Vol. 13, No. 2JOURNAL 0F CLINICAL MICROBIOLOGY, Feb. 1981, p. 335-3430095-1137/81/020335-09$02.00/0
Phospholipase D Activity of Corynebacteriumpseudotuberculosis (Corynebacterium ovis) and
Corynebacterium ulcerans, a Distinctive Marker Within theGenus Corynebacterium
LANE BARKSDALE,* REGINA LINDER, IOAN T. SULEA,t AND MARJORIE POLLICEtDepartment ofMicrobiology, New York University School ofMedicine and Medical Center, New York,
New York 10016
A search has been made for corynebacterial phospholipase D, "ovis toxin," asphingomyelinase (phosphatidylcholine phosphohydrolase, EC 3.1.4.4), among awide variety of corynebacteria. Phospholipase D activity has been found in strainsexhibiting the biochemical properties characteristic of Corynebacterium pseudo-tuberculosis or of Corynebacterium ulcerans and in no other species of Coryne-bacterium. Methods for the assay of phospholipase D as a sphingomyelinase andmethods for screening for phospholipase D in the presence of Corynebacteriumequi on washed sheep blood agar are discussed.
Certain strains of Corynebacterium pseudo-tuberculosis (Corynebacterium ovis), when ly-sogenic for a tox+-carrying phage, produce bothdiphtherial toxin (3, 30) and a second toxic sub-stance, a sphingomyelinase (phosphatidylcho-line phosphohydrolase, EC 3.1.4.4, a phospholi-pase D [PLD]) capable of hydrolyzing sphingo-myelins to N-acylsphingosyl phosphates (39,40).When the gene tox is absent from either of thesecorynebacteria, PLD is the characteristic toxinproduced. Over a period of 55 years what cameto be called "ovis toxin" gave rise to muchspeculation (2, 6, 28), because sometimes it con-sisted of a mixture of diphtherial toxin and PLD,but most often it consisted of only the latter(34). Once the matter ofthe two toxic substanceswas resolved (12, 26, 27), there was a need toinvestigate the relationship between the PLDactivity of the toxin and "associated substances"which under certain conditions caused the he-molysis of erythrocytes and which blocked thehemolytic activity of the fl-toxin ofStaphylococ-cus aureus. Over the last 15 years, through theefforts of Soucek and co-workers (36-38), Goeland Singh (17), and Lovell and Zaki (27), it hasbecome clear that the hemolytic activity (14, 15,46) of C. pseudotuberculosis (and by analogythat of Corynebacterium ulcerans), the capacityof products of these two corynebacterial speciesto block the hemolytic activity of the ,B-lysin(phospholipase C) of S. aureus (13-15, 18) andthat of the a-toxin of Clostridium perfringens
t Present address: Institute of Rehabilitation Medicine,New York University Medical Center, New York, NY 10016.
t Present address: Department of Medicine, New YorkHospital, Cornell Medical Center, New York, NY 10021.
(38), and their PLD activity are all one and thesame (27, 37, 39). More recently, Linder andBernheimer purified to near homogeneity thePLD from C. pseudotuberculosis, finding its mo-lecular weight to be around 31,000 with a pI ofabout 9.8. They assessed its activity as sphin-gomyelinase by employing radioactively labeledsphingomyelin (24, 25).The hemolytic activity early ascribed to C.
pseudotuberculosis required its growth on ablood agar plate in reasonably close proximityto a colony of Corynebacterium equi (14), andFraser suggested that the hemolysis observedresulted from the conjoint action on sheep eryth-rocytes of products diffusing from these twobacterial species (14, 15). Bernheimer et al. (8)have investigated the conjoint hemolysis ofsheep erythrocytes by the PLD of C. pseudotu-berculosis and a protein, equi factor, from C.equi. They have purified equi factor and foundit to contain a phospholipase C with a require-ment for activity of either Mg2e or Ca2", to havethe capacity to cleave sphingomyelin to cera-mide and phosphoryl choline, and, further, to becapable of hydrolyzing the phosphate groupfrom ceramide phosphate. The greatest activitywas found when Triton X-100 was included inthe assay mixture. Among the other phospholip-ids hydrolyzed by equi factor were phosphati-dylcholine, phosphatidylserine, and phospha-tidic acid. In cooperatively bringing about thelysis of sheep erythrocytes, PLDs of C. pseudo-tuberculosis make available, to equi factor, cer-amide phosphate split from sphingomyelin atthe surface of the erythrocyte. It has been sug-
335
336 BARKSDALE ET AL.
gested that phospholipase C of equi factor hy-drolyzes ceramide phosphate, modifying the in-tegrity of the cell membrane so that it becomessensitive to physical effects such as chilling, andwhen these occur, lysis ensues (8).There are available, then, two reliable meth-
ods for the detection of the PLDs of C. pseudo-tuberculosis and of C. ulcerans: (i) an assay forsphingomyelinase activity by using radiolabeledsphingomyelin and (ii) an assay for hemolyticactivity in the presence of C. equi. Up to thepresent only a few strains of C. pseudotubercu-losis and of C. ulcerans have been examined forPLD activity. We have employed these two sys-tems for detecting PLD activity among a num-ber of bacteria labeled as C. pseudotuberculosisand C. ulcerans and for ascertaining the distri-bution of PLD among representative speciesbelonging to the genus Corynebacterium. Unlikediphtherial toxin, which occurs in only about50% of field isolates of Corynebacterium diph-theriae (35, 42), PLD appeared to be character-istic of most (if not all) strains of C. pseudotu-berculosis and C. ulcerans, and it was not foundin any other corynebacteria.
MATERIALS AND METHODSBacterial strains. The sources of bacterial strains
employed in this study are listed in Table 1.Production of PLD. Cultures of bacteria were
inoculated into 500 ml of medium (in 2-liter flasks)containing: 220 ml of yeast extract diffusate (preparedas described by Bernheimer and Schwartz [9]), 20 g ofCasamino Acids (Difco Laboratories), 33 ,ug of thia-mine, 1.2 mg of nicotinic acid, and distilled water to 1liter (pH 7.2). Cells were grown to an optical densityof 6.0 at 650 nm, and the supernatant was collectedafter centrifugation at 8,000 x g for 1 h at 4°C. Thepooled culture supernatants were made 80% saturatedwith (NH4)2SO4 at room temperature and left over-night at 4°C. The centrifuged precipitate was washedwith and resuspended in 80% saturated (NH4)2SO4.The suspension from 680 ml of culture was centri-
fuged, the supernatant was discarded, and the pelletwas taken up in approximately 3 ml of 0.1 M sodiumphosphate buffer (pH 6.0) containing 0.25 M NaCl and5% (vol/vol) glycerol.
Estimation of PLD activity of C. pseudotuber-culsis. PLD (sphingomyelinase) activity was esti-mated with radioactively labeled sphingomyelin sus-pensions (1 mg/ml) containing 1 ,uCi of N-[methyl-'4C]sphingomyelin (60 Ci/mol, Radiochemical Centre,Amersham, United Kingdom) per ml as previouslydescribed (24). Incubation mixtures employed in esti-mating sphingomyelinase activity contained: 5 pl of['4C]sphingomyelin suspension (6.7 nmol), 20 pl of 0.1M tris(hydroxymethyl)aminomethane-hydrochloridebuffer (pH 9.2) containing 0.24 M NaCl-5.5 mM MgCl2and 10 p1 of diluted enzyme preparation. Incubationwas at 37°C for 30 min. After incubation, the mixtureswere spotted at the origin of chromatograms of What-
man no. 1 paper and developed for 5 h in ii-butanol-glacial acetic acid-water (2:1:1, vol/vol) to resolvesphingomyelin from free choline. ['4C]choline liber-ated in the reaction was quantified by cutting out theareas corresponding to sphingomyelin and free cholineand counting in a Nuclear Chicago Mark I scintillationspectrometer in aqueous counting scintillation fluidfrom Amersham Corp. (Arlington Heights, Ill.). Oneunit of activity is defined as the amount of enzymereleasing 1 nmol of choline in 30 min.
Cooperative production of hemolysis by PLDof C. pseudotuberculosis and equi factor. Wheneither C. pseudotuberculosis, C. ulcerans, or C. equiwas grown on a neopeptone agar (Difco) containing2% (vol/vol) washed sheep erythrocytes there wasvery little evidence of hemolysis: at the edge of thegrowth of C. pseudotuberculosis there was sometimesa faint clearing, and at the margins of the growth of C.ulcerans there was a slight greening. However, wheneither C. ulcerans or C. pseudotuberculosis was grownin the presence of C. equi, there appeared clear zonesof hemolysis between the two species (see Fig. 1A).This cooperative hemolysis was the result of the con-joint action of the equi factor and the PLDs of C.pseudotuberculosis and C. ulcerans.
The first step in carrying out the tests for coopera-tive hemolysis involved the growth of cultures over-night on chocolate agar slants. A mass of overnightgrowth was taken up on a sterile cotton swab anddeposited (smudged) on a sheep blood agar plate ac-cording to the patterns shown in Fig. 1A, where C.equi was inoculated in the center of the plate, and thecultures of C. pseudotuberculosis and C. ulceranswere inoculated radial to the center. The inoculatedplates were incubated for 48 h (37°C) and scored forhemolysis. As many as eight strains were tested perplate.
Inhibition of the cooperative hemolysis pro-duced by staphylococcal 8-lysin and a proteinfrom Streptococcus agalactiae. When fB-lysin-pro-ducing strains of Staphylococcus aureus are grown onsheep blood agar plates near a strain of Streptococcusagalactiae (at 37°C), an area of clear hemolysis de-velops between the two strains (the CAMP reaction,the name originating from the first letters of Christie,Atkins, and Munch-Petersen, 13). This cooperativehemolysis is subject to interference from the PLD ofC. pseudotuberculosis or that of C. ulcerans (39) (Fig.1B). Since PLD interferes with the CAMP reaction,such interference under controlled conditions offersanother means of demonstrating the presence of PLD.In Fig. 1B the CAMP interaction was set up in such away that a broad band of hemolysis occurred in theareas occupied by Staphylococcus aureus G-128 andStreptococcus agalactiae. Where growth of C. pseu-dotuberculosis and C. ulcerans impinged on the zoneof interaction between Staphylococcus aureus andStreptococcus agalactiae there was inhibition, indi-cated by a diminution in the width of the band ofhemolysis. Inocula for testing were from overnightgrowths on chocolate agar slants. Inoculum was takenup with a sterile swab and smudged on a sheep bloodagar plate by the pattern shown in Fig. 1B.Procedures employed for the biological char-
J. CLIN. MICROBIOL.
Species
Corynebacterium bovis
C. cystitidis
C. diphtheriae
C. diphtheriae subsp. gravis
C. diphtheriae subsp. intermedius
C. equi
C. fascians
C. flavescens
C. genitalium
C. kutscheri
C. liquefaciens
C. minutissimum
C. paurometabolum
C. pilosum
C. pseudodiphtheriticum
C. pseudotuberculosis (C. ovis)
C. renale
C. renale received as C. renale type I
C. ulcerans
ABLE 1. Strains used in this studyStrain no.
ATCC 7715
42(=ATCC 29593)
ATCC 2701053-A-16A8888
NCTC 3985G53-A-7NCTC 7289
NCTC 3987PW8(P)OX+EB79
ATCC 6939
ATCC 12974
ATCC 10340
392-1 type I (=ATCC 33030)
ATCC 15677NCTC 949, NCTC 1386, NCTC 3655C7813
ATCC 14929d
ATCC 23348NCTC 10284, NCTC 1028566-124
ATCC 15530
46 (=ATCC 29592)
ATCC 10700H823
ATCC 19410 (=NCTC 3450)A, LM, 21 (Romania)CSIR-1, CSIR-2, CSIR-5NCTC 4681, NCTC 4683, NCTC 4691NYS 245, NYS 32244
ATCC 10849, ATCC 19412NCTC 7449
ATCC 10848, FS113/63, H25, R/4, 8
CU 170DLC-Hd 81 (=DLC 1613/51), DLC-Hd 84(=DLC 1403/51)
DLC 603/50, DLC 842/50, DLC 903/50,DLC 1605/50, DLC 1661/50, DLC 1771/50
40C, 298G976(LZ)tox, 842(Z)5291, 37142, 39164,.48255, 51166, 51167,
51168, 51169, 521039304
Source (reference)ATCC
RY (45)
(3)bAFIP (5)EIP (33)
NCTCAFIP (5)NCTC
NCTCTL (22)NYU
ATCC
ATCC
(4)
GF (16)
ATCCNCTCJBN (32)
ATCC
ATCCNCTCMEM
ATCC
RY (45)
ATCC(43)AS (31)
ATCCAS (29)HRC (11)NCTCJMC
ATCCNCTC
RY (21, 44)
(19)DHH (20)
SDH (19)
ASS. B. Arden, TLJMC
AS (29)337
Ti
TABLE 1-ContinuedSpecies Strain no. Source (reference)
C. vitarumen ATCC 10234e ATCC12143e (23)
C. xerosis ATCC 7711 ATCCNCTC 9755 NCTC
Staphylococcus aureus G-158 (7)
Streptococcus agalactiae GT-71-890 (10)
'Abbreviations: ATCC, American Type Culture Collection, Rockville, Md.; AFIP, U.S. Armed ForcesInstitute of Pathology; AS, A. Saragea, Cantacuzino Institute, Bucharest, Romania; DHH, D. H. Howard; EIP,E. I. Parsons; GF, G. Furness; HRC, H. R. Carne; JBN, J. B. Nelson; JMC, J. M. Coffey, New York StateDepartment of Health, Albany, N.Y.; MEM, M. E. McBride, Baylor College of Medicine, Houston, Tex.; NCTC,National Collection of Type Cultures, Central Public Health Laboratory, London NW9 5HT, England; NYU,Collection of the Department of Microbiology, New York University School of Medicine, New York, N.Y.; RY,R. Yanagawa; SDH, S. D. Henricksen; TL, this laboratory. Reference numbers indicate sources of additionalinformation concerning a given strain.
b From L. Barksdale, in M. P. Starr et al., The Prokaryotes, in press.'Received as C. murium (kutscheri)." Received as Brevibacterium liquefaciens.Received as B. vitarumen.
acterization of corynebacteria received as C.pseudotuberculosis and C. ulcerans. The proce-dures for the biological characterization of corynebac-teria have been described recently (4): (i) presence ofpolyphosphate (metachromatic) granules, (üi) reduc-tion of potassium tellurite, (iii) catalase activity, (iv)glucan phosphorylase (a-1,4-glucan:orthophosphateglucosyl transferase) activity, (v) production of hydro-gen sulfide, (vi) deamidation of pyrazinamide, (vii)fermentation of carbohydrates, (viii) hydrolysis ofpolyoxyethylene sorbitan conjugates, (ix) hydrolysisof sodium hippurate, (x) decarboxylation of ornithineand lysine, (xi) fmal hydrogen ion concentration inbuffered peptone glucose broth, (xii) production ofacetoin, (xiii) gelatinase activity, (xiv) nitrate reduc-tase activity, (xv) hydrolysis of urea, (xvi) proteolyticaction on milk, (xvii) alkaline phosphatase activity,and (viii) phage susceptibility.
RESULTSIn Table 2 are shown the results of assays for
PLD (as sphingomyelinase) activity of severalstrains of C. pseudotuberculosis. All strainswhich gave biochemical reactions characteristicof the species exhibited PLD activity. Fivestrains which gave negative results proved tohave been wrongly identified as C. pseudotuber-culosis (see last column in Table 2). The eightstrains of c. pseudotuberculosis listed in the firstcolumn of Table 2 all produced marked hemol-ysis on sheep blood agar plates in the presenceof C. equi (Fig. 1). For reasons of economy andbecause of the sensitivity of the test, the PLDactivities of all other strains of C. pseudotuber-culosis and of C. ulcerans were determined inthe presence of C. equi on plates of washedsheep blood agar, and the results are given in
Table 3. As indicated in footnote a of Table 3,each of the PLD-positive strains inhibited thehemolysis resulting from the interaction of ,B-lysin of Staphylococcus aureus and the CAMPprotein of Streptococcus agalactiae with sheeperythrocytes. The strains of C. diphtheriae listedin Table 3 were without PLD activity, as wereall other corynebacteria listed in Table 1.
DISCUSSIONSoucek, Souckova, and their associates, in a
landmark series ofpapers concerning diphtherialtoxin, ovis toxin, and the PLDs of C. pseudotu-berculosis and C. ulcerans, employed the follow-ing strains: C. pseudotuberculosis (C. ovis)NCTC 4655 (36, 37, 39) and C. ulcerans ATCC9015 (39), B48/55, IC7210, and NCTC 7910 (40).We were interested in PLD both as a geneticmarker and as a property useful in the taxonomyof Corynebacterium spp. We assembled a num-ber of strains designated as C. pseudotubercu-losis (Table 2) and examined them for theircapacity to liberate [14C]choline from labeledsphingomyelin. Some of the strains failed toproduce detectable levels of PLD as sphingo-myelinase activity. Furthermore, ofthree strainsof C. ulcerans similarly tested, one producedjust detectable activity, and the others producednone. This cursory survey (data in column 1 ofTable 2) suggested that PLD was no more pe-culiar to all strains of C. pseudotuberculosis andC. ulcerans than was diphtherial toxin peculiarto all strains of C. diphtheriae; the latter werefound in only 50% of isolates during outbreaksof diphtheria (35, 42). However, this preliminary
338 BARKSDALE ET AL. J. CLIN. MICROBIOL.
PHOSPHOLIPASE D ACTIVITY IN CORYNEBACTERIA 339
o
o4 300a>'ea4> r
à.
à
zz z çjz ç
o00~~~ + + +++++ + ++ ++ +
|5@+ ++++++ +++ +++
+1+++1 ++I 111
Sæ +1 + +1 ++ III
ccIII + + +
! I1 I II ++lçi
c (X~~~~~~~~~~~~~~~~Io~~~~~~~~~~~~~~
+ ++++++ + ++ III
+1I+1I+1 ++ I +1 .
o ~~~~~~~~~~~~~cc
+ +++ + + + +
_ cat + ++++++ +++ ++ c
a>+ + + + + + +I ++I III
q
f, I1 I I I+++
X~~~ L
ciEÉ t ONOOO OOO OOO .c
0000do-' oo00
O a~~~~~~c~cy
.Q0Ça..
O o.c
Q.
O .~~~~~~~~~~~Out 4< c
H m e e ;eilei xeix QéE.cc Go Go m GO Uo E- E-
<~~ ~
w < <
>0
~
~o
-2c
D.
c
cc
VOL. 13, 1981
a..
L.
.0
a...
a..
.0
c..>
*0,
oQs
340 BARKSDALE ET AL.
A
B
FIG. 1. Effects of the PLDs of C. pseudotubercu-losis and of C. ulcerans which can be observed mac-
roscopically on sheep blood agar plates. (A) Conjointhemolysis of sheep erythrocytes by PLD and equifactor. The diagram is from a tracing of a platecontaining 2% washed sheep erythrocytes, inoculatedas follows and incubated for 48 h at 37C. C. equiwas inoculated at the center, and radiating from it(clockwise) were inoculated C. ulcerans 39164, C.pseudotuberculosis A, C. ulcerans 37142, and C. pseu-dotuberculosis 19140. The stippled area representsunlysed erythrocytes. The eight-"sided" zone of lysishas resulted from the diffusion of PLD from thegrowth of C. pseudotuberculosis and of C. ulceransinto an area also penetrated by equi factor diffusingfrom the central growth of C. equi. (B) The inhibitionof hemolysis resulting from the interaction of f-lysin
assessment regarding PLD activity in C. pseu-dotuberculosis and C. ulcerans proved to beerroneous for two reasons. First, careful bio-chemical characterization of the strains we hadexamined revealed that five of them werewrongly identified, as is clearly shown in Table2. Second, strain DLC603/50 of C. ulcerans,which failed to produce detectable PLD by thesphingomyelinase test, did yield positive resultson sheep blood plates. Souckova and Soucek(40) had pointed out that the strains of C. ulcer-ans with which they worked yielded about 1/100 or less sphingomyelinase activity than didstrains of C. pseudotuberculosis. Bernheimer etal. have demonstrated that a small amount ofpurified corynebacterial PLD (65 U) cross-streaked with a small amount of equi factor (5U) could produce a visible spot of lysis on asheep blood agar plate. Thus, the plate assay issuitable for detecting small amounts of PLD inthe presence of equi factor (8).
In Table 3 it is evident that all strains of C.pseudotuberculosis and C. ulcerans producedPLD as well as urease, and that among thepyrazinamidase-negative corynebacteria theyalone produced these two enzymes. Table 3 alsoindicates that these two species can readily beseparated one from the other on the basis ofstarch fermentation, and that within C. ulceransthere are strain differences with regard to thefermentation of trehalose and the hydrolysis ofTween 60.The value ofPLD activity in the identification
of C. pseudotuberculosis and C. ulcerans is evi-dent from results reported here and in recentliterature. The use of sheep blood agar plates fordetecting PLD activity is most convenient. As ameans of characterizing other corynebacteria,activities comparable to that of equi factorshould be sought. Additional specificity mightbe brought to such systems by the use of controlplates containing specific antibody directedagainst one of the agents of cooperative hemol-ysis, e.g., equi factor.
ACKNOWLEDGMENTSWe are grateful to A. W. Bernheimer for numerous helpful
discussions during the course of this study. We thank Diana
of Staphylococcus aureus and CAMP protein ofStreptococcus agalactiae with sheep erythrocytes.The diagram is from a tracing, as in (A). The bandof hemolysis shown resulted from the diffusion of thejl-lysin (phospholipase C) of Staphylococcus aureusG158 into a broad zone of CAMP protein diffusingfrom the two areas in which Streptococcus agalactiaeGT-71-890 has grown. The constriction in the band isin the zone of diffusion ofPLD from C. pseudotuber-culosis 19140 and C. ulcerans 37142. For further de-tails, see text.
J. CLIN. MICROBIOL.
VOL. 13, 1981 PHOSPHOLIPASE D ACTIVITY IN CORYNEBACTERIA 341
I I + Il++ +++ I 1
w~~~~~~~~~~~~~~~~~++ i!+ + ++ +++ + +o+
z Ma+ +~~~~~~++++ ++ ++t-coq1~~~~~~~~~~~
21 {1+~ + I+ III "'a5
i {g 83+ II, 3M .,| {IIIO~~~~~~Mc
~~o + +~î ++++m+
+~ + + ++ +
+ + ++ ++ +++ ++
+. ç ++ ++ ++ ++.1+ II
CfD z~~~~~~-
342 BARKSDALE ET AL.
Grant for her expert production of the tables and MichelleOwens for much help with the manuscript.
Part of this investigation was supported by Public HealthService grant AI-02874 from the National Institute of Allergyand Infectious Diseases to A. W. Bernheimer.
LITERATURE CITED
1. American Type Culture Collection. 1978. Catalogue ofstrains, 13th ed. American Type Culture Collection,Rockville, Md.
2. Arden, S. B. 1970. Comparative studies of Corynebacte-rium diphtheriae, C. ovis, C. ulcerans and related spe-
cies. University Microfilms, Ann Arbor, Mich.3. Barksdale, L. 1970. Corynebacterium diphtheriae and
its relatives. Bacteriol. Rev. 34:378-422.4. Barksdale, L., M.-A. Lanéelle, M. C. Pollice, J. Asse-
lineau, M. Welby, and M. Norgard. 1979. Biologicaland chemical basis for the reclassification of Microbac-terium flavum Orla-Jensen as Corynebacterium flaves-cens nom. nov. Int. J. Syst. Bacteriol. 29:222-233.
5. Barksdale, W. L., K. Li, C. S. Cummins, and H.Harris. 1957. The mutation of Corynebacterium py-ogenes to Corynebacterium hemolyticum. J. Gen. Mi-crobiol. 30:749-758.
6. Barratt, M. M. 1933. A group of aberrant members of thegenus Corynebacterium isolated from the human na-
sopharynx. J. Pathol. Bacteriol. 36:369-397.7. Bernheimer, A. W., L. S. Avigad, and K.-S. Kim. 1974.
Staphylococcal sphingomyelinase (ffl-hemolysin). Ann.N. Y. Acad. Sci. 236:292-306.
8. Bernheimer, A. W., R. Linder, and L. S. Avigad. 1980.Stepwise degradation of membrane sphingomyelin bycorynebacterial phospholipases. Infect. Immun. 29:123-131.
9. Bernheimer, A. W., and L. L. Schwartz. 1963. Isolationand composition of staphylococcal alpha toxin. J. Gen.Microbiol. 30:455-468.
10. Brown, J., R. Farnsworth, L. W. Wannamaker, andD. W. Johnson. 1974. CAMP factor of group B strep-tococci: production, assay, and neutralization by sera
from immunized rabbits and experimentally infectedcows. Infect. Immun. 9:377-383.
11. Carne, H. R. 1939. A bacteriological study of 134 strainsof Corynebacterium ovis. J. Pathol. Bacteriol. 49:313-328.
12. Carne, H. R. 1940. The toxin of Corynebacterium ovis. J.Pathol. Bacteriol. 51:199-212.
13. Christie, R., N. E. Atkins, and E. Munch-Petersen.1944. A note on a lytic phenomenon by group B strep-tococci. Aust. J. Exp. Biol. Med. Sci. 22:197-200.
14. Fraser, G. 1961. Haemolytic activity of Corynebacteriumovis. Nature (London) 189:246.
15. Fraser, G. 1964. The effect on animal erythrocytes ofcombinations of diffusable substances produced by bac-teria. J. Pathol. Bacteriol. 88:43-53.
16. Furness, G., and A. T. Evangelista. 1978. A diagnostickey employing biological reactions for differentiatingpathogenic Corynebacterium genitalium (NSU cory-nebacteria) from commensals of the urogenital tract.Invest. Urol. 16:1-4.
17. Goel, M. C., and I. P. Singh. 1972. Purification andcharacterization of Corynebacterium ovis exotoxin. J.Comp. Pathol. 82:345-353.
18. Hartwigk, H. 1963/1964. Antihamolysinbildung beimTier vorkommender f3-hamolytischer Corynebakterien.Zentralbl. Bakteriol. Parsitenkd. Infektionskr. Hyg.Abt. 1 Orig. 191:274-280.
19. Henriksen, S. D., and R. Grelland. 1952. Toxigenicity,serological reactions and relationships of the diphthe-ria-like corynebacteria. J. Pathol. Bacteriol. 64:503-511.
20. Howard, D. H., and G. J. Jann. 1954. The isolation andcharacterization of bacteriophage active against the
J. CLIN. MICROBIOL.
diphtheria-like corynebacteria. J. Bacteriol. 68:316-319.
21. Kumazawa, N., and R. Yanagawa. 1969. DNA basecompositions of the three types of Corynebacteriumrenale. Jpn. J. Vet. Res. 17:115-120.
22. Lampidis, T., and L. Barksdale. 1971. Park-Williamsnumber 8 strain of Corynebacterium diphtheriae. J.Bacteriol. 105:77-85.
23. Lanéelle, M.-A., and J. Asselineau. 1977. Glycolipidsof Brevibacterium vitarumen. Biochim. Biophys. Acta486:205-208.
24. Linder, R., and A. W. Bernheimer. 1978. Effect onsphingomyelin-containing liposomes of phospholipaseD from Corynebacterium ovis and the cytolysin fromStoichactis helianthus. Biochim. Biophys. Acta 530:236-246.
25. Linder, R., A. W. Bernheimer, and K.-S. Kim. 1977.Interaction between sphingomyelin and a cytolysinfrom the sea anemone Stoichactis helianthus. Biochim.Biophys. Acta 467:290-300.
26. Lovell, R., and M. M. Zaki. 1966. Studies on growthproducts of Corynebacterium ovis. I. The exotoxin andits lethal action on white mice. Res. Vet. Sci. 7:302-306.
27. Lovell, R., and M. M. Zaki. 1966. Studies on growthproducts of Corynebacterium ovis. Il. Other activitiesand their relationship. Res. Vet. Sci. 7:307-311.
28. Mair, W. 1928. A strain of B. diphtheriae showing unusualvirulence for guinea-pigs. J. Pathol. Bacteriol. 31:136-137.
29. Maximescu, P., A. Oprisan, A. Pop, and E. Potorac.1974. Further studies on Corynebacterium species ca-pable of producing diphtheria toxin (C. diphtheriae, C.ulcerans, C. ovis). J. Gen. Microbiol. 82:49-56.
30. Maximesco, P., A. Pop, A. Oprisan, and E. Potorac.1968. Relations biologiques entre Corynebacterium ul-cerans, Corynebacterium ovis et Corynebacteriumdiphtheriae. Arch. Roum. Pathol. Exp. Microbiol. 27:733-750.
31. Meitert, E., A. Saragea, A. Petrovica, and M. Ionescu.1972. Studies on the host phage systems of Corynebac-terium hofmanni species. Arch. Roum. Pathol. Exp.Microbiol. 31:31-37.
32. Nelson, J. B. 1973. Response of mice to Corynebacteriumkutscheri on footpad injection. Lab. Anim. Sci. 23:370-372.
33. Parsons, E. I. 1955. Induction of toxigenicity in non-toxigenic strains of C. diphtheriae with bacteriophagesderived from non-toxigenic strains. Proc. Soc. Exp. Biol.Med. 90:91-93.
34. Petrie, G. F., and D. MeClean. 1934. The inter-relationsof Corynebacterium otis, Corynebacterium diphthe-riae, and certain diphtheroid strains derived from thehuman nasopharynx. J. Pathol. Bacteriol. 39:635-663.
35. Saragea, A., P. Maximescu, E. Meitert, I. Stuparu, E.Vieru, V. Petrus, and C. Balteanu. 1966. Incidentasi distributia geografica a tipurilor fagice de Corynebac-terium diphtheriae. Microbiol. Parazitol. Epidemiol.11:351-362.
36. Soucek, A., C. Michalec, and A. Souckova. 1971. Iden-tification and characterization of a new enzyme of thegroup "phospholipase D" isolated from Corynebacte-rium ovis. Biochim. Biophys. Acta 227:116-128.
37. Soucek, A., and A. Souckova. 1974. Toxicity of bacterialsphingomyelinases D. J. Hyg. Epidemiol. Microbiol.Immunol. 18:327-335.
38. Soucek, A., A. Souckova, and F. Patocka. 1967. Inhi-bition of the activity of a-toxin of Clostridium perfrin-gens by toxic filtrates of corynebacteria. J. Hyg. Epi-demiol. Microbiol. Immunol. 11:123-124.
39. Souckova, A., and A. Soucek. 1972. Inhibition of thehemolytic action of a and ,B lysins of Staphylococcuspyogenes by Corynebacterium hemolyticum, C. ovis and
PHOSPHOLIPASE D ACTIVITY IN CORYNEBACTERIA 343
C. uléerans. Toxicon 10:501-509.
40. Souckova, A., and A. Soucek. 1974. Two distinct toxicproteins in Corynebacterium ulcerans. J. Hyg. Epide-miol. Microbiol. Immunol. 18:336-341.
41. Sulea, I. T., M. C. Police, and L Barksdale. 1980.Pyrazine carbosylamidase activity in Corynebacterium.Int. J. Syst. Bacteriol. 30:466-472.
42. Toshach, S., A. Valentine, and S. Sigurdson. 1977.Bacteriophage typing of Corynebacterium diphtheriae.J. Infect. Dis. 136:655-660.
43. Welby-Gieusse, M., M.-A. Lanéelle, and J. Asseli-
neau. 1970. Structure des acides corynomycoliques deCorynebacterium hofmanùi et leur implication bio-génétique. Eur. J. Biochem. 13:164-167.
44. Yanagawa, R. 1975. A numerical taxonomic study of thestrains of three types of Corynebacterium renale. Can.J. Microbiol. 21:824-827.
45. Yanagawa, R., and E. Honda. 1978. Corynebacteriumpilosum and C. cystitidis, two new species from cows.
Int. J. Syst. Bacteriol. 28:209-216.46. Zaki, M. M. 1965. Production of a soluble substance by
Corynebacterium ovis. Nature (London) 205:928-929.
VOL. 13, 1981
