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Plasmid content in Yersinia pestis strains of different origin
FEMS MicrobiologyLeners 67 0990) 45-48 Published by Elsevier
45
FEMSLE 03858
Plasmid content in Yersinia pestis strains of different origin A n d r e w A. Filippov i Nikolai S. Solodovnikov 2, Liubov M. K o o k l e v a ~ a n d Olga A. Protsenko I Department of Genetics and ~ Collection of bring Cultures, All-Union Research Anti-Plague Institule 'Microbe'. Saratov. U.S.S.R.
Received l l September 1989 Accepted 20 September 1989 Key words: Yersiniapeslis strains; Plasmid content
1. S U M M A R Y Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains. 172 (71%) wcrc shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 MDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.
2. I N T R O D U C T I O N It has been shown that Yersinia p e s t i s - - c a u s a rive agent of plague--carries, as a rule, three plasmids, namely 6-MDa replicon pPst, encoding synthesis of pesticin 1 bacteriocin as well as the production of fibrinolysin/coagulase [1-4]; a Correspondence to." Andrew A. Filippov, Department of Genetics, AIbUnion Re,arch Anti-Plague Institute "Microbe', 410071 S.aratov, Univcrsitetskaya46. U.S.S.R.
45-50-MDa plasmid, pCad. determining cell growth dependence upon Ca -'+ ions at 37°C. synthesis of the V antigen and a number of thermoinducible outer membrane proteins [2,3,5-7]; and a 60-MDa plasmid, pFra, encoding the production of capsular antigen known as fraction I and murine toxin [4]. Products of all three plasraids are implicated in expression of virulence. In the territory of the U.S.S.R.. there are 15 natural plague loci differing by geographical peculiarities and species of rodents and their ectoparasites, the bacterium carriers and vectors, respectively. There are a few communications which describe plasmid profile features of strains from diverse plague loci. it has been shown that Y. pestis isolates from voles obtained in the Caucasus lack pPst and carry pFra considerably increased in size [8.9 I. Strains from the Central-Caucasian focus (the carriers are susliks) which, besides the three above-mentioned plasmids, harbour an additional extrachromosomal replicon of about 1.5-2 MDa have been described [10]. Another additional cryptic plasmid (15-16 MDa) has recently been found in strains from the Tuvinian focus (the south of East Siberia) [11]. The purpose of our work was a comparative study of plasmid content in a considerable num-
0378-1097/90/$03.50 © 1990 Federation of European Microbiolo~cal .Societies
46 ber of Y. pestis strains from the majority of natural plague foci of the U.S.S.R. and also from several Asian, African and Latin American countries.
I 2 3 4 S 6 7 $ 9 I0t'112 q~5141516'ITlSq9
3. MATERIALS A N D M E T H O D S Y. pestis and Escherichia coil strains used were received from the Collection of Living Cultures of All-Union Research Anti-Plague Institute "Microbe' (Saratov). The plasmids from E. coil C600 (pBR327) (2.2 MDa), DH1 (pBR322) (2.9 MDa), C600 (pCR1) (7.5 MDa), J53(S-a) (23 MDa), C600 (R6K) (26 MDa), J53 (RP4) (36 MDa), K-12 (F +) (62 MDa), JC411 (R386) (79 MDa) and CSH2 (Rtsl) (140 MDa) were used as molecular mass markers. The main phenotypic determinants encoded by Y. pestis plasmids: calcium dependence, pesticin I and fraction I production were tested as described previously [4,12]. Plasmids from Y. pestis and E. coil were screened by the method of Kado and Liu
[13].
4. RESULTS A N D DISCUSSION We studied plasmid content in 242 Y. pestis strains from 14 natural foci of the U,S,S.R. as well as from Mongolia, China, Viet-Nam, India, Indonesia, Brazil and Africa. The majority of strains, 172 (71%), carried three plasmids: pPst, pCad and pFra. This conclusion is founded,, on typical plasmid sizes (with minor) variations~ see Fig, 1, lanes 5--11) and correlation with the relevant phenotypic markers. At the same time, 20 Y. pestis strains (8%) of diverse origin harboured additional extrachromoscmal rcplicons (Fig, 2), in particular, 18% of strains (7/38 tested) from the Volga-Ural Sandy natural plague focus, one strain (1/23) from the Pricaspian Steppe focus and 100% (4/4) of strains from the Tuvinian one carried an additional, the fourth, plasmid of about 22 MDa, A cryptic plasmid of 15-16 MDa in all strains (34 investigated) from the latter region has been described
Ill N
Fig. 1. Plasmids in Y. pestis strains of different origin. Lanes 1-3 and 17-19: the molecular mass markers (RP4. S-a, pCRI. F, R386 and Rtsl, r'-',++peclively:see MATERIALSAND~ O D S . Lanes 4-16: plasmids in Y. pearls slraills. 4 and ll: 1-3085and 1-2359, respectively
t
'1
3
4
S
6
7
S
9 4o 4t
~2 t 3 44
Fi& 2. Additional cryptic plasmids of ¥. pe~tl/~. Lanes 2 and 13:42 5 and 12: 14: the molecular mass markers (pBR322, R6K, S-a and pBR327, respectively;see ~TERIAI,S ^NOMFrn,
Ups. The rest of the lanes: plasmids in Y. peslis strains. 1: M-231 (the Ticn.Shan natural plague focus]; 3 and 6: S-62t and S-63l, respectively (bolh from the CcntrabCaucasian focus); 7: M-64t (th~ Volga-Ural Sandy tocus); 8: M-63S (The Prieaspian Steppe focus): 9.1771 (the Tuvinian focus): l0 and 11:A-1813 and A-1820(both from the Talas focus).
of Tsukano et al. [14] ~ho detected a 23-MDa replicon in Y. pestis Ireca strain. We consider all additional plasmids found to be cryptic ones since we could not determine correlation between their presence in bacterial cells and any phenotypic markers. We confirmed previous data [8] showing that the absence of pPst is a constant property of the vole Y. pestis strains from the Transcaucasian Alpine and the Daghestan Mountain natural loci (both in the Caucasian mountains). All the strains (29 tested) lacked it irrespective of their isolation year (Fig, 1, lanes 12-13). We showed that pPst from Y. peslis strains of diverse origin had practically identical size, about 6 MDa. Only one strain carried a slightly larger plasmid (ca. 6.2 MDa: Fig. 1). The molecular masses of pCad were about: 45 MDa (vaccine Y. pestis EV strain ~'rom the Madagascar island and
the majority of other glycerol negative oceanic strains), 47 MDa (the majority of glycerol positive continsr'al strains), 48 MDa (strains from the Central-Asian Desert focus) and 49 M D a (the mountain vole strains), respectively. Plasmid pFra displayed pronounced size variability (Fig. 1). These were approximately: 60 M D a (EV as well as other glycerol negative Y. pestis strains from Viet-Nam, India, Indonesia, Brazil, Africa), 65 M D a (the majority of glycerol positive strains), 68 M D a (strains from the Gissar and the Talas loci; the former is situated in the west of Tajikistan), 69 M D a (strains from the Mountain-Altai focus: the south of West Siberia), 80 MDa (the mountahl vole strains), 92 M D a (Y. pestis strain 924 from China) and even as large as 190 M D a (strain 679 from the Volga-Ural Sandy focus). Y. pestis strain Yava8 had typical morphologic and cultural properties but harboured three plasmids of unusual size: 10, 28 and 52 MDa, respectively (Fig. 1). One may conclude that the majority of Y. pestis strains tested carry the three "canonical' plasmids: pPst, pCad and pFra. At the same time, some peculiarities of plasmid content and size in strains from different natural plague loci are revealed: the presence of additional cryptic plasmids in a number of the Volga-Ural and the Talas strains; the molecular mass increase of pCad from the Central-Asian Desert and the mountain vole strains: and the characteristic sizes of pFra from the Gissar, the Talas, the Mountain-Altai and oceanic strains. These data are essential to intraspecific taxonomy of Y. pestis. The particular strains and plasmids detected can serve as objects for genetic analysis.
REFERENCES tit Popov,Yu.A.. Protscnko. O,A., Anisimov,P.I., Kokooshkin, A.M. and Mozharov, O.T. (1980) in Prophylaxis of Particular!v ~'~angerousInfections. pp. 20-25, Saratov. 12] Ferbar, D.M. and Brubaker. R.R. (1981) Infect. Immun. 31,839-841. [3l Bcn-Gurion. R. and Shefferman, A. (1981) Plasmid 5, 183-t87.
48 [4] Protsenko, O.A., Anisimov, P.I., Mozharov, O.T,, Konnov, N.P., Popov. Yu.A. and Kokooshkln, A.M. (1983) Genetika SSSR 19, 1081-1090. [5] Pormoy, D.A. and Falkow, S. (1981) J. Bacteriol. 148. 877-883. [6] Straley, S.C. and Bowmer, W.S. (1986) Infect. lmmun. 51. 445-454. [7] Perry. R.D.. Harmon. P.A., Bowmer, W.S. and Straley. S.C. (1986) Infect. Immun. 54, 428-434. [8] Fursov, V.V. and Popov, Yu.A. (1983) in Prophylaxis of Natural Focel Infeclions, pp. 326-327, Stavropol. [9] Zarenkov, M.I. and Goncharova, N.A. (1986) in Molecular Biology and Genetics of Plasmids, p. 18, Pushchino.
[]0] Gramotina, L.I., Protsenko, S.L. and l.opatkin, O.N. (1987) in Particularly Dangerous Infections in the Caucast]s, pp. ]1-13, Stavropol. [1|] Balahonov, S.V. (]989) Molekul. Genetika SSSR 4, 39-42. 112] Higuchi, K. and Smilh. J.L. 0961) J. Ba~leriol. 81, 615-618. 113] Kado, C.J. and Liu, S.-T. (1981) $. BacletloL 145. 1365-]373. [14] Tsukano, H.. Wake. A. and Sakakibara. Y. (1986) Microbiol. Immunol. 30. 837-848.
45
FEMSLE 03858
Plasmid content in Yersinia pestis strains of different origin A n d r e w A. Filippov i Nikolai S. Solodovnikov 2, Liubov M. K o o k l e v a ~ a n d Olga A. Protsenko I Department of Genetics and ~ Collection of bring Cultures, All-Union Research Anti-Plague Institule 'Microbe'. Saratov. U.S.S.R.
Received l l September 1989 Accepted 20 September 1989 Key words: Yersiniapeslis strains; Plasmid content
1. S U M M A R Y Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains. 172 (71%) wcrc shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 MDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.
2. I N T R O D U C T I O N It has been shown that Yersinia p e s t i s - - c a u s a rive agent of plague--carries, as a rule, three plasmids, namely 6-MDa replicon pPst, encoding synthesis of pesticin 1 bacteriocin as well as the production of fibrinolysin/coagulase [1-4]; a Correspondence to." Andrew A. Filippov, Department of Genetics, AIbUnion Re,arch Anti-Plague Institute "Microbe', 410071 S.aratov, Univcrsitetskaya46. U.S.S.R.
45-50-MDa plasmid, pCad. determining cell growth dependence upon Ca -'+ ions at 37°C. synthesis of the V antigen and a number of thermoinducible outer membrane proteins [2,3,5-7]; and a 60-MDa plasmid, pFra, encoding the production of capsular antigen known as fraction I and murine toxin [4]. Products of all three plasraids are implicated in expression of virulence. In the territory of the U.S.S.R.. there are 15 natural plague loci differing by geographical peculiarities and species of rodents and their ectoparasites, the bacterium carriers and vectors, respectively. There are a few communications which describe plasmid profile features of strains from diverse plague loci. it has been shown that Y. pestis isolates from voles obtained in the Caucasus lack pPst and carry pFra considerably increased in size [8.9 I. Strains from the Central-Caucasian focus (the carriers are susliks) which, besides the three above-mentioned plasmids, harbour an additional extrachromosomal replicon of about 1.5-2 MDa have been described [10]. Another additional cryptic plasmid (15-16 MDa) has recently been found in strains from the Tuvinian focus (the south of East Siberia) [11]. The purpose of our work was a comparative study of plasmid content in a considerable num-
0378-1097/90/$03.50 © 1990 Federation of European Microbiolo~cal .Societies
46 ber of Y. pestis strains from the majority of natural plague foci of the U.S.S.R. and also from several Asian, African and Latin American countries.
I 2 3 4 S 6 7 $ 9 I0t'112 q~5141516'ITlSq9
3. MATERIALS A N D M E T H O D S Y. pestis and Escherichia coil strains used were received from the Collection of Living Cultures of All-Union Research Anti-Plague Institute "Microbe' (Saratov). The plasmids from E. coil C600 (pBR327) (2.2 MDa), DH1 (pBR322) (2.9 MDa), C600 (pCR1) (7.5 MDa), J53(S-a) (23 MDa), C600 (R6K) (26 MDa), J53 (RP4) (36 MDa), K-12 (F +) (62 MDa), JC411 (R386) (79 MDa) and CSH2 (Rtsl) (140 MDa) were used as molecular mass markers. The main phenotypic determinants encoded by Y. pestis plasmids: calcium dependence, pesticin I and fraction I production were tested as described previously [4,12]. Plasmids from Y. pestis and E. coil were screened by the method of Kado and Liu
[13].
4. RESULTS A N D DISCUSSION We studied plasmid content in 242 Y. pestis strains from 14 natural foci of the U,S,S.R. as well as from Mongolia, China, Viet-Nam, India, Indonesia, Brazil and Africa. The majority of strains, 172 (71%), carried three plasmids: pPst, pCad and pFra. This conclusion is founded,, on typical plasmid sizes (with minor) variations~ see Fig, 1, lanes 5--11) and correlation with the relevant phenotypic markers. At the same time, 20 Y. pestis strains (8%) of diverse origin harboured additional extrachromoscmal rcplicons (Fig, 2), in particular, 18% of strains (7/38 tested) from the Volga-Ural Sandy natural plague focus, one strain (1/23) from the Pricaspian Steppe focus and 100% (4/4) of strains from the Tuvinian one carried an additional, the fourth, plasmid of about 22 MDa, A cryptic plasmid of 15-16 MDa in all strains (34 investigated) from the latter region has been described
Ill N
Fig. 1. Plasmids in Y. pestis strains of different origin. Lanes 1-3 and 17-19: the molecular mass markers (RP4. S-a, pCRI. F, R386 and Rtsl, r'-',++peclively:see MATERIALSAND~ O D S . Lanes 4-16: plasmids in Y. pearls slraills. 4 and ll: 1-3085and 1-2359, respectively
t
'1
3
4
S
6
7
S
9 4o 4t
~2 t 3 44
Fi& 2. Additional cryptic plasmids of ¥. pe~tl/~. Lanes 2 and 13:42 5 and 12: 14: the molecular mass markers (pBR322, R6K, S-a and pBR327, respectively;see ~TERIAI,S ^NOMFrn,
Ups. The rest of the lanes: plasmids in Y. peslis strains. 1: M-231 (the Ticn.Shan natural plague focus]; 3 and 6: S-62t and S-63l, respectively (bolh from the CcntrabCaucasian focus); 7: M-64t (th~ Volga-Ural Sandy tocus); 8: M-63S (The Prieaspian Steppe focus): 9.1771 (the Tuvinian focus): l0 and 11:A-1813 and A-1820(both from the Talas focus).
of Tsukano et al. [14] ~ho detected a 23-MDa replicon in Y. pestis Ireca strain. We consider all additional plasmids found to be cryptic ones since we could not determine correlation between their presence in bacterial cells and any phenotypic markers. We confirmed previous data [8] showing that the absence of pPst is a constant property of the vole Y. pestis strains from the Transcaucasian Alpine and the Daghestan Mountain natural loci (both in the Caucasian mountains). All the strains (29 tested) lacked it irrespective of their isolation year (Fig, 1, lanes 12-13). We showed that pPst from Y. peslis strains of diverse origin had practically identical size, about 6 MDa. Only one strain carried a slightly larger plasmid (ca. 6.2 MDa: Fig. 1). The molecular masses of pCad were about: 45 MDa (vaccine Y. pestis EV strain ~'rom the Madagascar island and
the majority of other glycerol negative oceanic strains), 47 MDa (the majority of glycerol positive continsr'al strains), 48 MDa (strains from the Central-Asian Desert focus) and 49 M D a (the mountain vole strains), respectively. Plasmid pFra displayed pronounced size variability (Fig. 1). These were approximately: 60 M D a (EV as well as other glycerol negative Y. pestis strains from Viet-Nam, India, Indonesia, Brazil, Africa), 65 M D a (the majority of glycerol positive strains), 68 M D a (strains from the Gissar and the Talas loci; the former is situated in the west of Tajikistan), 69 M D a (strains from the Mountain-Altai focus: the south of West Siberia), 80 MDa (the mountahl vole strains), 92 M D a (Y. pestis strain 924 from China) and even as large as 190 M D a (strain 679 from the Volga-Ural Sandy focus). Y. pestis strain Yava8 had typical morphologic and cultural properties but harboured three plasmids of unusual size: 10, 28 and 52 MDa, respectively (Fig. 1). One may conclude that the majority of Y. pestis strains tested carry the three "canonical' plasmids: pPst, pCad and pFra. At the same time, some peculiarities of plasmid content and size in strains from different natural plague loci are revealed: the presence of additional cryptic plasmids in a number of the Volga-Ural and the Talas strains; the molecular mass increase of pCad from the Central-Asian Desert and the mountain vole strains: and the characteristic sizes of pFra from the Gissar, the Talas, the Mountain-Altai and oceanic strains. These data are essential to intraspecific taxonomy of Y. pestis. The particular strains and plasmids detected can serve as objects for genetic analysis.
REFERENCES tit Popov,Yu.A.. Protscnko. O,A., Anisimov,P.I., Kokooshkin, A.M. and Mozharov, O.T. (1980) in Prophylaxis of Particular!v ~'~angerousInfections. pp. 20-25, Saratov. 12] Ferbar, D.M. and Brubaker. R.R. (1981) Infect. Immun. 31,839-841. [3l Bcn-Gurion. R. and Shefferman, A. (1981) Plasmid 5, 183-t87.
48 [4] Protsenko, O.A., Anisimov, P.I., Mozharov, O.T,, Konnov, N.P., Popov. Yu.A. and Kokooshkln, A.M. (1983) Genetika SSSR 19, 1081-1090. [5] Pormoy, D.A. and Falkow, S. (1981) J. Bacteriol. 148. 877-883. [6] Straley, S.C. and Bowmer, W.S. (1986) Infect. lmmun. 51. 445-454. [7] Perry. R.D.. Harmon. P.A., Bowmer, W.S. and Straley. S.C. (1986) Infect. Immun. 54, 428-434. [8] Fursov, V.V. and Popov, Yu.A. (1983) in Prophylaxis of Natural Focel Infeclions, pp. 326-327, Stavropol. [9] Zarenkov, M.I. and Goncharova, N.A. (1986) in Molecular Biology and Genetics of Plasmids, p. 18, Pushchino.
[]0] Gramotina, L.I., Protsenko, S.L. and l.opatkin, O.N. (1987) in Particularly Dangerous Infections in the Caucast]s, pp. ]1-13, Stavropol. [1|] Balahonov, S.V. (]989) Molekul. Genetika SSSR 4, 39-42. 112] Higuchi, K. and Smilh. J.L. 0961) J. Ba~leriol. 81, 615-618. 113] Kado, C.J. and Liu, S.-T. (1981) $. BacletloL 145. 1365-]373. [14] Tsukano, H.. Wake. A. and Sakakibara. Y. (1986) Microbiol. Immunol. 30. 837-848.