- Email: [email protected]
Evidence for the smallest nuclear genome (2.9 Mb) in the microsporidium Encephalitozoon cuniculi
MOLECULAR ii!&EMKAL PARASITOLOGY
Molecularand Biochemical Parasitology 74 (1995) 229-231
ELSEVfER
Short communication
Evidence for the smallest nuclear genome (2.9 Mb) in the microsporidium Encephalitozoon cuniculi Corinne Biderrea, Michel Pagkb, Guy MktCnier”, Elizabeth U. Canning’, Christian P. Viva&* a “Laboratoire
de Proiistologie
mokulaire
et celhdaire
des parasites,
63177 Aubke bLaboratoire
GPnome des Parasites.
URA
UniversitP
Montpellier
34000 Montpellier. ‘Department
of Biology,
Imperial
College of Science,
CNRS
1944. UniversirP
Blaise Pascal. Biologie
A,
Cedex. France I. Rue Augusre-Broussonnet,
France
Technology,
and Medicine.
London
5 W7 ZAZ.
England,
UK
Received 27 June 1995; revision received 7 September 1995; accepted 7 September 1995
Keywoxk
PFGE; Molecular karyotype; Protozoa; Microsporidia; Encephalirozoon cuniculi
Among unicellular eukaryotes, microsporidia are obligately intracellular amitochondrial parasites and are considered to be of very ancient origin as deduced from the prokaryotic features of their ribosomes [1,2] and rRNA phylogenies [3]. On the assumption that they are primitively amitochondrial, they have been included, with Metamonada and Archamoebae, in a new kingdom, the Archezoa. Conceivably, archezoan cells originated from a single prokaryotic ancestor 141. Pulsed-field gel electrophoresis (PFGE) studies on the karyotypes of several species have shown that the haploid genome size ranges from only 5.3 to 19.5 Mb (5-91, indicating that C-value paradox also exists for Microsporidia. The present work concerns the karyotyping of Encephalitozoon *Corresponding author, Tel.: t33 73407457: Fax: +33 73407670; E-mail: [email protected].
cuniculi which has been incriminated as an opportunistic parasite in AIDS patients [lo]. DNA separation was performed by contour-clamped homogeneous electric field (CHEF) electrophoresis. To get some information on ploidy of microsporidian nuclei, the total DNA content of spores was determined by a diphenylamine colorimetric procedure [ 111. Electrophoretic karyotypes of E. cuniculi are shown in Fig. 1. Eleven chromosomal DNA bands were finally resolved, the estimated molecular sizes being: I, 217 kb; II, 235 kb; III, 241 kb; IV, 247 kb; V, 251 kb; VI, 257 kb; VII, 266 kb; VIII, 272 kb; IX, 290 kb; X, 304 kb; XI, 315 kb. It should be noted that experiments of longer duration pulses did not provide evidence for unresolved DNA molecules beyond 2 Mb. The same banding pattern was observed when using samples of parasitized host cells, the higher molecuIar weight DNA of the host cells being unresolved within a compres-
0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)02495-Y
230
C. Biderre et al, /Molecular and Biochemical Parasitology 74 (1995) 229-231
E
XEX
1
E
kb
3aa-
291-
194-
a Fig.
I. CHEF electrophoresis
b of E. cuniculi chromosomal
DNA
(lanes E). Spores were purified as previously described [7] and inserted in 2”/0 low melting point agarose. Agarose blocks were incubated in I mM CaClz, 5% H,Oz at 37°C for 30 min to induce germination, before treatment with lysis solution. Electrophoresis was carried out using 0.5 x TBE buffer and I .S% agarose gels. (a) Separation at 130 V with a program of 30-s pulses for 24 h followed by 20-s pulses for 24 h. Encephaliro:oon DNA bands are seen in a gel zone between 4th and 7th bands of lambda DNA concatemers (lanes lambda); the 2nd and 3rd smallest size ones are doublets. A faint band high up corresponds to some residual DNA of host cells(MDCK cell line). (b) Separation at 130 V with a pulse program linearly incremented from 20 to 30 s for 48 h then from 30 to 40 s for 24 h. Eleven bands are visible and designated with consecutive roman numerals in increasing size order.
sion band (data not shown). In comparison with previously studied microsporidia, E. cuniculi therefore displays the narrowest size range of DNA molecules, the difference between the largest and the smallest ones being close to 100 kb. The largest molecules indeed exceed 600 kb, and frequently 1 Mb, in the other species. However, the chromosome number of E. cuniculi is higher than that of Vairimorpha sp. and Nosema costelytrae both of which have eight chromosomes [6]. The haploid genome size can be estimated to be 2.9 Mb, which is smaller than that of Escherichia co/i (4.7 Mb) [12] and within the range found for
Archaebacteria currently termed Archaea [ 131. It represents about half that of the previously smallest known microsporidian genome, i.e., 5.3 Mb in Nosema locustae [8] and less than a quarter that of Saccharomyces cerevisiae [14] and of Giardia lamblia, a non-microsporidian archezoan parasite [15]. The nucleomorph of the cryptomonad alga Pyrenomonas salina has a haploid genome size of only 660 kb [16], but this is the vestigial nucleus of an eukaryotic endosymbiont, P. salina appearing as a chimaeric organism with four different genomes. Thus, E. cum&h’ harbors the smallest nuclear genome known for a eukaryotic organism with a single nucleus. The total DNA content of spores of E. cuniculi is about 0.01 pg per cell (10.1 f 0.3 pg/109 spores; n = 11). Using the conversion 1 pg = 965 Mb, the ratio between the DNA content and the haploid genome size reaches 3.4. We can deduce that the number of chromosomal sets per nucleus is four. These spores may therefore be viewed as differentiated diploid cells arrested in G2 phase. Some cytophotometric data combined with cytological observations on the developmental cycle of the microsporidium Amblyospora suggest that karyogamy and a meiosis-like process occur in sporogony leading to the production of haploid spores [17]. This is probably not the case in Encephalitozoon for which sexual reproduction has never been observed. The success of the infestation might be enhanced by a late arrest of the cell cycle during spore differentiation, a G2-nucleus appearing to be able to undergo more rapidly its first division within a host cell. Obligate parasitism almost certainly involves a significant loss of genetic material. However, the current 7-fold variation in the PFGE-estimated genome size between microsporidian species cannot be explained entirely by differences in cellular complexity. If we accept the three-domains concept of the living world - Bacteria, Archaea and Eucarya [ 13]- the genomes of the microsporidian lineage should exhibit features closest to those of the common ancestor for Archaea and Eucarya. We think that the very low genome size in E. cuniculi is more relatable to the early divergence of microsporidia. Mapping and gene sequencing of this tiny genome are in progress. A partial se-
C. Biderre et al. /Molecular and Biochemical Parasitology 74 (199s) 229-231
quencing of randomly fragmented genomic DNA (about 12 kb) indicates that the GC content is close to 50%. Acknowledgements
We thank Pr. F. Derouin, Hopital St Louis, Paris, for welcoming C.B. in his laboratory during a short training period. Drs. C. Sarfaty and B. Bovais are ackowledged for their help. We are grateful to Pr. G. Prensier from Universite Blaise Pascal, Clermont-Ferrand, to Pr. A. Danchin, from Institut Pasteur, Paris and to Pr. C. Vago, from Institut de France (Academic des Sciences), for hepful discussion and comments on the manuscript. C.B. is supported by an allocation de recherche from the Ministere de I’Enseignement Superieur et de la Recherche. This study was supported by a grant from the GREG (no. 124/94). References
Ill Curgy, J.J., Vavra, J. and Viva&, C. (1980) Presence of ribosomal RNAs with prokaryotic properties in microsporidia, eukaryotic organisms. Biol. Cell. 38, 49-52. 121Hartskeerl, R.A., Schuitema, A.R.J. and de Wachter, R. (1993) Secondary structure of the small subunit ribosomal RNA sequence of the microsporidium Encephalirozoon cuniculi. Nucleic Acids Res. 21, 1489. CR., Maddox, J.V., Friedman, S., 131 Vossbrinck, Debrunner-Vossbrinck, B.A. and Woese, C.R. (1987) Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature, 326, 41 l-414. [41 Cavalier-Smith, T. (1991) The evolution of prokaryotic and eukaryotic cells. In: Fundamentals of Medical Cell Biology (Bittar GE., ed.), Vol. I, pp. 271-278, J.A.I. Press, Greenwich. 151Munderloh, U.G., Kurtti, T.J. and Ross, SE. (1990) Electrophoretic characterization of chromosomal DNA from two microsporidia. J. Invertebr. Pathol. 56, 243-248. 161Malone, L.A. and Mclvor, C.A. (1993) Pulsed-field gel electrophoresis from four microsporidian isolates. J. Invertebr. Pathol. 61. 203-205.
23
171 Biderre, C., Pages, M., Met&tier, G., David. D., Bata, J., Prensier, G.and Viva+ C. (1994) On small genomes in eukaryotic organisms : molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. C.R. Acad. Sci. Paris, 317, 399-404. 181 Street, D.A. (1994) Analysis of Nosema locusrue (microsporida: Nosematidae) chromosomal DNA with pulsed-field gel electrophoresis. J. Invertebr. Pathol. 63, 301-303. 191 Kawakami, Y., Inoue, T., Ito, K., Kitamisu, K., Hanawa, C., Ando, T., Iwano, H. and Ishihara, R. (1994) Identification of a chromosome harboring the small subunit ribosomal RNA gene of Nosema bombycis. J. Invertebr. Pathol. 64, 147-148. 1101 Hollister, W-S., Canning, E.U. and Willcox, A. (1991) Evidence for the widespread occurrence of antibodies to Encephalitozoon cuniculi (Microspora) in man provided by ELISA and other serological tests. Parasitology 107, 33-43. IllI Friesen, J.D. (1968) Measurement of DNA synthesis in bacterial cells. In: Methods in Enzymology (Grossman L. and Moldave K., eds.). Academic Press, Vol. 12, pp. 625-635, New York, NY. WI Smith, CL., Econome, J.G., Schutt, A., Klco, S. and Cantor, CR (1987) A physical map of theEscherichiu coli Kl2 genome. Science, 236, 1448-1453. 1131 Woese, C.R., Kandler, 0. and Wheelis, M.L. (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576-4579 I141 Oliver, S.G., James, CM., Gent, M.E. and Indge, K.J. (1993) Yeast genome organization and evolution. In: The Eukaryotic Genome Organisation and Regulation (Broda P.M.A., Oliver S.G. and Sims P.F.G., eds.), pp 1-17, Cambridge Univ. Press, Cambridge. [I51 Fan, J.B., Korman, S.H., Cantor, CR. and Smith, L.C. (1991) Giardia lumblia: haploid genome size determined by pulsed field gel electrophoresis is less than I2 Mb. Nucleic Acids Res. 19, 1905-1908. I161 Eschbach, S., Hoffmann, C.J.B., Maier, U-G., Sitte, P. and Hansmann, P. (1991) A eukaryotic genome of 660 kb: electrophoretic karyotype of nucleomorph and cell nucleus of the cryptomonad alga, Pyrenomonas salina. Nucleic Acids Res. 19, 1779-1781. iI71 Hazard, E.I. and Brookbank, J.W. (1984) Karyogamy and meiosis in an Amblyosporu sp. (Microspora) in the mosquito Cufex salinarius. J. Invertebr. Pathol. 44, 3-l I.
Molecularand Biochemical Parasitology 74 (1995) 229-231
ELSEVfER
Short communication
Evidence for the smallest nuclear genome (2.9 Mb) in the microsporidium Encephalitozoon cuniculi Corinne Biderrea, Michel Pagkb, Guy MktCnier”, Elizabeth U. Canning’, Christian P. Viva&* a “Laboratoire
de Proiistologie
mokulaire
et celhdaire
des parasites,
63177 Aubke bLaboratoire
GPnome des Parasites.
URA
UniversitP
Montpellier
34000 Montpellier. ‘Department
of Biology,
Imperial
College of Science,
CNRS
1944. UniversirP
Blaise Pascal. Biologie
A,
Cedex. France I. Rue Augusre-Broussonnet,
France
Technology,
and Medicine.
London
5 W7 ZAZ.
England,
UK
Received 27 June 1995; revision received 7 September 1995; accepted 7 September 1995
Keywoxk
PFGE; Molecular karyotype; Protozoa; Microsporidia; Encephalirozoon cuniculi
Among unicellular eukaryotes, microsporidia are obligately intracellular amitochondrial parasites and are considered to be of very ancient origin as deduced from the prokaryotic features of their ribosomes [1,2] and rRNA phylogenies [3]. On the assumption that they are primitively amitochondrial, they have been included, with Metamonada and Archamoebae, in a new kingdom, the Archezoa. Conceivably, archezoan cells originated from a single prokaryotic ancestor 141. Pulsed-field gel electrophoresis (PFGE) studies on the karyotypes of several species have shown that the haploid genome size ranges from only 5.3 to 19.5 Mb (5-91, indicating that C-value paradox also exists for Microsporidia. The present work concerns the karyotyping of Encephalitozoon *Corresponding author, Tel.: t33 73407457: Fax: +33 73407670; E-mail: [email protected].
cuniculi which has been incriminated as an opportunistic parasite in AIDS patients [lo]. DNA separation was performed by contour-clamped homogeneous electric field (CHEF) electrophoresis. To get some information on ploidy of microsporidian nuclei, the total DNA content of spores was determined by a diphenylamine colorimetric procedure [ 111. Electrophoretic karyotypes of E. cuniculi are shown in Fig. 1. Eleven chromosomal DNA bands were finally resolved, the estimated molecular sizes being: I, 217 kb; II, 235 kb; III, 241 kb; IV, 247 kb; V, 251 kb; VI, 257 kb; VII, 266 kb; VIII, 272 kb; IX, 290 kb; X, 304 kb; XI, 315 kb. It should be noted that experiments of longer duration pulses did not provide evidence for unresolved DNA molecules beyond 2 Mb. The same banding pattern was observed when using samples of parasitized host cells, the higher molecuIar weight DNA of the host cells being unresolved within a compres-
0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)02495-Y
230
C. Biderre et al, /Molecular and Biochemical Parasitology 74 (1995) 229-231
E
XEX
1
E
kb
3aa-
291-
194-
a Fig.
I. CHEF electrophoresis
b of E. cuniculi chromosomal
DNA
(lanes E). Spores were purified as previously described [7] and inserted in 2”/0 low melting point agarose. Agarose blocks were incubated in I mM CaClz, 5% H,Oz at 37°C for 30 min to induce germination, before treatment with lysis solution. Electrophoresis was carried out using 0.5 x TBE buffer and I .S% agarose gels. (a) Separation at 130 V with a program of 30-s pulses for 24 h followed by 20-s pulses for 24 h. Encephaliro:oon DNA bands are seen in a gel zone between 4th and 7th bands of lambda DNA concatemers (lanes lambda); the 2nd and 3rd smallest size ones are doublets. A faint band high up corresponds to some residual DNA of host cells(MDCK cell line). (b) Separation at 130 V with a pulse program linearly incremented from 20 to 30 s for 48 h then from 30 to 40 s for 24 h. Eleven bands are visible and designated with consecutive roman numerals in increasing size order.
sion band (data not shown). In comparison with previously studied microsporidia, E. cuniculi therefore displays the narrowest size range of DNA molecules, the difference between the largest and the smallest ones being close to 100 kb. The largest molecules indeed exceed 600 kb, and frequently 1 Mb, in the other species. However, the chromosome number of E. cuniculi is higher than that of Vairimorpha sp. and Nosema costelytrae both of which have eight chromosomes [6]. The haploid genome size can be estimated to be 2.9 Mb, which is smaller than that of Escherichia co/i (4.7 Mb) [12] and within the range found for
Archaebacteria currently termed Archaea [ 131. It represents about half that of the previously smallest known microsporidian genome, i.e., 5.3 Mb in Nosema locustae [8] and less than a quarter that of Saccharomyces cerevisiae [14] and of Giardia lamblia, a non-microsporidian archezoan parasite [15]. The nucleomorph of the cryptomonad alga Pyrenomonas salina has a haploid genome size of only 660 kb [16], but this is the vestigial nucleus of an eukaryotic endosymbiont, P. salina appearing as a chimaeric organism with four different genomes. Thus, E. cum&h’ harbors the smallest nuclear genome known for a eukaryotic organism with a single nucleus. The total DNA content of spores of E. cuniculi is about 0.01 pg per cell (10.1 f 0.3 pg/109 spores; n = 11). Using the conversion 1 pg = 965 Mb, the ratio between the DNA content and the haploid genome size reaches 3.4. We can deduce that the number of chromosomal sets per nucleus is four. These spores may therefore be viewed as differentiated diploid cells arrested in G2 phase. Some cytophotometric data combined with cytological observations on the developmental cycle of the microsporidium Amblyospora suggest that karyogamy and a meiosis-like process occur in sporogony leading to the production of haploid spores [17]. This is probably not the case in Encephalitozoon for which sexual reproduction has never been observed. The success of the infestation might be enhanced by a late arrest of the cell cycle during spore differentiation, a G2-nucleus appearing to be able to undergo more rapidly its first division within a host cell. Obligate parasitism almost certainly involves a significant loss of genetic material. However, the current 7-fold variation in the PFGE-estimated genome size between microsporidian species cannot be explained entirely by differences in cellular complexity. If we accept the three-domains concept of the living world - Bacteria, Archaea and Eucarya [ 13]- the genomes of the microsporidian lineage should exhibit features closest to those of the common ancestor for Archaea and Eucarya. We think that the very low genome size in E. cuniculi is more relatable to the early divergence of microsporidia. Mapping and gene sequencing of this tiny genome are in progress. A partial se-
C. Biderre et al. /Molecular and Biochemical Parasitology 74 (199s) 229-231
quencing of randomly fragmented genomic DNA (about 12 kb) indicates that the GC content is close to 50%. Acknowledgements
We thank Pr. F. Derouin, Hopital St Louis, Paris, for welcoming C.B. in his laboratory during a short training period. Drs. C. Sarfaty and B. Bovais are ackowledged for their help. We are grateful to Pr. G. Prensier from Universite Blaise Pascal, Clermont-Ferrand, to Pr. A. Danchin, from Institut Pasteur, Paris and to Pr. C. Vago, from Institut de France (Academic des Sciences), for hepful discussion and comments on the manuscript. C.B. is supported by an allocation de recherche from the Ministere de I’Enseignement Superieur et de la Recherche. This study was supported by a grant from the GREG (no. 124/94). References
Ill Curgy, J.J., Vavra, J. and Viva&, C. (1980) Presence of ribosomal RNAs with prokaryotic properties in microsporidia, eukaryotic organisms. Biol. Cell. 38, 49-52. 121Hartskeerl, R.A., Schuitema, A.R.J. and de Wachter, R. (1993) Secondary structure of the small subunit ribosomal RNA sequence of the microsporidium Encephalirozoon cuniculi. Nucleic Acids Res. 21, 1489. CR., Maddox, J.V., Friedman, S., 131 Vossbrinck, Debrunner-Vossbrinck, B.A. and Woese, C.R. (1987) Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature, 326, 41 l-414. [41 Cavalier-Smith, T. (1991) The evolution of prokaryotic and eukaryotic cells. In: Fundamentals of Medical Cell Biology (Bittar GE., ed.), Vol. I, pp. 271-278, J.A.I. Press, Greenwich. 151Munderloh, U.G., Kurtti, T.J. and Ross, SE. (1990) Electrophoretic characterization of chromosomal DNA from two microsporidia. J. Invertebr. Pathol. 56, 243-248. 161Malone, L.A. and Mclvor, C.A. (1993) Pulsed-field gel electrophoresis from four microsporidian isolates. J. Invertebr. Pathol. 61. 203-205.
23
171 Biderre, C., Pages, M., Met&tier, G., David. D., Bata, J., Prensier, G.and Viva+ C. (1994) On small genomes in eukaryotic organisms : molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. C.R. Acad. Sci. Paris, 317, 399-404. 181 Street, D.A. (1994) Analysis of Nosema locusrue (microsporida: Nosematidae) chromosomal DNA with pulsed-field gel electrophoresis. J. Invertebr. Pathol. 63, 301-303. 191 Kawakami, Y., Inoue, T., Ito, K., Kitamisu, K., Hanawa, C., Ando, T., Iwano, H. and Ishihara, R. (1994) Identification of a chromosome harboring the small subunit ribosomal RNA gene of Nosema bombycis. J. Invertebr. Pathol. 64, 147-148. 1101 Hollister, W-S., Canning, E.U. and Willcox, A. (1991) Evidence for the widespread occurrence of antibodies to Encephalitozoon cuniculi (Microspora) in man provided by ELISA and other serological tests. Parasitology 107, 33-43. IllI Friesen, J.D. (1968) Measurement of DNA synthesis in bacterial cells. In: Methods in Enzymology (Grossman L. and Moldave K., eds.). Academic Press, Vol. 12, pp. 625-635, New York, NY. WI Smith, CL., Econome, J.G., Schutt, A., Klco, S. and Cantor, CR (1987) A physical map of theEscherichiu coli Kl2 genome. Science, 236, 1448-1453. 1131 Woese, C.R., Kandler, 0. and Wheelis, M.L. (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576-4579 I141 Oliver, S.G., James, CM., Gent, M.E. and Indge, K.J. (1993) Yeast genome organization and evolution. In: The Eukaryotic Genome Organisation and Regulation (Broda P.M.A., Oliver S.G. and Sims P.F.G., eds.), pp 1-17, Cambridge Univ. Press, Cambridge. [I51 Fan, J.B., Korman, S.H., Cantor, CR. and Smith, L.C. (1991) Giardia lumblia: haploid genome size determined by pulsed field gel electrophoresis is less than I2 Mb. Nucleic Acids Res. 19, 1905-1908. I161 Eschbach, S., Hoffmann, C.J.B., Maier, U-G., Sitte, P. and Hansmann, P. (1991) A eukaryotic genome of 660 kb: electrophoretic karyotype of nucleomorph and cell nucleus of the cryptomonad alga, Pyrenomonas salina. Nucleic Acids Res. 19, 1779-1781. iI71 Hazard, E.I. and Brookbank, J.W. (1984) Karyogamy and meiosis in an Amblyosporu sp. (Microspora) in the mosquito Cufex salinarius. J. Invertebr. Pathol. 44, 3-l I.