Lecithinase Production by Clostridium Perfringens Grown in Synthetic and Complex Media

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Graduate Student Theses, Dissertations, & Professional Papers Graduate School

1968

Lecithinase production by Clostridium perfringens grown in synthetic and complex media

William Richard Cross The University of Montana

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LECITHINASE PRODUCTION BY CLOSTRIDIUM PERFRINGENS

GROWN IN SYNTHETIC AND COMPLEX MEDIA

William R. Cross

B. A«, Eastern Washington State College, 1966

Presented in partial fulfillm ent of the requirements for the degree of

Master of Science

UNIVERSITY OF MONTANA

1968

Approved by:

Chairman, Board of Examiners

Dean,/Graduate School

may 1 1 I960 D ate

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: EP37625

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

I wish to express xny sincere thanks and appreciation to Dro

Mo Nakamura, Professor and Chairman, Department of Microbiology,

University of Montana, for his advice, guidance, and encouragement

throughout the course of this investigationo I am also indebted to my

wife for her devotion and understanding during the period of this

investigation*

A major portion of this investigation was supported by a research

grant (UI-00291-02) awarded to Dro Nakamura from the National Center

for Urban and Industrial Health, Public Health Service*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TAELE OF CONTENTS

PAGE

Ackn.0wl©d.gcïll©ïï'ts 0000000000000000000000000«00ft000000«000000 ix

XiXSi^ o f TclblLOS qo^i^oooooooooooooooooooooooooooqoooooooooooo XV

LxS t o f Fx^UPOS oeooooooooooooooodoooooooooooooooooooooeooo V

CHAPTER

X o XN 'JXCOOUC TXOH «ooooooooooooooooooooeooooooooooooooeo X

XXo STATEMENT OF TtlE PROBLEM ooodoooooooooooooooooeoooo 12

XXX* METHODS AND MATERIALS aoooooo*ooooo«>o*oooooo0 *0 0 * 0 0 13

XV * RESUL TS 000»«09000000000000000000000000*000000e00*0 2 V

V* DISCUSSION AND CONCLUSIONS ooooooooooooooooooooooooUH

VI* SUMMARY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOQOOOOOOOOOO 36

BIBLIOGRAPHY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 5s

AUTOBXOGRAPHY OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 63

XXX

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

TABLE PAGE

! • Ttie toxins of Clostridiim perfringens oooooooooooooooooooo 2

2o Composition of the medium for lecithinase production ooooo 15

3« Composition of medium NCTC 109 ooooooooooooooooooooooooooo l6

Uo Scheme for performing the lecithovitellln assay «ooooooooo 19

5* Lecithinase activities of e i^ t selected strains of

ClOS^Ua^iUm JQerfin^gene ooooooooooooooooooooooooooeoaooooo 29

6. Lecithinase activity of cell fractions of Clostridium

^erfllngena ooaoeoooooooooooooooooooooooosoooooooooooeooeo 30

7* Effect of £H on commercial lecithinase Ooooaoooooooooooooo 32

So Effect of on the hydrolysis of egg yolk saline by

lecithinase ooooooeooooooooooooooooooooooooooooooooooooooo 33

9# Effect of dialysis on lecithinase activity in protoplasm

of Clostridium nerfringens BP6K oooooooooooooooooooooooooo 34

10* Effect of dialysis on lecithinase in culture filtrates of

Clostridium perfringens EP6K oooooooooooooooooooooooeooooo 36

l i e Effect of selected chemical agents on the activity of

lecithinase oooooooooooooooooooooooooooooooooooooooooooooo 37

12* Peptides that stimulated lecithinase production by

Clostridium perfringens BPéK 00000000000000000000000000000 39

1 3o Peptides that failed to stimulate lecithinase production

by Clostridium perfrlngens BP6K oooooooooooooooooooooooooo 40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

FIGURE PAGE

lo The action of phospholipase C on lecithin oooooooooooooo 4

II» Standard assay curve for the détermination of lecithinase

a c t X V l t y OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOSOOQOOOOOOO»»» 2S

IIIo The effects of jgH on the growth and lecithinase activity

of Clostridium perfringens BP6K ooooeoooooooooooooooeoo» 42

IV» The effects of on the growth and lecithinase activity

of Clostridium perfringens Hobbs 3 o»oooooo<.oooooooooooo 43

Vo The inactivation of lecithinase by heat at 60 C oooooooo 44

VEo The inactivation of lecithinase by heat at 90 C oooooooo 45

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

INTRODUCTION

Clostridium perfringens is an anaerobic, gram positive rod-shaped

bacterium, measuring 2-4 p, in length and Oo8-lo5 in width* It is

nonmotile, encapsulated, and forms central to subterminal spores* Six

types, A-F, are differentiated on the basis of the toxins produced*

Ç.» perfringens was first isolated and named Bacillus aero genes

capsulatus by Welch and Nuttal in 1892 (58)* _C* perfringens is best

known as the primary etiological agent of gas gangrene* Other diseases

caused by C* perfringens are dysentery of newborn lambs, an entero-

toxemic disease of sheep known as "struck", and enteritis necroticans,

an enterotoxemic disease of man* Certain strains of type A JC* per

fringens have been shown by McClung (33) and Hobbs (17) to cause a

mild gastroenteritis in man following the ingestion of food contaminated

with C_* perfringens* Nygren (42) has postulated that the lecithinase of

£« perfringens was directly related to the clinical symptoms of food

p o ison ing*

Numerous toxins are produced by £* perfringens* "Toxins which are

not lethal may be detected by their hemolytic properties or by their

enzymatic activity on a variety of substrates (Table l)*

AH strains of type A JC* perfringens do not produce large amounts

of a-toxin* Hobbs et ai* (18) described isolates from food that pro

duced small amounts of a-toxin* Hobbs* isolates did not produce ©-

toxin and differed from classical type A strains by forming spores that

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. table lo The toxLns of Clostridium perfringens (5l)

Type

Toxin A B 0 D E F Properties

A lpha ♦+ + ♦ + * L e th a l, hemolytic, lecithinase

B eta — - — L e th a l, necrotizing

Gamma - + ♦ — - + L e th a l

D e lta — + + - — - L e th a l, h e m o ly tic

E p silo n - - ♦ - — L e th a l, formed as prototoxin

E ta v ^ L e th a l

T heta + * + 4- L e th a l, hemolytic O2 l a b i l e

I o t a — — - - - L e th a l, formed as prototoxin

Kappa + — * V - L e th a l, collagenase

Lambda - - V — Nonlethal, proteinase

Mu V — V —- Nonlethal, hyaluronidase

Nu ♦ + 4 <*■ * 4 Nonlethal, deoxyribonuclease

^ v a r ia b le

i**variable, rare

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. could remain viable after being heated for 1 hr at 100 Co Weiss and

S tro n g ( 56) have reported tiiat heat-resistant strains of _Co perfringens

produced less a-toxin than did heat-sensitive strainso

Lecithinase activity has been reported in other species of Clos

tridia* Macfarlane (2Ô) studied the lecithinase activity in cultures

of Clostridium oedematlens and Clostridium sordelH i and concluded that

they were distinct from the lecithinase of C.o perfringens on the basis

of the hemolytic action on erythrocytes* Noyse and Easterling (41)

detected a hemolysin that appeared to have properties similar to £*

perfringens lecithinase, but no ensyme-substrate reactions were carried

out*

The mechanism of erythrocyte hemolysis by lecithinase has been

postulated* Macfarlane (29) demonstrated that hemolysis of sheep and

horse erythrocytes was always preceded by the hydrolysis of somei phos

pholipid fractions in the cell stroma, and that the rates of hemolysis

were different in red cells from these animals* Matsuraoto (31) reported

that sheep and bovine red cells contain only small amounts of lecithin

and have sphingomyelin as their major choline-carrying component* On

(the other hand, rabbit erythrocytes, which are readily hemolyzed, con

tain large amounts of lecithin* Presumably, the rate of hemolysis is

dependent on the kind and amount of the phospholipids in the cell

strom a*

In 1 9 4 1, Macfarlane and Knight (30) showed that the a-toxin of

Co perfringens was a lecithinase 0 i^Aiich hydrolyzed lecithin with the

release of phosphorylcholine and a,3-diglyceride (Fig* X)* From these

studies they concluded that the lecithinase of Ç,* perfriniprens was

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o ro ro CD X O CL z + CM X C l O S CM X 5 a. •HCJ o ai c o o o GJco fij o. I— o o o= co o o ÜJ .CÜ

l o o a o I 4-3•rl C.)

X CJ Ht o X I—I o ë

O: o

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. distinct from a phosj^odiesterase because it did not hydrolyze mono=-

phenyl-, diphenyl-, or g-glycerophosphate* Lecithinase was also in active against ribonucleic acid© Macfarlane (27) reported that cephalin

was not hydrolyzed by lecithinase, but that sphingomyelin was hydrolyzed

very slowly* Zamecnik et ai* (60) later reported that perfringens

lecithinase did not hydrolyze a-phosphatidylethanolamine, lysolecithin,

cephalin or sphingomyelin* Macfarlane (26) conclusively demonstrated

that spJhingomyelin was hydrolyzed if the amount of lecithinase was in

creased and the incubation period prolonged* Matsumo to (32), using

bacterial filtrates, showed that lecithinase hydrolyzed lecithin,

sphingonQrelin, and Tween 80* Long and Maguire (25) reported that only

lecithins with unsaturated side chain fatty acids were hydrolyzed by

_G« perfringens lecithinase*

£* perfringens lecithinase has been shown to hydrolyze lecithin

and phospholipids of sim ilar structure* Using the nomenclature of

Z e l le r (6l), the a-toxin of _C* perfringens has been designated phos

pholipase C« This name is indicative of the general class of compounds

that are hydrolyzed by it* Phospholipids, termed phosphatides in some

textbooks, have resulted in the name phosphatidase C for Co perfringens

lecithinase* When associated with Ç,* perfringens. any of the preceding

names refer specifically to the a-toxin produced by this organism*

Calcium ions are essential for enzymatic activity of lecithinase*

Oakley and Warrack (43) reported that the minimal indicating dose of

a-toxin, regardless of the assay system employed, was dependent on the

amount of free ionized calcium available for the reaction* Smith (5l)

regarded the calcium ions as links between the enzyme and substrate*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This conclusion was based on the observation that when lecithinase and

red blood cells were placed in a calcium-free medium, the toxin did not

absorb to the red cells* Macfarlane and Knight (30) reported that the

affinity of lecithinase for lecithin was increased twofold in the pres

ence of calcium ions* Their studies alèo showed that the amount of

calcium ion necessary for maximum reaction velocity was inversely pro

portional to the amount of enzyme present*

Macfarlane and Knight (30), Smith and Gardner (52), Weiss and

S tro n g ( 56), and Card (6) reported that C.* perfringens lecithinase was

relatively heat-stable, losing only one-half of its activity when heated

for 10 min at 100 C* Smith and Gardner (52) reported that when leci

thinase was heated for 10 min at 60 C in the presence of calcium and

phosphate, complete inactivation of the enzyme resulted* However, if

the same solutions were heated to 100 C the enzyme could be reactivated

t o 50% of its original activity* Apparently, toxin molecules reacted

with calcium ion at 60 0 to form an inactive coinplex* Heating at a

higher temperature resulted in dissociation of the complex with the lib

eration of active lecithinase and the precipitation of calcium phosphate»

On the other hand, Kushner (22) reported that crude filtrate material

r e ta in e d 63% of its activity in egg yolk saline after having been heated

for 5 min at 100 C* If subjected to the same treatment, partially puri

fied lecithinase retained only 6% of its activity in egg yolk saline*

Although resistant to heat, lecithinase is easily denatured by

chemicals* Salts of heavy metals, halogen gases, cysteine, glutathione,

thioglycolic acid, hydrogen sulfide, and sodium bisulfite have been

shown to irreversibly inactivate lecithinase (51, 19)« Smith ( 51) h as

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. postulated that the mechanism of inactivation by reducing agents may be

due to the reduction of disulfide linkages that contribute to the struc

tural integrity of the lecithinase molecule® Ispolatovskaya and

Klimacheva (19) reported total inactivation of lecithinase by ferric

ion, cupric ion, cobaltic ion, iodine gas, and ethylenediaminetetra-

acetic acid (EDTA)o Dialysis to remove metal ions or their complexes

failed to restore enzymatic activLtyo

Co perfringens lecithinase can be assayed by several methods®

Weiss and Strong ($6), van Heyningen (55), and Kushner (22) assayed the

activity of lecithinase by measuring the turbidity produced in egg yolk

saline by its enzymatic action (this is known as the lecithovitellin

reaction)® Zamecnik et a^l® (60) assayed lecithinase activity manometri-

cally by determining the amount of carbon dioxide released from a cal

cium carbonate buffer when the products of lecithin hydrolysis were re

leased into the medium® Macfarlane and Knight (30) and Card (6)

assayed lecithinase activity by measuring the amount of acid-soluble

phosphorus produced by the hydrolysis of purified lecithin® Burrows (5)

assayed lecithinase activity by determining the amount of hemoglobin

released from hemolyzed erythrocytes® Hemoglobin was determined as the

ferri hemic acid and expressed as its equivalent of hemin® Assay of

lecithinase activity need not be carried out in an aqueous medium®

Hanahan and Vercamer (ll) have shown that lecithinase D (from cabbage)

hydrolyzed lecithin in a medium of 9&% ether and 2% ethyl alcohol® It

has been reported that Bacillus cereus lecithinase could be detected

only after the addition of organic solvents (22)®

The production of culture filtrates with high yields of lecithinase

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8

requires the use of special media» Robertson and Keppie (45) produced

up to 100 mouse minimum lethal doses (MLD)/ml in a medium consisting

of peptone and normal horse serum» Macfarlane and Knight (30)

produced 100-200 mouse M^D/ml using a peptone base medium which was

supplemented with glucose and protein free extracts of horse muscle»

Pappenheimer and Shaskan (44) produced high yields of lecithinase using

a basal medium of casein hydrolyzate which was supplemented with various

peptones. Of the casein hydroly^ates employed by these workers, a pan

creatic digest of casein stimulated lecithinase production quite well. Adams and Hendee (l) investigated different lots of pancreatic digests

of casein and reported that the stimulation of lecithinase production

varied from lot to lot. Adams ^ jl. (2) reported that autolyzed hog

stomach was capable of supporting lecithinase production as well as

pancreatic digests of casein and that enhancement of lecithinase pro

duction may have been due to the presence of glycerophosphorylcholine

in this digest. Logan et al. (24) reported yields of lecithinase that

ranged between 800-1000 mouse KLD/ml when perfringens was grown in a

basal medium containing pancreatic digests of beef heart.

Stimulation of lecithinase production by iron has been reported by

Rogers and Knight (46), Logan et al. (24), Murata et al. (38), and

Tamura et al. (53)» The amounts of iron reported essential for leci

thinase production vary, but the maximum concentration of iron was

2 pg/ml. Murata ^ al. (38) reported that lecithinase production was

inhibited if the concentration of iron was greater than this.

Lecithinase production is affected by the carbohydrate that is used

in the culture medium* Logan et al. (24) and Adams and Hendee (l)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reported that dextrin supported lecithinase production* Other poly

saccharides and glucose supported growth of C* perfringens. but lessened

the yield of lecithinaseo Smith (5l) reported that glucose inhibited

lecithinase production in the presence of starch* In what form(s) dex

trin and starch are utilized by perfringens for the biosynthesis of

lecithinase is not known, but glucose-1-, or glucose-6-phosphate did not

support lecithinase as well* For £* perfringens type D, dextrin was

superior to glucose in supporting lecithinase production lAen the pH of

the culture medium was not controlled ( l 6 ) o However, if the pH of the

medium was maintained at 7*0, both carbohydrates supported lecithinase

production equally* Murata and Yamamoto (37) reported that fructose was

able to replace dextrin as a source of carbohydrate, Lecithinase pro

duction was also stimulated by amino sugars (46)* Glucosamine and N-

acetylglucosamine were active in stimulating lecithinase production,

while chondrosamine was not*

Attempts have been made to stimulate the production of large

amounts of lecithinase in synthetic (chemically defined) media* Logan

e t _gl* ( 2 4) developed a semi-synthetic medium that consisted of casein

hydro lysate, inorganic salts, and amino acids* Boyd ^ (3, 4) dev

eloped a synthetic medium which supported good growth of jC* perfringens

and used this medium for microbiological assay of amino acids. No

lecithinase was produced in this medium. Strains of perfringens

grown in this synthetic medium did not lose their ability to prpduce

lecithinase, because when they were transferred to a suitable complex

medium, the enzyme was produced* Arginine, cystine, glutamic acid,

histidine, leucine, lysine, methionine, threonine, phenylalanine.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10

serine, tyrosine, and valine were essential for the growth of nei—

fringenso Vitamins and glucose stimulated growth* Shankar and Bard

(48) reported that magnesium ions were essential for cell division by

C.® perfringens. and that the cation could not be replaced by manganese

or cobalt* Mirata and Yamamoto (37) developed a synthetic medium that

was capable of supporting the production of up to 23 egg units (69 MLD)

of lecithinase/ml* %ese workers reported that cystine, arginine, and

aspartic acid were essential for lecithinase production* The concen

tration of cystine was shown to be a critical factor in lecithinase pro

duction by Murata ^ al* (38), and Gooder and Gehring (lO)* If the con

centration of cystine was greater than 0*0$ mg/ml, lecithinase activity

d ecreased *

Peptides have been shown, to st:lmulate lecithinase production in

strains of C* perfringens * Mxrata et jJ,* (36) reported that the dialyz-

able fraction of peptone (possibly small, peptides) supported lecithinase

production very well* Ha.us.-.nild (l2) showed that peptides Wiich consis

ted of 12, 4*5, and 2*$ amino a, . Id residues were capable of supporting

good growth and lecithinase production by G.* perfringens type D* Jayko

and Lichstein (20) report.ed that lecithinase production was enhanced by

the addition of glycyl-L~asparagine to the synthetic medium of Boyd

e t a l* (3)* The st:imula,to:ry activity of this peptide could not be re

placed by glycyl-D-asparagine or alanyl-L-asparagine* Tsukamoto ^ al*

(54) were unable to cordirm the work of Jayko and Lichstein* However,

it was reported that 8 egg 'units (24 îïïiD) of lecithinase/m l were formed

in Boyd^s medium if sucrose was substituted for glucose, thioglycolic

acid for ascorbic acid, and by the addition of glutamine ($4)*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11

The optimum pH for lecithinase production has been shown to be

within the lim its of 6o 5~7o 8 depending upon the strain of C» nerfrlneens

employedo Several workers reported that the optimum temperature for

lecithinase production was 43-46 C (56, 26), but this factor may be de

pendent upon the strain of 0<, perfringens employed (53 , 30 )» It has

been shown that lecithinase production reaches a maximum within 6 h r

after inoculation and then declines rapidly ( 56)0

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPUR I I

STATEMENT OF THE PROBLEM

Although the lecithinase activity of classical type A strains of

Clostridium -perfringens has been extensively studied, very little is

known about the factors that influence the production of lecithinase

and the activities of food poisoning strains of this organism*

This investigation consisted of a comparative study of the leci

thinase found in classical type A and in food poisoning strains of

jC* perfringens. with special emphasis on the factors affecting its pro

duction in complex and synthetic media* In addition, the physical and

chemical factors affecting lecithinase acti-vity were studied»

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I I I

METHODS AND MATERIALS lo General Methods and Pfa.terla.ls

(1) Strains of Clostridium perfringens employed

E i^ t strains of Clostridium perfringens type A were used th rcu t

out this investigationo Strains BP6K, PB6H, and FIO 6 were obtained

from the National Institutes of Health, Bethesda, Maryland* Strains

A48, A91, and Hobbs serotype 3 were obtained from Dr* H* E* H all,

Robert A* Taft Sanitary Engineering Center, Cincinnati, Ohio* Strain

NCTC 8246 was obtained from R* Fuller, National Institute for Research

in Dairying, Berkshire, England* Strain UM707 was obtained from

St* Patrick Hospital, Missoula, Montana* Cultures were maintained in

Bacto Cooked Meat Medium (Difco) and were routinely checked for purity

employing biochemical, mcrpholigical, and hemolytic criteria*

( 2) Routine media

The solid medium used for making plate counts was Brain Heart

Infusion Agar (Difco)* Fifteen ml volumes of medium were poured into

petri dishes and stored at 4 C*

Rapidly growing cultures of Co perfringens were obtained by inocu

lating 0*1 ml of a cooked meat culture into Bacto Fluid Tiioglycollate

medium (Difco)* Tubes were incubated for 18 hr at 37 C* "Die medium was

dispensed into test tubes and sterilized*

The complex medium for lecithinase production (Table 2) was inocu

lated with 1*0 ml of an 18 hr fluid thioglycollate culture and incubated

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14

for 5 hr at 46 C» lîie materials for this mediiim, with the exception of

ferric sulfate and soluble starchy were weighed and dissolved in 750 ml

of distilled water* Soluble starch was dissolved in boiling water and

added to the medium* Ferric sulfate was dissolved in 2 ml of IN HCl and

added to the medium* The jgH was adjusted in 6*7 with 12N HGl and the

total volume was adjusted to 1 lite r with distilled water* Twenty ml

volumes of this medium were dispensed into 25 x 150 mm screwcapped test

tubes and autoclaved at 121 C for 20 min* "Die sterile medium was stored

a t 4 C*

Medium NCTC 109 was prepared by dissolving 9*40 g of the dehydrated

medium (Grand Island Biological Co*, Table 3) in 950 ml of glass dis

tilled water* One g of L ascorbic acid and 1*4 g of NaHCO^ were then

added* The was adjusted to 6*6 and the total volume was adjusted

to 1 liter with glass distilled water* This medium was sterilized by

pressure filtration employing a De Laval (Model L-14) pressure filter*

Ten ml volumes of this medium were aseptically dispensed into sterile,

19 X 150 mm screwcapped test tubes and stored at 4 C*

(3) Transfer of C* perfringens in the synthetic medium

Co perfringens strains BP6K and A48 were transferred in the

synthetic medium a minimum of 20 times before assay experiments were

performed* One ml of each culture in the synthetic medium was trans

ferred to a fresh tube of medium every 12 hr until 20 transfers had

been made*

(4) Preparation of culture filtrates

Culture filtrates of C* perfringens were prepared by transferring

the entire contents of a culture to sterile **Nalgene** (polypropylene)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15

TABLE 2. Composition of the medium for

lecithinase production

Ingredients Amount per liter

T ry p tic a se 20,0 g

Yeast extract 5,0 g

Soluble starch 2 ,5 g

S u cro se 1 .0 g

K2HP04»3H20 1 ,0 g

MgSO^-THgO 0 ,1 g

Fe2 (S0 4) 3 <>nH20 0 ,1 g

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16

'DIHjE 3 o Composition of medium NCTC 109*

Ingredients per lite r (mg)

L -A lan in e 31o48 Choline chloride 1 .2 5 L-a-Amino butyric a c id 3o51 p-Aminobenzoic acid 0 ,1 2 5 L -A rg in in e 25o76 V itam in A 0 .2 5 L-Asparagine 8 .0 9 C a lc if e r o l 0 .2 5 L-Aspartic acid 9o91 M enadione 0 ,0 2 5 D-Glueo samine 3o20 a-Tocopherol phosjiiate 0 .0 2 5 L-Glutamic acid 8 .2 6 Vitamin B-] g 1 ,0 G ly c in e 13o51 Glutathione 1 0 ,1 L -C y stin e 1 0 .4 9 Ascorbic acid 4 9 .9 L-Histidine 19o73 Diphosphopyridine Hydroxy-L-proline 4 .0 9 n u c le o tid e 7 .0 L-Isoleucine 1 8 .0 4 Triphosphopyridine L -L eu cin e 2 0 .4 4 n u c le o tid e 1 ,0 L -L y sin e 3 0 .7 5 Coenzyae A 2 ,5 L-Methionine 4 .4 4 Cocarboxylase 1 .0 L-Omithine 7.38 Flavine adenine L-Phenylalanine 16.53 dinucleotide 1 ,0 L - P r o lin e 6.13 Uridine triphosphate 1 ,0 L -S e rin e 1 0 .7 5 Deoxyad enosine 1 0 ,0 L -T a u rin e 4 .1 8 Deoxycytidine 1 0 ,0 L-Threonine 1 8 .9 3 Thym idine 1 0 ,0 L-Tryptophane 1 7 .5 0 5-Methylcyto sine 0 ,1 L -T y ro sin e 1 6 .4 4 Glueuronolactone 1 .8 L-Valine 25.00 Sodium gluorronate 1 ,8 Tween SO 1 2 .5 3 G lutam ine 135.73 T hiam ine 0 .0 2 5 Sodium acetate 5 0 .0 Riboflavine 0 .0 2 5 Sodium chloride 6800 P y rid o x in e 0 .0 6 2 5 Potassium chloride 400 P y rid o x a l 0 .0 6 2 5 Calcium chloride 200 N ia c in 0.0 6 2 5 Magnesium sulfate.THgO 200 Niacinamide 0 .0 6 2 5 Mono sodium phosphate 125 Pantoth enat e 0 .0 2 5 D ex tro se 1000 B io tin 0 .0 2 5 Phenol red 20 F o lic a c id 0 .0 2 5 Sodium bicarbonate 2200

^Formula taken from Tissue Culture„ A Manual of M aterials and Methods ; published by B-D Laboratories^ Inc®

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17

bottles and centrifuging each culture for 45 M.n at 2500 rpm» The

supernatant liquid was then pressure filtered to insure complete re

moval of the cellso Each culture filtrate was then adjusted to 7o2

by the addition of lU NaOH<, The filtrate was transferred to test tubes

and stored at 4 Co The lecithinase activity of each preparation was

assayed no later than 5 hr after cell removal*

(5) Counting method

The serial dilution method using sterile lecithinase production

medium cooled to 10 C as diluent, was used throughout this investiga

t i o n .

One-tenth ml of the desired dilution was pipetted onto the surface

of an agar plate® Bent glass rods were used to spread the aliquot over

the entire surface of the agar® Ihe plates were inverted and incubated

in a Gaspak Anaerobic Jar (Baltimore Biological Laboratory) for 24 hr

at 37 Co The number of colonies was counted at 24 hr (with the aid of

a Cenco colony counter) and the number of viable cells/m l was calculated

and recorded®

(6) Lecithovitellin assay of lecithinase activi.tv

(a) Preparation of reagents

Egg yolk saline (the substrate) was prepared by ^ulsifying

the yolk of 1 egg in 500 ml of 0*9% NaCl» Twenty g of Kaolin

(Merck) were stirred in, and the preparation was filtered through

a double-thickness of filte r paper (Whatman no* l) at 4 C® The

filtrate was further clarified and steriHzed by pressure filtra

tion through an asbestos sterilizing pad® The sterile filtrate

was stored at 4 C® Fresh egg yolk saline was prepared every 7

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. l ô

days* Calcium chloride was prepared by dissolving 1*47 g of rea

gent grade CaClgoaH^O in 100 ml of distilled water* Tris buffer

(0*05M) was prepared by dissolving 6*0 g of Trishydroxymethyl-

aminomethane (N utritional Biochemicals Corp*) in 500 ml of glass

d istilled water* Four hundred and fifty;.ml,6f'O^IN* HCl'were-addéd

and the jgH was adjusted to 7*2 with IN HCl* The to tal volume of

the buffer solution was adjusted to 1 liter with glass distilled

w ater*

Type A antitoxin was obtained from Burrougb s-Wellcome Labora

tories* This was used in the control tube to inhibit lecithinase

a c tiv ity *

(b) Assay procedure

Assay tubes were prepared according to the scheme listed in

Table 4* After all the materials had been added, the tubes were

incubated for 15 min at 46 C* When incubation was complete, 6,0

ml of cold (4 C) distilled water were added to stop the reaction*

The contents of each assay tube were then transferred 19 x 105

mm Coleman cuvettes* The optical density of each tube was read at

650 mp on a Coleman Junior II (Model 6/35) spectrophotometer* The

spectrophotometer was set at 100^ transmittance against tube no* 1

(T ab le 4 )»

A standard assay curve (Fig* II) was prepared by plotting the

optical densities (OD) of the ••known” tubes (nos, 1-6 of Table 4)

against the pg of phospholipase G (NBC) added* Ihe lecithinase

activity of the culture filtrates was interpolated from this curve

and expressed as pg lecithinase/ml of culture filtrate, A standard

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19

TABLE 4o Scheme for performing the

lecithovitellin assay

Tube No,

M aterials added 1 2 3 4 5 6 Unknowns*

Egg yolk saline 2 o 0 2 ,0 2 ,0 2 ,0 2 ,0 2 ,0 2 ,0

O o l M CaCl2«2H20 Oo4 0 , 4 0 ,4 0 ,4 0 ,4 0 ,4 0 ,4

iÿpe A antitoxin 0,1 0 0 0 0 0 0

Culture filtrate 0 0 0 0 0 0 1 ,0

Phospholipase C C o l 0 ,2 0 ,1 5 0 , 1 0 ,0 5 0 ,0 2 0

0®05M T r is b u ff e r 2 o 8 2 , 8 2 ,8 5 2 ,9 2 ,9 5 2 ,9 8 2 ,0

Final volume (ml) 5o4 5 , 4 5 , 4 5 ,4 5 ,4 5 , 4 5 , 4

*Tubes that contain culture filtrates of Go perfringenso

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assay curve was prepared for each lecithinase deterailnationo

The lecithinase activity in culture filtrates prepared from

the synthetic medium was determined in a sim ilar manner* The in

cubation period of the assay tubes was increased to 60 min because

of the lower and more widely varied activities produced in this

medium* Eight-tenths ml of OolM CaCl2®2H20 was added to increase

the affinity of lecithinase for its substrate*

( 7) Determination of the location of lecithinase activity in C* per-

fringens strains EP6K and Hobbs 3

(a) Inoculation, incubation, and cell counting procedures

Treatment of the vegetative cells was the same as previously

described* Spores of £, perfringens were prepared according to

the method of Schneider et al* (47)o

(b) Preparation of the cells to be fractionated

Cultures of Co perfringens grown in complex medium for leci

thinase production were centrifuged to remove the cells* The cul

ture filtrate was assayed for lecithinase activity* The packed

cell pellet was washed by suspending it in 10 ml of 0*05M Tris

buffer and recentrifuging* This procedure was repeated 5 times*

The final cell suspension (to be fractionated) was made by sus

p en d in g 17 g (wet w ei^t) of vegetative cells in 16O ml of sterilej

c o ld (4 C), 0*855^ NaClo Cells were fractionated 1 hr after the

final suspension was made*

The cell suspensions were connected to the intake line of a

Servall Ribi Cell Fractionator (Model RF-l)* Approximately 15O ml

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of the cell suspensions were fractionated at 25,000 pounds per

square inch (psi)o Five samples of the fractionated material were

collected aseptically and gram stained to determine the efficiency

of fractionation* This material was also cultured in fluid thio-

glycollate to determine the presence of any viable cells*

(d) Separation of the fractionated material into soluble and

particulate fractions

The fractionated suspensions were transferred to 50 ml poly

ethylene centrifuge tubes and centrifuged for 30 min at 17,000 rpm

enç)loying a Servall Superspeed Centrifuge fitted with an SS-34

head* The soluble fraction was decanted and stored at 4 C* The

packed particulate fraction was suspended in 5 ml of 0*05M Tris

buffer £H 7<»2 and re centrifuged* This procedure was repeated 5

times* The particulate fraction was resuspended in 0*05M Tris

buffer and stored at 4 0* The washings of the particulate fraction

were pooled and stored at 4 C* Each component was assayed for

lecithinase activity* A special control had to be prepared for the

particulate fraction due to its turbid nature* This was done by

autoclaving a sample of the particulate fraction for 30 min to

inactivate any lecithinase that was present* An equal volume of

the inactivated sample was added to an assay tube which was used to

set the spectrophotometer at 100^ transmittance*

(S) Testing the effects of pH on the enzymatic activity of lecithinase

Sanples of lecithinase (100 pg) were exposed to various jpH values

for 1 hr by dissolving phospholipase C (NBC) in distilled water and then

adjusting the to the desired value by the addition of 0*1N HCl or

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22

OolN NaOHo At the end of the incubation period the of each sample

was adjusted to 7* 2, the materials for the assay of lecithinase activity

were added, and the tubes were incubated for 15 min at 46 Co A control

tube with the jpH adjusted to 7oO was employed as the sample of 100%

activity* The activity remaining after exposure was calculated by

dividing the OD of the control sample into the OD of the test sample and

multiplying by 100^ o

To determine the effects of jpH on the enzyme-substrate reaction,

the tubes containing all the materials for the assay of lecithinase,

e x c e p t O0O5M Tris buffer, were preparedo In place of the buffer, OolN

HCl or OolN NaOH was added and the to tal volume of the solutions was

adjusted to 5o4 ml with distilled water* A control tube with the jgH

adjusted to 7o2 was employed to determine maximum activity* The enzyme

concentration was 100 p.g/ml*

( 9) Testing the effects of dialysis on lecithinase activity

Ten ml volumes of phospholipase C (NBC), fractionated m aterial, and

culture filtrate material were pipetted into dialysis tubing (average

pore diameter of 24 &)* The ends of the tubing were tied in knots to

form a dialysis bag, which were placed into 20 ml of 0*05M Tris buffer

jgH 7*2 or glycerol* Lecithinase preparations were dialyzed for 24 hr

at 4 C* After the completion of dialysis, the volume inside the bag

was determined by transferring its contents to a 10 ml graduated

cylinder* One ml of each preparation was assayed for lecithinase

activity* The effects of dialysis were determined by comparing the

original activity of the sample with its activity after dialysis*

In order to determine if the increased lecithinase activity was due

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23

to the removal of inhibitors^ 3 ml of the dialysate was mixed with the

nondialyzable m aterial, and the activity of the mixture was determined»

(10) Testing the effects of selected chemical agents on lecithinase

(a) Preparation of the test compounds

All inorganic salts were dissolved in distilled water to a

final concentration of 0o2Kt> Ascorbic acid and sodium thioglyco-

late were dissolved in distilled water to final concentrations of

loO mg/ml* L cysteine was dissolved in distilled water to a final

concentration of 1»0 mg/ml» Trypsin was dissolved in distilled

water to final concentration of 0o05^o

(b) One ml (lOO pg) of phospholipase C was transferred to test

tubes that contained loO ml of each reagent» The tubes were mix

ed and incubated for 1 hr at 37 C» After incubation was complete^

laO ml of each sanple was withdrawn and assayed for lecithinase

a c tiv ity ® A c o n tr o l tu b e iÆi.ich was n o t exposed t o any re a g e n t was

employed to determine 100^ activity® The effects of each reagent

were determined by dividing the OD of the control sample into the

OD of each test sample and multiplying by 100^®

Metals that were inhibitory (Table ll) were removed by dia-

lyzing the preparations against 0®05M Tris buffer 7o2 for 48 hr

at 4 C» Tris buffer was replenished every 8 hr to insure removal

of the metal by this procedure® After dialysis was complete, 1®0

ml of each sample was assayed for lecithinase activity®

(11) Testing the effects of chemically defined additives on lecithinase

production in Medium NCTC 109

(a) Preparation of test materials

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Each peptide was dissolved in glass distilled water to a

final concentration of 1©0 mg/mlo Each peptide solution was auto-

claved at 121 G for 15 mino Ten mg of L-a-lecithin were suspended

in 10 ml of NCTG 109 and shaken in order to obtain an almost clear

suspension* To test the effects of all the peptides, 40 ml of

double-strength NCTG 109 were prepared© To the double-strength

preparation, 1*0 ml of each of the 40 peptide solutions was added©

After the addition of any reagent to the synthetic basal medium,

the tubes were incubated overnight to detect contamination* To

test the effects of each peptide individually, 0©5 ml of each

stock peptide solution was added to 10 ml of NGTC 109* This re

sulted in a final peptide concentration of approximately 0*05

mg/ml*

(b) Inoculation and incubation procedures

One ml of a -perfringens culture grown in NGTG 109 for 12

hr was inoculated into the test medium© "Die inoculated tubes were

placed into a Gaspak Anaerobic Jar (BBL) and incubated anaerobic

ally for 12 hr at 37 C© A complex medium control was also includ

ed with each experiment©

( c ) G el l -rmmnvA,]

Cells of C* perfringens were removed by c entrif ugat ion * The

supernatant fluid was transferred to test tubes and the ^ was

adjusted to 7*2 with 0©IN NaOH© Lecithinase activity was assayed

as previously described© Bie effect of each peptide was determin

ed by comx>aring the lecithinase activity in ihe synthetic medium

without the addition of peptides©

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25

(12) The effects of pH on lecithinase production and a.ctlvltv One ml of an 18 hr culture of Co perfringens grown in fluid

thioglycollate was inoculated into 50 ml of the complex medium for

lecithinase production* The pH of each volume had been preadjusted

to pH 4*5, 5*5, 6*5, 7*5, 8*5, 9*5 or 10*5, by the addition of 12tJ

HCl or ICM NaOH* All tubes were incubated for 5 hr at 46 C* When

incubation was complete, the number of viable cells/m l was determined*

the cells were removed by centrifugation and the pH of the supernatant

fluid was adjusted to 7*2* Each preparation was then assayed for leci

thinase activity*

( 13) The effects of heat on commercial and culture filtrate lecithinase

Phospholipase C (NBC) was dissolved in 0«05M Tris buffer pH 7*2

to a final concentration of 100 pg/ml* Five ml volumes of this solution

were pipetted into I 6 x 125 mm screwcapped test tubes and the tubes were

placed in a waterbath (National Appliance Co*, #8725) preheated to the

desired temperature* When the lecithinase solutions had been exposed

to heat for the required length of time, they were removed and cooled

by immersion in an icebath* After cooling, each tube was stored at

4 C until alT the tubes had been removed and cooled* One ml of each

preparation was assayed for lecithinase activity* An unheated sample

of lecithinase was employed as a control*

To determine the internal temperature of the tubes that contained

the lecithinase solution, a control tube with a thermometer which con

tained 5*0 ml of 0*05M Tris buffer was placed into the waterbath* When

the thermometer in this tube recorded the desired temperature, timing

of heat exposure was initiated*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 6

Ihe preceding method was used to determine the effects of heat on

commercial phosjhoHpase C (NBC) dissolved in 0o05M Tris buffer jgH 7o2,

and dissolved in the complex medium for lecithinase production» The

effects of heat were also determined on the lecithinase present in

culture filtrates of perfringens strain PB6H*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV

RESULTS

(1) Lecithinase activities of the selected strains of C. perfringens

"Die method employed for the assay of lecithinase was rapid and re

producible* A standard assay curve (Fig© II) prepared for each determi

nation enabled the quantitation of the lecithinase activity present in

culture filtrates of different strains of Co perfringens©

The lecithinase activities of the 8 test strains of £© perfringens

are listed in Table 5o There was considerable variation from strain to

strain in the amounts of lecithinase produced© The classical infectious

strains, BP6K, NCTC 8246, and PB6H produced more lecithinase than did

the other strains* On the other hand, there was considerable variation

in the activity of these strains© The standard deviation values indi

cated that there was more random error and a greater scatter of data

around the mean by the strains that possessed high lecithinase activi

ties* The strains that produced small quantities of lecithinase, name

ly, strains Hobbs 3, A91, and F106, were consistent in the amounts of

lecithinase produced as evidenced by the range of lecithinase produc

tion and the standard deviation values (Table $)©

(2) Localization of lecithinase activity in C© perfringens

Lecithovltellin assay of ruptured cell fractions demonstrated the

presence of active lecithinase in the soluble fractions of iJ© perfrin

gens strains BP6K and Hobbs 3 (Table 6)© No activity could be demon

strated in the particulate fractions obta:lned from either strain©

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CM r ' j -P •H -PO 00 rj 0)Ul ctI •f-i

'rHO Cj <1-1 o CD •H0 -P CM >- 1(L 4-) > P

W :p c ixi . CD < C) Ü >Cl crj a Cl

C T l -P CO CM

CD CM o ro S CM M

(rfW 0 S 9 ) AXISN3Q OVOIldO

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TABLE 5o Lecithinase activities of eight selected

strains of Clostrldinm perfringens

Lecithinase activity (pg/ml) Colony count S t r a in (I0l&/ml)* Range Mean** SD

BP6K Ool3 38-145 9 8 .6 4 0 .6

NCTC 8246 0 .8 $ 56-139 78.8 3 1 .3

PB6H 0 .9 5 37-107 65 o 6 23.3

A48 0 .9 9 19-30 2 5 .8 4 .1

UM707 0 .8 3 10-28 2 0 .2 7 .9

Hobbs 3 13 16-20 1 8 .8 1 .6

A91 91 12-15 1 3 .8 1 .1

F106 39 12-15 1 3 .5 1 .1

«Calculated on the basis of 4 or more déterminâtions«

««Mean value, calculated on the basis of 4 or more determinations„

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TABLE 60 Lecithinase activity of cell fractions of

Clostridium perfringens

Lecithinase activity in pg/ml

F r a c tio n b p 6k* Hobbs 3**

Culture filtrate 74*0 1 2 .0

Soluble fraction 16*0 1 5 .5

Particulate fraction 0*0 0*0

Pooled washings of 8 .0 t r a c e particulate fraction

Total activity 98*0 27*5

*3*1 X 10^ viable cells per ml*

**2*6 X 10^ viable cells per ml.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31

Strain BP6K had a higher lecithinase activity in its culture filtrate

than in the soluble fraction obtained from disintegrated vegetative

cells* On the other hand, strain Hobbs 3 had approxinately equal

activities in its soluble and culture filtrate fraction* A measurable

lecithinase activity was found in the pooled washings of the particu

late fraction from strain EP6K, but only a trace of activity was found

in the pooled washings of strain Hobbs 3o

A lecithinase activity of 18 pg/ml was found in the soluble frac

tion prepared from the spores of each strain* The particulate spore

fractions, however, were without activity*

(3) The effects of oH on the enzymatic activity of lecithinase

Lecithinase was inactivated by exposure to pH values of 1-3 for

1 hr (Table 7)® However, its enzymatic activity was not as markedly

affected by alkaline pHs* After e^qjosure to pH 10 for 1 hr, 3é*8^ of

the activity remained* Maximum enzyme activity was observed at pH 7*0,

and the activity decreased rapidly as the pH was lowered by the addition

o f a c id *

The data presented in Table 8 indicate that the hydrolysis of egg

yolk saline by lecithinase is dependent upon a narrow range of pH

values for appreciable activity to occur* Maximum activity occurred at

pH 7*0 (Table 8)* The reaction was practically ■unaffected at pH 8*0,

but the completeness of hydrolysis rapidly declined at pH 9*0 and 10*0*

The same effect was observed for lecithinase prepared from culture fil

trates of Ç.O -perfringens strains BP6K, Hobbs 3$ and PB6H*

( a ) Effect of dialysis on lecithinase acti-vitv

The activity of lecithinase present in the cell fractions of strain

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TAlHLE 7 o Effect of £H on commercial lecithinase

Absorbance A ctivity remaining pH (650 mp.) (p e rc e n t)

1 .0 0 .0 0 0 0

2 .0 OoOOO 0

3 .0 0 .0 0 0 0

4 .0 0 .0 1 0 3 .0

5 .0 0 .1 7 0 50.0

6 .0 0 .3 2 0 9 7 .0

7 .0 * 0 .3 4 0 100

8 .0 0.333 9 8 .0

9 .0 0.333 9 8 .0

1 0 .0 0 .125 3 6 .8

*TJsed as the control to determine 100% a c t i v i t y

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TABLE 80 Effect of on the hydrolysis of

egg yolk saline by lecithinase

A bsorbance pH (650 mpi)

1 .0 0 .0 0 0

2 .0 0.0 0 0

3 .0 0 .0 0 0

4 .0 0 .0 0 0

5 .0 0 .0 0 0

6 .0 0 .0 7 2

7 .0 * 0 .2 6 0

8 .0 0 .2 1 0

9 .0 0 .0 3 0

1 0 .0 0 .0 0 8

•îîüsed as a control to determine 100% a c t i v i t y

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TABLE 9« Effect of dialysis on lecithinase activity in

protoplasm of Clostridium -perfringens BP6K

D ia ly z e d # Final volume lecithinase F r a c tio n a g a in s t (m l) (pg/m l)

u n tr e a te d - - 8 .3 0

d i a l y s a t e g ly c e r o l 2 2 .0 OoOO

nondialyzable g ly c e r o l 3 .0 61*4 f r a c t i o n

d i a l y s a te Tris buffer 1 5 .0 0 .0 0 0

nondialyzable Tris buffer lOoO 3 0 .0 f r a c t i o n

* A 1 1 samples dialyzed for 24 hr at 4 Co

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BP6K was markedly increased by dialysis against OoO^M Tris buffer

(£H 7*2) and gylcerol (Table 9)* The activity of lecithinase in pre parations of cytoplasm increased from 8.30 pg/ml to 6l.4 pg/ml when

dialyzed against glycerol and increased to 30.0 pg/ml iniien dialyzed

against 0.05M Tris buffer. When the preparations were dialyzed against

glycerol, the volume of the nondialyzable fraction was reduced three

fold. On the other hand, there was no change in the final volume of

the nondialyzable fraction vÆien 0.05K Tris buffer was employed (Table

9 ) . When culture filtrates of strain H*6K were subjected to dialysis,

the same increase in lecithinase activity was observed (Table 10). The

original activity of the culture filtrate was 66.0 pg/ml. After dialy

sis against glycerol the activity increased to 205.5 pg/ml. When 0.05M

Tris buffer was used the activity increased to 154.0 pg/ml.

When commercial phospholipase C (NBC) was dialyzed, no increase in

its activity was observed.

Mixing equal volumes of dialysate and the nondialyzable fraction

did not result in a decreased lecithinase activity.

( 5) The effects of selected chemicals on lecithinase activity

Lecithinase was inactivated by cobaltic sulfate, aluminum chloride,

L cysteine, and trypsin, whereas the other test compounds had no apprec

iable affect on lecithinase activity (Table ll). Sodium thioglycolate

did not inactivate lecithinase, however. Smith (51) reported that this

compound did inactivate lecithinase.

Dialysis to remove these compounds or their complexes did not re

store enzymatic activity. The same compounds that were active against

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TA.BLE 10o Effect of dialysis on lecithinase in

CTilture filtrates of Clostridium perfringens BP6K

D ia ly z e d ^ Final volume Lecithinase F ra c tio n a g a in s t (m l) (prg/ml)

u n tr e a te d - - 66oO

d i a l y s a te glycerol 22.0 0 .0 0 0

nondialyzable g ly c e r o l 3 .0 205.5 f r a c t i o n

d i a l y s a t e Tris buffer 1 5 .0 0 .0 0 0

nondialyzable Tris buffer 10 oO 154.0 f r a c t i o n

* A 1 1 samples dialyzed for 24 hr at 4 Co

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TABLE 11.0 Effect of selected chemical agents on

the activity of lecithinase

A bsorbance A ctivity remaining Compound (650 mpi) (p e rc e n t)

Control (untreated) 0 . 2 6 0 100

Ascorbic acid 0 . 2 5 8 9 9 . 2

Sodium thioglycolate 0 .2 6 0 100

HgCl 2 0 .2 6 0 100

CoSOi^ 0 .0 0 0 0

CoClg 0 .2 5 8 9 9 .2

AICI3 O o O O O 0

M g C l g 0 .2 6 0 100

Ca(N0 3)g 0 . 2 5 9 9 9 .6

T ry p sin 0 .0 0 0 0

L c y s te in e 0 .0 0 0 0

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38

commercial phospholipase C were also active against the lecithinase pro

duced by jC« perfringens strains BP6K and Hobbs 3«

(6) Stimulation of lecithinase production by various cheinicallv defined

a d d itiv e s

Tables 12 and 13 list the effects of hO chemically defined peptides

on lecithinase production by Co perfringens strain EP6Ko Twenty-one

peptides stimulated lecithinase production by this strain (Table 12)«

However, much variation in the amount of stimulation was observed.

DL-benzoyl-alanine supported the minimum activity 15 pg/ml, ■while maxi

mum activity of SO pg/ml was supported by the addition of glyeyl-DL-

norvaline and D-leucyl-glycyl-glycine.

Lecithinase activity in the peptide supplemented medium was

consistently less than the complex medium control® On the other hand,

no lecithinase was produced in the basal synthetic medium devoid of any

p e p tid e .

Stimulation of lecithinase production was quite specific in some

instances® D-leucyl-glycyl-glycine supported an activity of 80 pg/ml

while L-leucyl-glycyl-glycine supported the production of only 45 pg/ml®

Peptides consisting only of glycine showed an inverse stimulatory

effect. As the chain length of these peptides increased, activity de

creased® Glycyl-glycine ethyl ester“HCl supported an activity of 78

pg/ml, wtiile glycyl-glycine methyl ester»HCl supported only 26 pg/ml

(Table 12).

Co perfringens strain A4S was stimulated by only 2 peptides®

Glycyl-L-tryptophan supported an activity of 10 pg/ml, and DL-benzoyl-

alanine supported fan activity of 15 pg/ml® In both strains, the

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TABLE 12» Peptides that stijnvuLated lecithinase production by

Clostridium perfringens BPéK

A c tiv ity P e p tid e * (j^g/m l)

Control (NCTC 109 devoid of any peptide) 0,0 DL-alanyl-glycine 70,0 DL-alanyl-DL -methionine 2 0 ,0 DL-alanyl-DL-phenylalanine 3 0 ,0 DL-alanyl-DL-valine 3 0 .0 DL-benzoyl-alanine 1 5 .0 G lycyl-glycine-Ethyl Est er » HC1 7 8 ,0 Glycyl-glycine-Methyl Ester*HCl 26.0 Glycyl-glycine 72.0 Glycyl-glycine» HCl 6 8 .0 Glycyl-glycyl-glycine 30.0 Glycyl-DL-leuc ine 60.0 Glycyl-DL-norvaline 8 0 .0 Glycyl-DL-phenylalanine 70.0 Glycyl-DL- serine 6 9 .0 Glycyl-DL-valine 78.0 D-leucyl-glycine 7 1 .0 DL-leuc yl-DL -glycyl-glyc ine 2 2 .0 L-leucyl-glycyl-glycine 4 5 .0 D-leucyl-glycyl-glycine 8 0 .0 L-leucyl-L-tyro sine 75.0 L-leucyl-DL-alanine 20.0

^Peptides tested at a concentration of 0»05 mg/ml»

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TABLE 13o Peptides that failed to stimulate lecithinase

production by Clostridium perfringens BP6K

Peptide-?*-

Grlycyl-EL-a-andno-n-butyric acid

Glycyl-glycyl-glycyl-glycine

L-leuc yl-glyc ine

Glycyl-glycyl-DL-ph enylalanin e

Glycyl-glycyl-L-alanine

Glycyl-L-leuc ine

Glycyl-L-threonine

DL-leucyl-glyc ine

Glycyl-DL-alanine

DL-alanyl-DL-norvaline

DL-alanyl-DL-norleuc ine

Glycyl-L-tyro sine

Glycyl-L-asparaglne

D-leucyl-L-tyrosine

Glycyl-DL-norleucine

Glycyl-L-valine

DL-alany 1-DL-al anin e

DL-alanyl-glyoyl-glycine

Glycyl-D-asparagine

■«Peptides t e s t e d a t a c o n c e n tra tio n o f 0o05 mg/ml<.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41

addition of L-a-lecithin failed to support lecithinase production,, The

free andno acids, glycine, asparagine, leucine, alanine, norvaline,

serine, and phenylalanine did not support lecithinase production»

(7 ) The effects of p H on lecithinase production and growth of C» per fringens in complex medium

Ç® -perfringens strains BP6K and Hobbs 3 produced lecithinase maxi

mally at £H 6o5 (Figs* III and IV)» Considerable variation in growth and lecithinase production as functions of was observed for both

strains* Strain BP6K had a measurable lecithinase activity at 5*5

-8*5 (Fig, III). On the other hand, strain Hobbs 3 produced measurable

lecithinase activity only at ^H 6.5 and 7,5 (Fig, 17), For both

strains, lecithinase production was directly proportional to the cell

density at a given ^H©

(8) Dénaturation of lecithinase by heat at 60 or 90 C

The lecithinase present in the culture filtrates of strain PB6H

was relatively heat resistant, losing 2^% of its original activity when

heated for 20 min at 60 C (Fig, V), However, there was a sharp loss in

the lecithinase activity of this preparation when heated for 30 min.

Commercial lecithinase dissolved in the complex medium for lecithinase

production showed a sharp loss in acti-vLty during the first 5 min of

heating at 60 C (Fig, V), However, the residual activity of this prep

aration was somewhat resistant to further inactivation. Commercial

lecithinase dissolved in 0,05M Tris buffer was very heat sensitive

( F ig . 7 ) ,

The effects of heat on lecithinase activity at 90 C are shown in

Figure 71, Lecithinase obtained from strain PB6 h was more active at

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 2

8 0

>- h~ > (/) h- _l W S o V. d 4 0 tu S LÜ z < o m — ^ < § a

0*- 4.5 6.5 7.55.5 9.58.5

pH OF GROWTH MEDIUM LOG NO. OF CELLS/M L • ------• LECITHINASE ACTIVITY (JUG/ML)

FIG'aE I I I . T-; , e ffe c ts of on the groivth and le cith in ase

activd-tj of Clostridium perfringens BP6K

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20

> c/5 h-

LU O ^ 3 O LU IS W z < CD CD Z = l < X > 3 I— S

pim: ------4.5 5.5 6.5 7 58.5 9.5

pH OF GROWTH MEDIUM • LOG NO. OF CELLS/ML LECITHINASE ACTIVITY (UG/ML)

FIGURE IV . The effects of jjH on the grov/th and lec ith in a se

activity of Clostridium perfringens Hobbs 3

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 .4 0 - LECITHINASE DISSOLVED IN 0.05 M TRIS BUFFER CULTURE FILTRATE OF 0 .3 5 STRAIN PB6H LECITHINASE DISSOLVED IN MEDIUM FOR ex TOXIN 0 .3 0 PRODUCTION

O LO CD 0 .2 5 -

CO 0.20 - Z LU Û 0.15 - < O 0.10 - \ CL O

0.05 -

TIME (MIN)

FIGUHE 7. The Inacti/ation oT lecith-naae by heat at T C

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 40 . , LECITHINASE DISSOLVED IN 0 .0 5 M TRIS BUFFER

0 .3 5 ^ CULTURE FILTRATE OF STRAIN PB6H k LECITHINASE DISSOLVED 0 .3 0 IN MEDIUM FOR cx TOXIN o \ PRODUCTION LO iS 0 .2 5 > -

œ 0.20 LU Q 0.15 < O \ 0.10 CL O

0 .0 5

0.0 o 10 20 30 40 50 60 TIME (MIN)

VI, TV.: inactivation of lecithinaso Vy heat at 90 C

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46

90 C than at 60 C (Figs* V & Tl)* The inactivation of this preparation

was gradual, with no sharp losses in activity* Commercial lecithinase

dissolved in 0*05M Tris buffer was subject to dénaturation at this

temperature (Fig* VI)* Commercial lecithinase dissolved in the complex

medium for lecithinase production was heat sensitive, but it was more

active at this temperature than at 60 C (Figs* V & Vl)«

(9) Growth of C* oerfringens in NCTC 109

_C. perfringens strains BP6K and A4â grew profusely in medium NCTC

109* However, growth was not as extensive as in the complex medium,

rarely surpassing 10? viable cells/ml* Cell morphology was typical in

this medium and gram positive rods were readily observed* Subcultiva

tion in this medium has been successful auid 86 transfers were achieved*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 7

DISCUSSION AND CONCLUSIONS

The lecithinase activity of the strains of Clostridium perfringens

was compared with the data of Nakamura and Converse (39, 40) on the

heat resistance of spores of these strains. In general, the strains

that exhibited high lecithinase activity (Table 5) also produced spores

of relatively low heat resistance (destroyed at 90 C in less than 30

min). Strains that had low lecithinase activity produced spores of high

heat resistance (requiring more than 150 min at 90 C for destruction).

There was one exception, namely, strain F106, a strain that had low

lecithinase activity, produced spores of low heat resistance. This in

verse relationship between heat resistance of spores and lecithinase

activity is in general agreement with other reported data (56, 59) o

However, it is interesting to note that among the strains that possessed

high lecithinase activity, there was considerable variation in the .

amounts of lecithinase produced, Ihis was not the case for the strains

of low lecithinase activity.

Although the heat resistance of spores of Ç, perfringens has been

reported to be genetically stable (8), Weiss and Strong (57) reported

that spores of selected strains of Ç, perfringens varied considerably

in their heat resistance when different sporulation media were employ

ed, From the data in Table 5 it appears that lecithinase activity, at

least in the strains that exhibited high lecithinase activity, is also

a variable property, Ihe employment of a complex medium may be a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48

predisposing factor for this variation, as nutritional requirements

for lecithinase production have been reported to vary from strain to

strain (46, 5l)o Therefore, in examination of strains that produce

high yields of lecithinase, at least several determinations should be

made to determine a mean lecithinase activity for each strain,, A

single experiment may yield false conclusions, namely, that a strain is

a low or intermediate producer of lecithinase*

Although the heat resistance of the spores and the level of leci

thinase activity are two criteria used to distinguish food poisoning

from classical type A strains of C* perfringens. the present study

indicated that these two criteria cannot be relied upon completely for

the identification of food poisoning strains*

Lecithinase activity in the cytoplasmic and culture filtrate

fractions of C* perfringens vegetative cells and spores was demonstrated

by Meisel ^ âl* (34)» In a later study, these investigators demon

strated the presence of kappa toxin (collagenase) in the cytoplasm of

the vegetative cells and spores of Ce perfringens type D (35)* It has

also been shown that lecithinase is synthesized ^ novo within the veg

etative cells of C* perfringens and is then partially excreted into the

surrounding medium (40)*

The lecithinase activity of the soluble fractions prepared from the

spores of perfringens strains BP6K and Hobbs 3 may be related to the

pathogenicity of perfringens and its ability to cause gas gangrene*

The liberation of lecithinase into the tissues of a susceptible host

during sporulation may cause necrosis and trauma, therby creating

suitable anaerobic conditions for the growth and m ultiplication of the

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vegetative form of C® perfringens®

The intracellular lecithinase activity of food poisoning strains of

jC, perfringens may contribute to their ability to cause the disease® If

the production of lecithinase is related to the ability of C® perfrin- •

gais to cause food poisoning in man, strains of high activity should

constitute the majority of those implicated, but this has not been

demonstrated® However, if intracellular lecithinase is liberated in an

active form when vegetative cells lyse, the resulting **total** activity

may be sufficient to cause the symptoms of food poisoning®

Ihe effects of on lecithinase activity may be explained in three

ways® The jgH of the culture medium may have inactivated the enzyme

molecule once it was liberated into the medium® Secondly, the number

of viable cells was reduced because of the adverse condition, vtiich

may have resulted in decreased lecithinase production and activity®

Thirdly, the jgH of the culture medium caused a decreased absorption of

the amino acid(s) necessary for lecithinase production® This has been

shown for perfringens type D (14)» The second condition is appli

cable to C® perfringens strains BP6K and Hobbs 3, as a decrease or

increase in the number of viable cells/m l resulted in a corresponding

decrease or increase in lecithinase activity® The differences observed

between strain Hobbs 3 and BP6K at the _pH range investigated was

probably due to the method used for determining lecithinase activity®

Since strain Hobbs 3 produced a maximum activity that was only 20^ of

the maximum activity of strain BP6K, a substantial decrease in the leci

thinase activity of this strain would result in an extremely low

activity® Due to the difficulty of measurement, a report of no activity

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50

would result where actually a trace of activity may have existed»

Therefore, the data presented imply that lecithinase activity may be a direct function of the number of viable cells present in the culture

medium»

The irreversible inactivation of lecithinase by acid ( < 5) £H

values was noted in this study® This result suggests that active leci

thinase cannot directly cause food poisoning symptoms» The jpH of the

stomach during digestion is close to 1»0 (21), and dénaturation of the

ingested enzyme would result if it was directly e]

tion* On the other hand, lecithinase molecules may be protected by

other ingested material» If these active molecules passed into the

small intestine (piH 7»5-8o5), they may be able to contribute to the

symptoms of food poisoning» This hypothesis is somewhat supported by

the data of Hauschild et al» (15) who produced enteric disorders in more

lambs by surgically implanting £» perfringens in the duodenum of these

animals than by feeding them viable cultures»

No mechanism has been suggested as to the role of lecithinase in

contributing to the clinical symptoms of food poisoning» However, a

possible mechanism may be suggested on the basis of the report by Slein

and Logan (50) inrtio demonstrated that Escherichia coli strain M.35 was

lysed by the phospholipase produced by Bacillus cereus» If ingested

lecithinase reaches the gut, or if it is produced by viable _Ç» perfrin

gens. it may cause a substantial decrease in the E» coli population*

Food poisoning symptoms may result from a proliferation of £» perfrin

gens in the gut due to the decrease in the normal flora of the gut»

The increased activity of lecithinase Tmàiich resulted from dialysis

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was probably due to concentration of the enzyme and to the removal of

inhibitors* When dialyzed against glycerol, the preparations of leci

other workers to obtain preparations of higher activity (30, 55)o The

increased activity of the lecithinase preparations dialyzed against

O0O5M Tris buffer pH 7o2 where no volumetric changes occurred, was

prepared lecithinase, which has been precipitated from culture fil

trates, did not gain any appreciable activity from dialysis, it appears

Some enzymes are inactivated by dialysis (9)o The xanthine oxi

dase in milk and aldehyde oxidase in liver lost their ability to reduce

cytochrome c, n itrate, or ferrocyanide, if molybdenum was removed by

The irreversible inactivation of lecithinase by metallic salts,

trypsin, and L-cysteir.e is in agreement with the results obtained by

other workers (l9)o However, the resistance of lecithinase to dénatura

tion by sodium thioglycolate does not agree with the results of others

( 1 9, 51)® Ispolatovskaya and Klimacheva (l9) reported that lecithinase

was probably due to the length of time that lecithinase was exposed to

sodium thioglycolate has been postulated to be a reduction of disulfide

linkages (5l), it would seem that an exposure time of 1 hr would not

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incubation should be sufficient time for complete reduction and dénat

enough* lecithinase may not contribute to the symptoms of food poison

with lecithinase as substrates for trypsin, it may not be appreciably

Dialysis to remove cationic or anionic inhibitors from the leci

that inhibitors of this type may become strongly bound to the charged

protein molecule was evidently sufficient to resist their removal by

probably resulted in a change in the structure of the protein, dialysis

was not expected to restore its activity after dénaturation by this

chem ical©

Die heat stability of lecithinase was probably dependent upon the

for 20 min, whereas the same enzyme heated in a complex medium was not

ers (7* 52, 6) and explained by Smith (5l)

Purified lecithinase dissolved in the conplex medium was not as

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heat stable as cijlture filtrate preparations of C_o perfringens strain

PBôHo The lecithinase molecules in the culture filtrate may have been

stabilized by cellular metabolites that were lacking in the uninoculated

mediumo Furthermore, the structure of the culture filtrate lecithinase

may be different than the commercial preparations of lecithinase which

have been partially purifiedo

The heat stability of lecithinase, when present in food, would en

able it to retain most of its activity after a mild reheating process*

Active lecithinase could then be ingested with the food and cause the

symptoms of food poisoning* However, it has been repoit-ed that heating

food or culture filtrates to 100 C destroyed their toxic factor (51,

15)* These data seem to rule out the possibility that ingested leci

thinase alone causes food poisoning*

Lecithinase was produced in a chemically defined medium supplement

ed with small peptides (4 amino acid residues or less)* Since the

addition of single amino acids or substrate (L-a-lecithin) did not sti

mulate lecithinase activity, it appears that peptides or larger mole

cules are necessary for its production* The requirement of peptides for

lecithinase, hemolysin, and epsilon prototoxin production by _C* perfrin

gens type D, was demonstrated by Hauschild (12)*

The effects of small peptides on lecithinase production were stud

ied by Jayko and Lichstein (20)* Only glycyl-L-asparagine stimulated

lecithinase production* In the present study, glycyl-L-asparagine did

not stimulate lecithinase production by strains BP 6K and A48* The

inability of this peptide to stimulate lecithinase production probably

resulted from the fact that nutritional requirements for lecithinase

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54

production may vary from strain to strain, as was shown in this study

with strain BP6K and A48» Furthermore, the basal medium NCTC 109 was

much more complete than the one employed by Jayko and Lichstein (20),

W&irata and Yamamoto (37) reported on the production of h i^ yields

of lecithinase by £, perfringens strain PB 6K grown in a medium devoid of

peptides. From their studies, they concluded that high (lO mg/ml) con

centrations of L-arginine were necessary for lecithinase biosynthesis

. by this strain. Conceivably, variations in strains and their metabolic

pathways for lecithinase biosynthesis may account for the differences

noted. Furthermore, it is possible that the requirements for lecithin

ase production are somewhat different for all strains of perfringens

type A, and individual strain requirements w ill have to be worked out.

The mechanism of stimulation of lecithinase production by £« per-

fingens by peptides has not been elucidated. However, it was shown that

p eptides were incorporated into the cells of Co perfringens type D at a

higher rate than were free amino acids (13), Leach and Snell (23)

postulated that the function of peptides in growth is one of providing

amino acids, which, in their free fom , may either be taken up inade

quately or be rapidly broken down by the cell® Inadequate uptake was

explained by them as being due to either the low activity of a specific

permease or to an imbalance of amino acids. Possibly this explanation

may account for the differences found between C® perfringens strain

BP6K and A 48® Further studies to determine the uptake of C^-labeled

peptides by these 2 strains may prove useful in explaining the differ

ences observed.

The factors affecting lecithinase production by _C* perfringens are

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only partially understood» However, the synthetic medium, NCTC 109,

■vdiich supports good growth of £» perfringens. is the most suitable

medium used thus far in the study of lecithinase production* In addi

tion to the experiments that involve the addition of chemically defined

substances to this medium, deletion experiments would contribute materi

ally as to the minimal requirements for lecithinase production*

One criticism of the studies by other workers ( 20, 37) is that

transfers in the synthetic medium were not attempted before lecithinase

activity was assayed» It seems essential that transfers be made in any

synthetic medium to eliminate the possibility of a carry over of complex

nutrients* Any products carried over during inoculation may lead to

erroneous conclusions»

Prom the data presented, it appears that ingested lecithinase does

not cause food poisoning in man* However, the production of lecithinase

by cells of ^» perfringens growing in the gut may contribute greatly to

the symptoms seen in man» Ihe demonstration of intracellular lecithi

nase in strain Hobbs 3 may explain the ability of this low toxigenic

strain to cause food poisoning»

Althougji peptides have been demonstrated to be essential fo r le c i

thinase production in strain EP6K and AJ+8, other factors, as yet unde

term ined, probably are also involved* Further studies dealing with such

factors as ferm entable carbohydrate source, the presence of large poly

peptides, and the oxidation-reduction potential of the medium w ill be

necessary before an explanation of the factors affecting lecithinase

production can be given*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER VI

SUMMARY 1* The factors affecting the activity and production of lecithinase by

Clostridium perfringens were studied*

2o Classical infectious strains of C* perfringens were separated from

food poisoning strains of this organism on the basis of the amount

of lecithinase produced* However, much variation in the lecithinase

production by the strains of h i^ activity was observed*

3* Assay of cell fractions obtained from disintegrated vegetative cells

of jC* perfringens indicated that a large proportion of the lecithin

ase formed by classical infectious strains was excreted into the

culture medium* However, more than half of the lecithinase activity

associated with the food poisoning strains was located within their

c e lls *

4o Lecithinase was detected in the soluble fractions prepared from dis

integrated spores of C* perfringens*

5* The enzymatic activity of lecithinase increased after dialysis

against glycerol or 0*05M Tris buffer _pH 7*2*

6* Lecithinase was inactivated after exposure to cobaltic sulfate,

aluminum chloride, L-cysteine, and trypsin*

7* Acid ( < 5) inhibited growth and lecithinase production by C*

perfringens*

8* jg* perfringens was successfully grown and subcultured in a synthetic

tissue culture medium, NCTC 109*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57

9o Varions chemically defined compounds, including 40 small peptides

(4 amino acid residues or less), were tested for their action on

lecithinase production by Ça perfringens grown in the synthetic

mediumo

10o Twenty-one peptides stimulated lecithinase production by £o per

fringens strain BP 6K, a classical type A organism, Wiereas, only 2

peptides stimulated lecithinase production by strain A48, a food

poisoning strain of C,® perfringens »

He Lecithinase was resistant to inactivation at 90 C* However, it was

readily inactivated at 60 Go

12o Ihe significance of the results is discussed*

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BIBLIOŒAPHY

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15© Hauschild, A© H* W©, L© N ilo, and W© J© Dorward© 1968© Experi mental enteritis with food poisoning and classical strains of Clostridium perfringens type A in lambs© J© Infect© Diseases, 117s379-386©

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38o M irata, Ro, A® Yamamoto, S® Soda, and A® Ito® 1965® Nutritional requirements of Clostridium -perfringens PB6K for alpha toxin production® Japan® J® Med® Sci® Biol® 18gl89-202®

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AUTOBIOGRAPffr

W m iAM Ro CROSS

Birthplaceg Spokane^ Washington

Birthdateg July 26g 1944

Citizenshipg USA

Social Security Numbers 536 42 0 1 7 2

Eastern Washington State College^ Cheney, Washington

Bo kog Major: Biology KB.nor: Chemistry 1 9 6 2 - 1 9 6 6

University of Montana, ÎO.ssoula, Montana

Mo So, Major: Microbiology 1966-1968 Student Numbers 25340664

E x p erien c e

Research A ssistant, Depto of Microbiology,

University of Montana, M.ssoula, Montana 1966-1967

Graduate A ssistant, D e p to of MLcrobiology.

University of Montana, Missoula, Montana 1 9 6 7 - 1 9 6 8

Membership in Professional Societies

American Society for îE.crobiology 1966-d a te

Montana Acad^ny of Science 1968- d a te

M ilit.^rv Status

2 L t, C hem ical C orps (USAR) 1966- d a te

Honors Member of Phi Sigma (National Biological Honorary Society) 1967- d a te

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.