Clostridium tetani Is a phospholipase (lecithinase)-producing bacterium.

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Title
Clostridium tetani is a phospholipase (Lecithinase)-producing bacterium
[1]
Author(s)Temaru, Emi; Shimura, Satoshi; Karasawa, Tadahiro
Citation Journal of Clinical Microbiology, 43(4): 2024-2025
Issue Date2005
TypeJournal Article
Text versionpublisher
URLhttp://hdl.handle.net/2297/6940
Right
http://dspace.lib.kanazawa-u.ac.jp/dspace/

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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2005, p. 2024–2025
0095-1137/05/$08.00?0doi:10.1128/JCM.43.4.2024–2025.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 4
Letters to the Editor
Clostridium tetani Is a Phospholipase (Lecithinase)-Producing Bacterium
Many Clostridium spp. produce phospholipases (lecithi-
nases). The phospholipase of Clostridium perfringens is known
as alpha-toxin (Cpa), which is the best characterized of all
clostridial phospholipases. C. tetani, the causative organism of
tetanus, has been recognized as a phospholipase-negative clos-
tridium. In the course of our research on clostridial phospho-
lipases (2, 3), a BLAST homology search revealed that the C.
tetani E88 genome (1) contained a cpa-related gene (gene
identifier, 1058703; locus tag, CTC00990). Cpa consists of two
domains, the N-terminal and C-terminal domains (2). The
N-terminal domain has phospholipase activity, while the C-
terminal domain is a calcium-dependent putative phospholipid
binding domain. According to the E88 genome sequence, the
phospholipase gene of C. tetani E88 comprising 242 amino acid
residues has similarity with the N-terminal domain of Cpa and
does not have a domain corresponding to the C-terminal do-
main. The presence of this gene prompted us to examine phos-
pholipase production by C. tetani.
All tested strains—NCTC 279 (the type strain of C. tetani),
NCTC 5404, CN 655 (Pasteur Institute strain 105554), Ramon
(Pasteur Institute strain 60.28), KZ 1180, KZ 1186, KZ 1189,
and KZ 1199—had been identified as C. tetani. The strains KZ
1180, KZ 1186, KZ 1189, and KZ 1199 were isolated from soil.
In this study we confirmed these strains as C. tetani by using a
16S rRNA gene bacterial sequencing kit (Microseq 500; Ap-
plied Biosystems). C. perfringens KZ 221 (3) and C. butyricum
NCTC 7423 were used for phospholipase-positive and -nega-
tive controls. An anaerobic culture using mixed gases (85% N2,
10% CO2, and 5% H2) was carried out at 37°C for 2 days.
We found that C. tetani NCTC 5404 expressed a positive
FIG. 1. Phospholipase production by C. tetani strains on PRYG agar medium containing 5% egg yolk. (A to D) A phospholipase reaction
characterized by opacity around a colony is observed. Yellow arrowheads indicate representative phospholipase-positive colonies. (E to H) A
phospholipase reaction mixture is distributed on the medium plates. (A) Ramon; (B) KZ 1189; (C) NCTC 5404; (D) CN 655; (E) NCTC 279;
(F) KZ 1180; (G) KZ 1186; (H) KZ 1199; (I) C. perfringens KZ 221 (phospholipase-positive control); (J) C. butyricum NCTC 7423 (phospholipase-
negative control). For C. butyricum, 0.5% glucose was added to the medium.
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phospholipase reaction, characterized by opacity around a col-
ony, on a medium, the basal formula of which had been used
for spore formation of C. tetani in our laboratory. We therefore
examined medium conditions to enhance phospholipase pro-
duction. The alterations were based on the knowledge that C.
tetani possesses an extensive sodium ion bioenergetics and a
sodium-glucose or galactose cotransporter (gene identifier,
1059145; locus tag, CTC01237) (1) and that several C. tetani
strains are also able to utilize glucose (4, 5). Finally, we devel-
oped a peptone-rich yeast extract glucose (PRYG) agar me-
dium containing 5% (vol/vol) egg yolk. PRYG agar medium
consisted of the following per liter of water: 40 g of Bacto
proteose peptone 2 (Difco Laboratories), 10 g of yeast extract
(Difco Laboratories), 1 g of glucose, and 20 g of agar (pH 7.4).
In brief, PRYG agar medium contained a high concentration
of peptone and a small amount of glucose but no sodium
chloride. All strains clearly showed the phospholipase reaction
on PRYG agar medium containing 5% egg yolk (Fig. 1). It was
noted that not all colonies produced phospholipase. Research
on the regulation system of the gene expression is awaited.
Additionally, we tested a commercial brain heart infusion agar
(Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) for NCTC
279, NCTC 5404, KZ 1180, KZ 1186, KZ 1189, and KZ 1199.
Growth of all strains on brain heart infusion agar containing
5% egg yolk was poor, but the NCTC 5404, KZ 1186, KZ 1189,
and KZ 1199 strains exhibited the phospholipase reaction after
4 days of incubation.
These results demonstrated that under the appropriate cul-
ture conditions, C. tetani is a phospholipase-producing bacte-
rium. C. tetani phospholipase may be produced in vivo and
contribute indirectly to tetanus by helping nutrient acquisition
for cell growth and multiplication.
We are grateful to S. Nakamura for providing C. tetani strains; to X.
Wang and Y. Sakaguchi for critical discussion; to C. Ishiyama, Y.
Ohiwa, and C. Nabeta for technical assistance; and to J. J. Ferretti for
reviewing the manuscript.
REFERENCES
1. Bru ¨ggemann, H., S. Ba ¨umer, W. F. Fricke, A. Wiezer, H. Liesegang, I. Decker,
C. Herzberg, R. Martinez-Arias, R. Merkl, A. Henne, and G. Gottschalk.
2003. The genome sequence of Clostridium tetani, the causative agent of
tetanus disease. Proc. Natl. Acad. Sci. USA 100:1316–1321.
2. Clark, G. C., D. C. Briggs, T. Karasawa, X. Wang, A. R. Cole, T. Maegawa,
P. N. Jayasekera, C. E. Naylor, J. Miller, D. S. Moss, S. Nakamura, A. K.
Basak, and R. W. Titball. 2003. Clostridium absonum alpha-toxin: new insights
into clostridial phospholipase C substrate binding and specificity. J. Mol. Biol.
333:759–769.
3. Karasawa, T., X. Wang, T. Maegawa, Y. Michiwa, H. Kita, K. Miwa, and S.
Nakamura. 2003. Clostridium sordellii phospholipase C: gene cloning and
comparison of enzymic and biological activities with those of Clostridium
perfringens and Clostridium bifermentans phospholipase C. Infect. Immun.
71:641–646.
4. Society of American Bacteriologists. 1923. Bergey’s manual of determinative
bacteriology, 1st ed. Williams & Wilkins, Baltimore, Md.
5. Yamagishi, T. 1969. Biological properties of Clostridium tetani: (II) glucose
utilization by Clostridium tetani. J. Juzen Med. Soc. 5:504–508 (In Japanese
with English abstract.)
Emi Temaru
Satoshi Shimura
Tadahiro Karasawa*
Department of Laboratory Sciences
School of Health Sciences
Kanazawa University
Kodatsuno
Kanazawa, Japan
*Phone: 81 (76) 265-2597
Fax: 81 (76) 234-4369
E-mail: karasawa@mhs.mp.kanazawa-u.ac.jp
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