMAN, C. E. (1957). Recent observations on type E botulism. Can. J . publ. Hlth 48, 187. DUFF, J. T., WRIGHT, G. G. & YARINSKY, A. (1956). Activation of Clostridium botulinum type E

HAZEN, E. L. (1937). A strain of B. botulinw not classified as type A, B or C. J . infect. Dis. 60,

KUSHNIR, E. D., BRUN, T. M. & PAIEINA, S. S. (1937). The source of Bacillus botulinus infection

MILES, A. A. & MISRA, S. S. (1938). The estimation of the bactericidal power of the blood. J . Hyg.,

OHYE, D. F . & SCOTT, W. J. (1957). Studies in the physiology of Clostridium botulinum type E.

PEDERSEN, H. 0. (1955). On type E botulism. J . appl, Bact. 18,619. SCHMIDT, C. F., NANK, W. K . & LECHOWICH, R. V. (1962)

toxin by trypsin. J . Bact. 72, 455.

260.

in sturgeons. Zh. Mikrobiol. Epidem. Immunobwl, U.S.S.R. p. 19, English abstr. p. 85.

Camb. 38, 732.

A m . J . bwl. Sci. 10, 85.

Radiation sterilization of food, 11. Some aspects of growth, sporulation and radiation resistance of spores of Clostridium botulinum type E. J . Food Sci. 27, 77.

SLOCUM, G . G. (1964). Problems of isolation and identification of C. botulinum. In Botulism: Proceedings of a Symposium a t the Robert A. Taft Sanitary Engineering Centre, Cincinnati, Ohio. Eds K. H. Lewis & K . Cassel. Washington: U.S. Public Health Service Publication No. 999-FP1.