FEMS Microbiology Letters 4 (1978) 55-58 © Copyright Federation of European Microbiological Societies Published by Elsevier/North-Holland Biomedical Press

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CR YP TIC PLASMIDS IN C L O S T R I D I U M B O T U L I N U M AND C. B O T U L I N U M - L I K E O R G A N I S M S

V.N. SCOTT * and C.L. DUNCAN **

Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706, U.S.A.

Received 11 April 1978

1. Introduction

The presence of extrachromosomal deoxyribonu- cleic acid (DNA) in a wide variety of bacteria has been reported [1 ]. Recently, plasmids have been demonstrated in Clostridium perfringens [2-4] . To our knowledge there have been no reports of plasmids in other clostridia. This paper presents the first evi- dence of plasmids in Clostridium botulinum and C. botulinum-like organisms.

2. Materials and Methods

All strains were obtained from Dr. H. Sugiyama (Food Research Institute, University of Wisconsin).

Plasmids were isolated from cells grown in 100 ml oftrypticase-peptone-glucose broth [5] containing 0.4% (w/v) yeast extract. Lysis was according to the method of Rood et al. [5] except that incubation with lysozyme was for 60 min and sodium dodecyl sufate, Brij-desoxycholate or Triton X-100 were used instead of sodium lauryl sarcosinate. The DNA was not sheared. When 500-ml cultures were used DNA was concentrated by precipitation with polyethylene- glycol 6000 [6]. Plasmids were isolated from crude lysates using cesium chloride-ethidium bromide gra- dients. Plasmid bands were visualized infrequently using a long-wave UV light; they were isolated by extracting an area 5 -10 mm beneath the chromo- somal band or by pooling the satellite DNA fractions

* To whom correspondence should be addressed. ** Present address: Campbell Institute for Food Research, Campbell Place, Camden NJ 08101, U.S.A.

from a gradient containing radioactively labelled DNA [5]. The isolation of plasmids by the various lysis techniques used was somewhat inconsistent, both between strains and even with the same strain. The reasons for this inconsistency are not known. Use of diethylpyrocarbonate [7] in the lysis procedure improved the consistency of plasmid isolation, whereas the addition of pronase, ribonuclease or Pro- teinase K did not.

Plasmid preparations were nicked using ethidium bromide and light or X-rays (5 min, 1800 rad/min). DNA'was prepared for electron microscopy by the Kleinschmidt technique [8]. The contour lengths of individual plasmid molecules were determined relative to a calibration grid and converted to molecular weights using the formula 1 wn = 2.07 megadaltons (Mdal). Plasmids R6K and ColE1 were measured as controls. The molecular weights obtained (26.2 -+ 0.4, 10 measurements and 4.3 -+ 0.2, 13 measurements) agree well with the literature values [9,10].

3. Results and Discussion

Fig. 1 shows electron micrographs of some of the plasmids isolated. Table 1 summarizes the data ob- tained from histograms of the plasmid measurements. The E-like, boticinogenic strains each contained three 2 -5 Mdal plasmids. There were 3.6-3.8 Mdal and 2.3-2.6 Mdal plasmid size classes common to all foul E-like boticinogenic strains examined, but it is not yet known if these represent identical plasmids com- mon to these strains. It is of interest to note that the strains were isolated from different fish and sediment samples ([11 ], K.L. Anastasio, 19 69, M.S. thesis,

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Fig. 1. Electron micrographs of plasmids from strains of Clostridium botulinurn and C. botulinurn-like organisms: (A) 6.9 Mdal plasmids from strain CBW 24; (B) 2.4 Mdal plasmids from strain CBW 18; (C) 3.7 and 4.9 Mdal plasmids from strain CBW 20; (D) 22.9 Mdal plasmid from strain CBW 2. Bar represents 0.5/~m.

TABLE 1

Summary of plasmic measurements from strains of C. botu- linum and C. botulinum-like organisms

Strain a Previous Molecular Number of designation weight measure-

(Mdal) ments

CBW 2 6d

CBW 20 A3759

CBW 18 S-5

CBW 19 28-2

2.3±0.2 145 3.8±0.2 25 5.5±0.5 6 7.4 1 8.8 1

22.3±1.9 9 41.7 1

2.3±0.3 141 3.7±0.2 24 4.9±0.2 43 6.5 1 8.4 1

23.4 1

2.4±0.2 63 3.6±0.2 17 4.7±0.2 5

18.0±0.0 2

1.9±0.1 31 2.6±0.1 113 3.7±0.2 33 5.2 1 6.2 1

19.4 1

CBW 24 A62 6.9 ± 0.2 85 18.9 1

CBW 13 Minnesota 1.9 1 18.3 1

a Strains were non-toxic, E-like and produce boticin E except strain CBW 24 which was a toxic type A and CBW 13 which was a toxic type E.

University o f Wisconsin). It is possible that one o f these plasmids may determine the product ion o f boticin E [12].

In all strains examined larger plasmids ( 1 8 - 2 5 Mdal) were observed but only a few measurements were obtained. Evidence o f these larger plasmids was also occasionally obtained by agarose gel electropho- resis [5].

Plasmid preparations cut by EcoRI restriction

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endonuclease (Rood, J.I., V.N. Scott , and C.L. Dun- can, in preparation) showed discrete bands on agarose gels (data not shown) indicating that the covalently closed circular DNA molecules seen by electron mi- croscopy were not generated randomly from chromo- somal fragments.

Addit iogal strains of C. botulinum and C. botuli-

num-like organisms are currently being examined for the presence of plasmids. The relationship o f plasmids to boticinogenici ty, toxigenicity and antibiotic resis- tance in these strains is also being examined. Isolation or plasmids from C. botulinum types C and D should also be possible if the bacteriophage responsible for toxic i ty in these toxigenic types exists in a pseudo- lysogenic state as has been proposed [13,14].

Acknowledgements

This research was supported by the College of Agri- cultural and Life Sciences, University of Wisconsin, Madison, and by contributions to the Food Research Insti tute by member industries. C.L.D. was the reci- pient o f Public Health Service Research Career Deve-

lopment Award AI-70721-03 from the National Insti- tute of Allergy and Infectious Diseases. We wish to thank Dr. H. Sugiyama for the strains used in this s tudy and Dr. M.B. Yatvin for his assistance with X- ray nicking.

References

[1] Schlessinger, D. (Ed.) (1975) Microbiology-1974, pp. 7-26. American Society for Microbiology, Washington, D.C

[2] Sebald, M., Bouanehaud, D. and Bieth, G. (1975) C.R. Acad. Sci. Paris (D) 280, 2401-2404.

[3] Ionesco, H., Bieth, G., Dauguet, C. and Bouanchaud, D. (1976) Ann. Microbiol. (Inst. Past.) 127,283-293.

[4] Duncan, C.L., Rokos, E.A., Christenson, C.M. and Rood, J.I. (1978) in: Microbiology-1978 (Schlessinger, D., Ed.), pp. 246-248. American Society for Micro- biology, Washington, D.C.

[5 ] Rood, J.I., Maher, E.A., Somers, E.B., Campos, E. and Duncan, C.L. (1978) Antimicrnb. Agents Chemother. 13.

[6] Humphreys, G.O., WiUshaw, G.A. and Anderson, E.S. (1975). Biochim. Biophys. Acta 383,457-463.

[7] Worcel, A. and Burgi, E. (1972) J. Mol. Biol. 71,127- 147.

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[8] Kleinschmidt, A.K. (1968) in: Methods in Enzymology, Vol. XII, part B (Grossman, L. and Moldave, K., Eds.), pp. 361-377, Academic Press, New York.

[9] CleweU, D.B. and Helinski, D.R. (1969). Proc. Natl. Acad. Sci. USA 62, 1159-1166.

[10] Kupersztoch-Portnoy, Y.M., Miklos, G.L.G. and He- linski, D.R. (1974) J. Bactedol. 120, 545-548.

[11] Kautter, D.A., Harmon, S.M., Lynt Jr., R.K. and Lilly

Jr., T. (1966) Appl. Microbiol. 14,616-622. [12] Anastasio, K.L., Soucheck, J.A. and Sugiyama, H.

(1971) J. Bacteriol. 107,143-149. [13] Eklund, M.W., Poysky, F.T. and Reed, S.M. (1972)

Nature New Biol. 235, 16-17. [14] Eklund, M.W., Poysky, F.T., Reed, S.M. and Smith, C.A.

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