jeanflop/post_ocr_correction2 huggingface dataset:
recently established a physical and genomic map of S. citri and this map is among those with the greatest number of identified loci in the mollicutes [19]. Because S. melliferum has the highest genomic similarity with S. citri, we have constructed its physical and genomic map and have compared it with that of S. citri. Materials and methods Preparation and separation of restriction fragments S. melliferum BC-3 (ATCC 33219) [20] was grown in BSR medium at 32 °C [21]. Genomic DNA was prepared after inclusion of pellets of the organisms in Low Melting Point (LMP) agarose plugs as previously described [19]. The genomic DNA was subsequently digested with restriction enzymes ApaI, BssHII, and SauI (Gibco-BRL, New England Biolabs), and separated in 1 % agarose gel using pulsed field gel electrophoresis (PFGE) (TAFE System, Geneline TM II, Beckman), with 3 steps, each of 12 h, with pulse times of 60, 120, and 180 s, respectively, at an amperage of 370 mA, in 0.25X TBE buffer, at 8°C. The size of the separated fragments was evaluated by comparison with Saccharomyces cerevisiae chromosomal DNAs (Biorad). Hybridization procedures After staining with ethidium bromide and photography under UV light, the agarose gels were treated twice for 10 min with 0.25 N HCl. Then the DNA fragments were transferred onto Hybond N+ nylon membranes (Amersham) according to the standard Southern blot procedure, using the alkaline method [22, 23]. The DNA fragments used as probes in hybridization experiments are listed in Table I. They were labeled from α-32P-dATP, using the random primer labeling kit from Gibco-BRL. Hybridizations were carried out at 42 °C in a buffer containing 50 % formamide and washes were performed at 50 °C, twice 10 min in 2XSSC, 0.1 % SDS, twice 15 min in 0.1 X SSC, 0.1 % SDS, and twice 5 min in 2X SSC. Results and discussion The restriction fragments separated by PFGE are shown in Figure 1 and summarized in Table II. The genome size obtained by summing up the values of the individual restriction fragments is close to 1 460 kbp. The physical maps for enzymes ApaI,
for 10 min with 0.25 N HCl. Then the DNA fragments were transferred onto Hybond N+ nylon membranes (Amersham) according to the standard Southern blot procedure, using the alkaline method [22, 23]. The DNA fragments used as probes in hybridization experiments are listed in Table I. They were labeled from α-32P-dATP, using the random primer labeling kit from Gibco-BRL. Hybridizations were carried out at 42 °C in a buffer containing 50 % formamide and washes were performed at 50 °C, twice 10 min in 2XSSC, 0.1 % SDS, twice 15 min in 0.1 X SSC, 0.1 % SDS, and twice 5 min in 2X SSC. Results and discussion The restriction fragments separated by PFGE are shown in Figure 1 and summarized in Table II. The genome size obtained by summing up the values of the individual restriction fragments is close to 1 460 kbp. The physical maps for enzymes ApaI, BssHII and SauI have been established from reciprocal double digestion products according to the method of Bautsch [11], as already described for the S. citri map [19]. Southern blot analyses were carried out with the probes listed in Table I. As an illustration, the results obtained with fib and pyrG-purA-purB as the probes are shown in Figure 2. The linearized physical and genomic map of S. melliferum is shown in Figure 3. Table I Genes used as probes for the establishment of S. melliferum genomic map Gene(s) Product(s) Source Reference rpsB-tsf-x-spi Ribosomal protein S2, Elongation factor Ts, protein X, Spiralin S. citri [24] 6-phosphofructokinase, Pyruvate kinase fibrillar protein S. citri [25] Elongation factor Tu M. gallisepticum [26] CTP synthetase, Adenylosuccinate synthetase and lyase S. citri [27] RNA polymerase α subunit S. citri [28] valine tRNA synthetase S. citri [28] DNA replication protein A, S. citri [29] Chromosomal replication origin rRNA 16S S. citri [30] trpJ-trpU-ser1 tRNAs Trp(UCA), Trp(CCA), Ser(CGA) S. 392 citri [31] trnD tRNAs Asn(GUU), Clu(UUC), Val(UAC) Thr(UGU), Lys(UUU), Leu(UAG) S. citri [27] msr 4.5S RNA A4, mycoides [32] SpV1-S102 Spiroplasma virus 1 S. citri unpublished SpV1-R8A2B Spiroplasma virus 1 S. citri [33] C. R. Acad. Sci. Paris, Sciences
as the probes are shown in Figure 2. The linearized physical and genomic map of S. melliferum is shown in Figure 3. Table I Genes used as probes for the establishment of S. melliferum genomic map Gene(s) Product(s) Source Reference rpsB-tsf-x-spi Ribosomal protein S2, Elongation factor Ts, protein X, Spiralin S. citri [24] 6-phosphofructokinase, Pyruvate kinase fibrillar protein S. citri [25] Elongation factor Tu M. gallisepticum [26] CTP synthetase, Adenylosuccinate synthetase and lyase S. citri [27] RNA polymerase α subunit S. citri [28] valine tRNA synthetase S. citri [28] DNA replication protein A, S. citri [29] Chromosomal replication origin rRNA 16S S. citri [30] trpJ-trpU-ser1 tRNAs Trp(UCA), Trp(CCA), Ser(CGA) S. 392 citri [31] trnD tRNAs Asn(GUU), Clu(UUC), Val(UAC) Thr(UGU), Lys(UUU), Leu(UAG) S. citri [27] msr 4.5S RNA A4, mycoides [32] SpV1-S102 Spiroplasma virus 1 S. citri unpublished SpV1-R8A2B Spiroplasma virus 1 S. citri [33] C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 392-8 393 F. Ye et al. Figure 1. S. melliferum (BC-3) genomic DNA restricted by enzymes ApaI (Ap), BssHII (Bs) and Salf (SI), and separated by pulsed field gel electrophoresis. Figure 3 in comparison with that of S. citri. The genome of S. melliferum BC-3 is some 360 kbp shorter than that of S. citri R8A2-HP. The gene order in the vicinity of oriC is quite similar for the two genomes: tuf, oriC, trpE-trpF-trpH, frpD, spi. However, the distance between frpE and spi seems to be at least 300 kbp shorter in S. melliferum. The region containing fib, msr, rrn, rpoD, valS, pyrG-purA-purB, seems to be conserved except that the gene order is inverted in S. melliferum with respect to oriC when compared to S. citri. Concerning pyrG-purA-purB, these genes are involved in the inversion but the distance between them and rpoD is clearly larger in S. melliferum Table II Number and size (in kbp) of fragments obtained by restriction enzyme digestion of the S. melliferum genome Apa BssHII Salf Fragment Size Fragment Size Fragment Size Apa 640 BsA 370 SIA 310 ApB 240 BsB 320 SIB 200 ApC 240 BsC 180 SIC 175
BC-3 is some 360 kbp shorter than that of S. citri R8A2-HP. The gene order in the vicinity of oriC is quite similar for the two genomes: tuf, oriC, trpE-trpF-trpH, frpD, spi. However, the distance between frpE and spi seems to be at least 300 kbp shorter in S. melliferum. The region containing fib, msr, rrn, rpoD, valS, pyrG-purA-purB, seems to be conserved except that the gene order is inverted in S. melliferum with respect to oriC when compared to S. citri. Concerning pyrG-purA-purB, these genes are involved in the inversion but the distance between them and rpoD is clearly larger in S. melliferum Table II Number and size (in kbp) of fragments obtained by restriction enzyme digestion of the S. melliferum genome Apa BssHII Salf Fragment Size Fragment Size Fragment Size Apa 640 BsA 370 SIA 310 ApB 240 BsB 320 SIB 200 ApC 240 BsC 180 SIC 175 ApD 240 BsD 175 SlD 170 ApE 100 BsE 145 SIE 150 BsF 115 SIF 110 BsG 90 SIC 100 BsH 55 SIH 85 SU 80 SIJ 30 SIK 25 SIL 25 Total 1460 1450 1460 394 C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 392-8 Spiroplasma melliferum genomic map ( than in S. citri. We have no explanation for this distance difference. It is known that certain spiroplasmas are infected by nonlytic SpV1 type viruses with single stranded, circular DNA (Inoviridae, Plectrovirus), the replication of which involves a double stranded replicative form (RF) of 8 kbp [4, 34]. SpV1-R8A2B and SpV1-S102 are two such SpV1 S. citri viruses, the genomes of which do not share sequence homology. The genome of S. citri contains multiple regions homologous to the RF of SpV1-R8A2B, and only 2 regions homologous to the SpV1-S102 RF [19, 34]. Similarly, when the RF of virus SpV1-R8A2B was used as the probe on BssHII restricted S. melliferum genomic DNA, multiple regions of homology were revealed, as all 8 restriction fragments hybridized (Fig. 4). In the case of Salf, several fragments also hybridized but not SIJ. This fragment contains the single rrn operon of
Spiroplasma melliferum genomic map ( than in S. citri. We have no explanation for this distance difference. It is known that certain spiroplasmas are infected by nonlytic SpV1 type viruses with single stranded, circular DNA (Inoviridae, Plectrovirus), the replication of which involves a double stranded replicative form (RF) of 8 kbp [4, 34]. SpV1-R8A2B and SpV1-S102 are two such SpV1 S. citri viruses, the genomes of which do not share sequence homology. The genome of S. citri contains multiple regions homologous to the RF of SpV1-R8A2B, and only 2 regions homologous to the SpV1-S102 RF [19, 34]. Similarly, when the RF of virus SpV1-R8A2B was used as the probe on BssHII restricted S. melliferum genomic DNA, multiple regions of homology were revealed, as all 8 restriction fragments hybridized (Fig. 4). In the case of Salf, several fragments also hybridized but not SIJ. This fragment contains the single rrn operon of S. melliferum. The same observation applies also to S. citri where fragment ApF which contains the single rrn (Fig. 3) does not hybridize with the SpV1-R8A2B RF [19]. When the RF of SpV1-S102 was used as the probe, three regions of the S. melliferum genome showed hybridization (as compared to two regions in S. citri). Two of the regions gave strong hybridization signals; the third region yielded a weak signal (Fig. 4) and its position on the genomes of the two spiroplasmas was similar, in the vicinity of spi (Fig. 3). The similarities between the genomes of S. melliferum and S. citri were expected on the basis of their close phylogenetic relationship and the homology of their genomic DNAs (68 %). The strong hybridization signals obtained, at high stringency, between S. citri DNA fragments used as probes and S. melliferum restriction fragments also indicate that individual genes have similar nucleotide sequences, as already demonstrated by sequence analysis for spi and fib [24, 35]. Even though 68 % DNA homology between two species is high, it nevertheless represents 32 % divergence. Apart from this divergence, the main differences between the two genomes reside in genome size and inversion of a large
two regions in S. citri). Two of the regions gave strong hybridization signals; the third region yielded a weak signal (Fig. 4) and its position on the genomes of the two spiroplasmas was similar, in the vicinity of spi (Fig. 3). The similarities between the genomes of S. melliferum and S. citri were expected on the basis of their close phylogenetic relationship and the homology of their genomic DNAs (68 %). The strong hybridization signals obtained, at high stringency, between S. citri DNA fragments used as probes and S. melliferum restriction fragments also indicate that individual genes have similar nucleotide sequences, as already demonstrated by sequence analysis for spi and fib [24, 35]. Even though 68 % DNA homology between two species is high, it nevertheless represents 32 % divergence. Apart from this divergence, the main differences between the two genomes reside in genome size and inversion of a large part of the chromosome. Therefore, it can be speculated that deletions and chromosomal rearrangements were 1820 kbp S. citri R8A2-HP 1460 kbp S. melliferum BC-3 Figure 3. Physical and genomic maps of S. melliferum (BC-3) and S. citri (R8A2-HP). Genetic markers are described in Table I. Restriction fragments for ApaI (Apa), BssHII (Bss) and Salf (Sal) are labeled alphabetically from the largest to the smallest + or -: strong or weak hybridization signal. The map is numbered (in °) with oriC as the starting point. The position of certain genes on the physical map is known with precision because they hybridize with adjacent restriction fragments from the same restriction enzyme. For instance, rrn of S. melliferum is such a gene: it hybridizes with Salf fragments F and J. Other genes only hybridize with overlapping fragments from different restriction enzymes. Their position is less precise and is indicated by a bar (I). C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 392-8 395 F. Ye et al. Figure 4. Hybridization between S. melliferum restricted DNA and RF of Spiroplasma virus SpV1-R8A2B and SpV1-S102. Figure 5. Schematic representation of the events (deletions and rearrangements) which may have led from
(Apa), BssHII (Bss) and Salf (Sal) are labeled alphabetically from the largest to the smallest + or -: strong or weak hybridization signal. The map is numbered (in °) with oriC as the starting point. The position of certain genes on the physical map is known with precision because they hybridize with adjacent restriction fragments from the same restriction enzyme. For instance, rrn of S. melliferum is such a gene: it hybridizes with Salf fragments F and J. Other genes only hybridize with overlapping fragments from different restriction enzymes. Their position is less precise and is indicated by a bar (I). C. R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1994; 317: 392-8 395 F. Ye et al. Figure 4. Hybridization between S. melliferum restricted DNA and RF of Spiroplasma virus SpV1-R8A2B and SpV1-S102. Figure 5. Schematic representation of the events (deletions and rearrangements) which may have led from the genome of the putative common ancestor to the genomes of S. citri and S. melliferum. common ancestor (Fig. 5). Deletions and inversions have also been observed in the case of Mycoplasma mycoides subsp. mycoides, Mycoplasma capricolum and Mycoplasma sp. PG50 [14, 15], three related species with DNA homologies of 40 to 60% [36]. These Mycoplasma species belong to the same phylogenetic group than S. citri [37]. When the genomic maps of twelve different S. citri strains were compared, we observed a variation of the genome sizes from 1650 to 1910 kbp but, in every instance, the gene order was conserved (Ye et al., submitted for publication). The same observation was made for different strains of Mycoplasma mycoides. Interestingly, the region which is shortened in S. melliferum corresponds to the region which has been shown to be variable in size among different S. citri strains. Thus, a part of this region might be nonessential. The presence of viral-like sequences and their distribution along the two genomes, as well as the fact that all S. citri and S. melliferum strains examined so far contain the viral sequences, suggest that the occurrence of these sequences is not the consequence of a recent
40 to 60% [36]. These Mycoplasma species belong to the same phylogenetic group than S. citri [37]. When the genomic maps of twelve different S. citri strains were compared, we observed a variation of the genome sizes from 1650 to 1910 kbp but, in every instance, the gene order was conserved (Ye et al., submitted for publication). The same observation was made for different strains of Mycoplasma mycoides. Interestingly, the region which is shortened in S. melliferum corresponds to the region which has been shown to be variable in size among different S. citri strains. Thus, a part of this region might be nonessential. The presence of viral-like sequences and their distribution along the two genomes, as well as the fact that all S. citri and S. melliferum strains examined so far contain the viral sequences, suggest that the occurrence of these sequences is not the consequence of a recent integration of viral DNA. Because these viral sequences are present in large numbers and because it is known that repeated sequences may be involved in genetic rearrangements in bacterial chromosomes, it can be speculated that these viral sequences have played a role in the evolution of the genomes of S. citri and S. melliferum. No conserved gene markers have been mapped so far around the region of replication termination (map positions 140 to 210 on S. citri genome and 170 to 250 on S. melliferum genome). These regions deserve further investigations. Spiroplasma melliferum genomic map VERSION ABRGÉE Les spiroplasmes sont des eubactéries sans paroi caractérisés par une morphologie hélicoïdale, une motilité en milieu liquide et un génome de petite taille possédant une faible proportion de bases G+C (24-31 mol %). Ils appartiennent à l'ordre des Entomoplasmatales au sein de la classe des Mollicutes. Plus de 250 isolats de spiroplasmes ont été obtenus à partir de plantes et d'arthropodes et ils ont été classés en 24 groupes. Spiroplasma citri, agent de la maladie du "stubborn" des agrumes, appartient au groupe I, tout comme Spiroplasma melliferum qui est pathogène pour les abeilles. Les génomes de ces deux spiroplasmes ont 68 % d'homologie.
citri and S. melliferum. No conserved gene markers have been mapped so far around the region of replication termination (map positions 140 to 210 on S. citri genome and 170 to 250 on S. melliferum genome). These regions deserve further investigations. Spiroplasma melliferum genomic map VERSION ABRGÉE Les spiroplasmes sont des eubactéries sans paroi caractérisés par une morphologie hélicoïdale, une motilité en milieu liquide et un génome de petite taille possédant une faible proportion de bases G+C (24-31 mol %). Ils appartiennent à l'ordre des Entomoplasmatales au sein de la classe des Mollicutes. Plus de 250 isolats de spiroplasmes ont été obtenus à partir de plantes et d'arthropodes et ils ont été classés en 24 groupes. Spiroplasma citri, agent de la maladie du "stubborn" des agrumes, appartient au groupe I, tout comme Spiroplasma melliferum qui est pathogène pour les abeilles. Les génomes de ces deux spiroplasmes ont 68 % d'homologie. C'est grâce à leur sexualité que les bactéries, au cours de la conjugaison, échangent du matériel génétique. Ce phénomène a été mis à profit pour initier, dans les années 1950, la carte génétique d'Escherichia coli. Des études similaires n'ont pas pu être entreprises chez les mollicutes car chez ces organismes sexualité et conjugaison n'ont jamais pu être mises en évidence. Cependant, récemment, la technique d'électrophorèse en champ pulvérisé, qui permet la séparation de fragments d'ADN de grande taille, a conduit à l'obtention de cartes physiques et génomiques pour plusieurs mollicutes. C'est ainsi qu'il a été démontré que la taille du génome de ces organismes était variable, que ce soit au sein du même genre, voire de la même espèce. Nous avons récemment établi la carte physique et génomique de S. citri. Etant donné le fort pourcentage d'hybridation (68 %) entre le génome de S. citri et celui de l'espèce voisine S. melliferum, nous avons déterminé la carte génomique de cette dernière pour la comparer à celle de la première. La carte du génome de S. melliferum, souche BC-3, a été réalisée au moyen de trois enzymes de restriction (Apal, BssHII et Sali) et de l'électrophorèse en champ pulvérisé. Les fragments
sexualité et conjugaison n'ont jamais pu être mises en évidence. Cependant, récemment, la technique d'électrophorèse en champ pulvérisé, qui permet la séparation de fragments d'ADN de grande taille, a conduit à l'obtention de cartes physiques et génomiques pour plusieurs mollicutes. C'est ainsi qu'il a été démontré que la taille du génome de ces organismes était variable, que ce soit au sein du même genre, voire de la même espèce. Nous avons récemment établi la carte physique et génomique de S. citri. Etant donné le fort pourcentage d'hybridation (68 %) entre le génome de S. citri et celui de l'espèce voisine S. melliferum, nous avons déterminé la carte génomique de cette dernière pour la comparer à celle de la première. La carte du génome de S. melliferum, souche BC-3, a été réalisée au moyen de trois enzymes de restriction (Apal, BssHII et Sali) et de l'électrophorèse en champ pulvérisé. Les fragments obtenus ont été ordonnés par des doubles digestions réciproques et des expériences d'hybridation moléculaire avec des gènes connus, isolés de S. citri ou d'autres mollicutes. La taille du génome de S. melliferum, souche BC-3, est voisine de 1 460 kpb. Cette taille est inférieure de 360 kpb à celle du génome de S. citri, souche R8A2HP. L'ordre des gènes sur les génomes des deux organismes est très conservé, en particulier à proximité de l'origine de replication (oriC). Cependant la distance entre trpT et spi (gène de la spiraline, protéine majeure de la membrane) semble être nettement inférieure chez S. melliferum. La région contenant les gènes fib (protéine de fibrilles), msr, rrn, rpoT), valS et pyrG-purA-purF, est aussi conservée mais l'ordre des gènes est inversé chez S. melliferum. Certains spiroplasmes sont infectés par des viruses non lyrics, de type SpV1, à ADN monocaténaire circulaire (Inoviridae, Plectrovirus) dont la replication passe par une forme réplicative (FR) bicaténaire. Plusieurs séquences homologues à la FR de deux virus SpV1 sont présentes dans le génome de S. citri. Il en est de même chez S. melliferum. Chez les deux spiroplasmes, ces séquences sont réparties tout le long du génome, à l'exception de la région contenant
génome de S. citri, souche R8A2HP. L'ordre des gènes sur les génomes des deux organismes est très conservé, en particulier à proximité de l'origine de replication (oriC). Cependant la distance entre trpT et spi (gène de la spiraline, protéine majeure de la membrane) semble être nettement inférieure chez S. melliferum. La région contenant les gènes fib (protéine de fibrilles), msr, rrn, rpoT), valS et pyrG-purA-purF, est aussi conservée mais l'ordre des gènes est inversé chez S. melliferum. Certains spiroplasmes sont infectés par des viruses non lyrics, de type SpV1, à ADN monocaténaire circulaire (Inoviridae, Plectrovirus) dont la replication passe par une forme réplicative (FR) bicaténaire. Plusieurs séquences homologues à la FR de deux virus SpV1 sont présentes dans le génome de S. citri. Il en est de même chez S. melliferum. Chez les deux spiroplasmes, ces séquences sont réparties tout le long du génome, à l'exception de la région contenant l'unique opéron ribosomique. La présence et la répartition similaire de ces séquences chez deux spiroplasmes distincts mais phylogénétiquement proches indiquent que l'intégration de séquences virales n'est probablement pas un événement récent. Les différences majeures entre les deux génomes portent sur la taille du génome et l'inversion d'une grande partie du chromosome. Il est intéressant de noter que la région qui est plus courte chez S. melliferum correspond à une région variable en taille chez différentes souches de S. citri. D'autre part, aucun des gènes conservés que nous avons étudiés ne se positionne autour de la région où la replication se termine (positions 140 à 210 et 170 à 250 chez S. citri et S. melliferum, respectivement). Cette région pourrait être spécifique de l'espèce. REFERENCES 1. Tully J. G., Bové J. M., Laigret F., Whitcomb R. F. 1993. Revised taxonomy of the class Mollicutes: proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales, ord. nov.), with provision for familial rank to separate species with non-helical morphology (Entomoplasmataceae, fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43: 378-85. 2. Williamson D. L, Tully J. G., Whitcomb R.