Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6–12 copies of ISMbov1, 11–15 copies of ISMbov2 and 4–10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4–8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.
Mycoplasma bovis is the most important mycoplasma species in cattle in countries free of contagious bovine pleuropneumonia (CBPP). It is widespread in North America [[, and in Europe where it is associated with bronchopneumonia and arthritis in calves, and with mastitis and genital infections in adult cattle [[,,,.
A large number of insertion sequences (IS) has been described in mollicutes [[,,,, and are useful genetic markers for diagnosis and epidemiological analysis. IS elements are mobile DNA fragments (<2.5 kb), often present in multiple copies and causing a significant degree of plasticity of prokaryotic genomes, thus leading to the appearance of variants and subtypes of bacterial species.
M. bovis is characterized by its antigenic variation associated with DNA-recombinations [. Recent studies [ have emphasized the presence of recombinase genes in several mycoplasmas, including M. bovis. Moreover, a gene encoding a putative IS30-like protein in M. bovis type strain PG45 was identified [. In addition, Mycoplasma agalactiae, which is phylogenetically closely related to M. bovis, presents an IS element, ISMag1, whose probe reacted with DNA of M. bovis [. Interestingly, the insertion sequence ISMmy1 identified recently in Mycoplasma mycoides subsp. mycoides small colony (SC) type seemed to be present also in M. bovis [. M. mycoides subsp. mycoides SC harbours indeed two additional IS elements, IS1296 [ and IS1634 [, that were referred to be absent in M. bovis. Thus, the presence of multiple, heterologous IS elements in the genomes of pathogenic mycoplasma species is not unusual.
The present study reports the presence of three IS elements in M. bovis, ISMbov1, ISMbov2 and ISMbov3, which are phylogenetically related to ISMag1, ISMmy1 and IS1634, respectively, and shows the distribution of these three plus a further IS element (IS30-like) [ in eleven M. bovis isolates differing in pathogenic and cultural features.
Materials and methods
Mycoplasma strains, growth conditions and DNA extraction
The mycoplasma strains used in this study are listed in the Table 1, according to their origin, their pathological and cultural features. All M. bovis strains were grown in modified Hayflick broth medium during 24–48 h [. Mycoplasma strains 2610, 0435 and 9585 were also subcultured several times (116, 80 and 98 times, respectively), through liquid medium at intervals of 48 h. Cells were harvested by centrifugation at 8000×g for 15 min, washed with phosphate-buffered saline (PBS) solution (140 mM NaCl, 2.7 mM KCl, 15 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) and re-suspended in PBS. Mycoplasmal genomic DNA was extracted by phenol/chloroform method as described previously [. DNA concentration was determined spectrophotometrically with GeneQuantI (Amersham Pharmacia Biotech). All M. bovis strains tested were confirmed to belong to this species by sandwich ELISA [ and the uvrC specific PCR [.
|Strain designation||Passage number||Country of origin||Sourcea||Disease||Adherence rates (%) to EBL cellsb|
|Strain designation||Passage number||Country of origin||Sourcea||Disease||Adherence rates (%) to EBL cellsb|
aBAL, bronchoalveolar lavage.
bEBL, embryonic bovine lung. See Ref. [.
cPG45, type strain of M. bovis.
dThe passage number is however >15.
eML1, rabbit isolate; all other strains are of bovine origin.
fND, not determined.
PCR amplification and preparation of DNA probes for Southern blotting
Polymerase chain reaction (PCR) was performed in a DNA thermal cycler Gene Amp 9600 (Applied Biosystems) in a 50-μl reaction mixture [50-mM Tris–HCl, pH 9.2, 1.75 mM MgCl2, 16 mM (NH4)2SO4, 350 μM of each dNTP] which contained approximately 50 ng of genomic template DNA, 300 nM of each primer, and 1.75 U of a mixture of Taq and Pwo DNA polymerases (Expand Long Template PCR System kit, Roche Diagnostics). The mixtures were subjected to 2 min denaturation at 94 °C followed by 30 cycles of amplification with the parameters: 30 s at 94 °C, 30 s at 48 °C, and 2 min extension at 68 °C. Digoxigenin-11-dUTP (DIG)-labelled probes were produced by PCR as described above in the presence of 50 μM DIG (Roche Diagnostics).
The ISMag1 specific probe was prepared with primers Maga-IS-L and Maga-IS-R [, using DNA from M. agalactiae strain 3990. The ISMbov1 specific probe was constructed using the oligonucleotide primers MBOV-IS-L and MBOV-IS-R (Table 2), and DNA from M. bovis strain PG45. The ISMmy1 specific probe was prepared with primers 5im_ismmy1 and 3im_ismmy1 [, using DNA from M. mycoides subsp. mycoides SC strain PG1. The ISMbov2 specific probe was constructed using the oligonucleotide primers ISMBO-L and ISMBO-R (Table 2), and DNA from M. bovis strain PG45. The IS1634 specific probe was prepared with primers IS1634(in)L and IS1634(in)R [, using DNA from M. mycoides subsp. mycoides SC strain PG1. The ISMbov3 specific probe was constructed using the oligonucleotide primers IS1634(in)L2 and IS1634(in)R2 (Table 2), and DNA from M. bovis strain PG45. The IS30-like specific probe was constructed using the oligonucleotide primers MBO-ISYOG-L and MBO-ISYOG-R (Table 2), and DNA from M. bovis strain PG45.
|Primer||Sequence (5′–3′)||Melting temperature (°C)a|
|Primer||Sequence (5′–3′)||Melting temperature (°C)a|
aObtained with the “Oligonucleotide Properties Calculator” at the website http://www.basic.nwu.edu/biotools/oligocalc.html, using the nearest neighbour method and the parameters 300 nM primer and 50 mM salt (Na+).
Identification and isolation of ISMbov1, ISMbov2 and ISMbov3
A genomic library was obtained from the isolate 2610 (passage 7) (Table 1). Genomic Sau3AI fragments of sizes between 1.0 and 8.0 kb were cloned into the BamHI site of pBluescriptII SK+ (Stratagene). Ligation products were transformed into XL1-Blue MRF′Escherichia coli (Stratagene) and transformants were grown on LB agar plates containing ampicillin (50 μg/ml), X-gal (80 μg/ml) and IPTG (20 mM). Colony hybridization at 56 °C was performed with the genomic library previously transferred onto Whatman paper filters (540, VWR International) using the probes for ISMag1, ISMmy1 and IS1634 following the standard protocol [. Plasmid DNA of the selected positive colonies was isolated using the Plasmid Midi kit (Qiagen AG). The clones obtained by ISMag1 hybridization harboured the M. bovis insertion element designated ISMbov1, those obtained by ISMmy1 hybridization harboured the insertion element designated ISMbov2 and those obtained by IS1634 hybridization harboured the insertion element designated ISMbov3.
Sequence analysis of the IS genes
DNA sequencing was performed with a DNA Sequenator AB 3100 and the Taq Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems). In the first step, oligonucleotide primers containing the T3 and T7 promoter sequences flanking the cloning site of the pBluescriptII SK+ vector were used. The sequences were completed by “primer walking” using synthesized oligonucleotides. For the analysis of the complete cloned segments, the deletion technique was employed by using exonuclease III of the double-stranded Nested Deletion kit (Amersham Pharmacia Biotech). The DNA sequences were assembled using the Sequencher 3.0 (GeneCodes) software. Alignments were done with PILEUP and sequence comparisons with FASTA and BESTFIT from the GCG Wisconsin package (Genetics Computer Group, Inc., Madison, WI). The deduced amino acid sequences were analysed with the program PROSITE [.
Nucleotide sequence Accession Numbers
The EMBL/GenBank Accession Nos. for the nucleotide sequences of one representative copy of each insertion element of M. bovis determined in this work are: AJ564386 for ISMbov1, AJ536157 for ISMbov2 and AJ829923 for ISMbov3.
Genomic mycoplasmal DNA was digested by EcoRV, a restriction enzyme not cutting within the sequences used as probes, submitted to electrophoresis on 0.7% (w/v) agarose gels and transferred onto positively charged nylon membranes (Roche Diagnostics) following standard protocol [. The membranes were pre-incubated with 10 ml hybridization buffer [5 X SSC (1 X SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.7), 0.1%N-lauroylsarcosine, 0.02% SDS and 1% (w/v) blocking reagent (Roche Diagnostics)] per 100 cm2 membrane at 68 °C for 2 h and then hybridized over night at 68 °C with 5 ml hybridization buffer containing 1 μg DIG-labelled probes (for IS30-like, ISMbov1, ISMbov2 and ISMbov3) per 100 cm2 membrane. The membranes were washed twice for 5 min at room temperature with 2 X SSC containing 0.1% SDS, and twice for 15 min at room temperature with 0.2 X SSC containing 0.1% SDS. The DIG-labelled probes were detected using phosphatase-labelled anti-digoxigenin antibodies and CDP-Star (Roche Diagnostics) according to the manufacturer's instructions.
Characterization of ISMbov1
While reacting a genomic library from M. bovis with the ISMag1 specific probe derived from the DNA of the ovine mycoplasma M. agalactiae, a new insertion element was discovered in the bovine pathogen. This IS element of 1521 bp, named ISMbov1, shows high homology (92%) with ISMag1 and contains inverted repeats of 3 bp. The encoded transposase (nucleotides 333–1361) is composed of 342 amino acids (Fig. 1(a)). An integrase core domain (Pfam accession number PF00665) was localized between bases 192 and 339.
Characterization of ISMbov2
From the above library, a positive clone reacting with a gene probe derived from ISMmy1 of M. mycoides subsp. mycoides SC was retained and analyzed in detail. A full IS element of 1671 bp was evidenced, named ISMbov2, that shows a very high homology (97%) with ISMmy1 and contains inverted repeats of 30 bp and a gene encoding the putative transposase on a single ORF (Fig. 1 (b)). The encoded transposase (nucleotides 235–1647) is composed of 470 amino acids. A transposase DDE domain (Pfam accession number PF01609) [ was detected between amino acids 172 and 385.
Characterization of ISMbov3
The M. bovis genomic library reacted also with the IS1634 specific probe. Sequencing of a positive clone revealed the presence of an IS element of 1873 bp, named ISMbov3, that shows 97% identity with IS1634 of M. mycoides subsp. mycoides SC and contains inverted repeats of 13 bp and a gene encoding a transposase (nucleotides 184–1785) of 533 amino acids (Fig. 1 (c)). A conserved integrase C1 signature sequence of IS4 family transposases was detected in the C-terminal half of the transposase between amino acids 416 and 430.
Distribution of IS30-like, ISMbov1, ISMbov2 and ISMbov3 in M. bovis strains
Genomic DNA from 11 M. bovis isolates (Table 1), strain PG1 of M. mycoides subsp. mycoides SC and strain 3990 of M. agalactiae digested by EcoRV was subjected to Southern blotting with probes for the IS elements IS30-like, ISMbov1, ISMbov2 and ISMbov3. The four typing experiments led to the identification of different hybridization patterns with heterogeneous profiles. Among the 11 M. bovis isolates, the band patterns revealed the presence of different copy numbers of the four IS elements.
IS30-like typing evidenced the presence of 4–8 copies, depending on the strain (Fig. 2). PG45 had a particular profile, whereas the other M. bovis strains formed three similarity clusters. The IS30-like specific probe also reacted with an EcoRV DNA fragment from M. agalactiae strain 3990 (Fig. 2). No pattern similarity was observed among the 11 M. bovis isolates by ISMbov1 typing (Fig. 3). The number of fragments reacting with the ISMbov1 specific probe varied from 8 to 12. Due to the high similarity between ISMag1 and ISMbov1, also the M. agalactiae strain 3990 presented several bands reacting with the ISMbov1 specific probe (Fig. 3). Twelve to 18 DNA fragments from the M. bovis strains tested reacted with the ISMbov2 specific probe (Fig. 4). Seven isolates presented unique profiles. Strains 86p and 39G presented a same band pattern. Both isolates of strain 0435 (passage 7 and passage 80) presented a similar hybridization profile with few differences. As expected, the M. mycoides subsp. mycoides SC strain PG1 reacted with the ISMbov2 specific probe. The bands observed were five (Fig. 4). On the contrary, the ISMbov2 specific probe did not react with the M. agalactiae strain 3990. ISMbov3 typing evidenced the presence of 4–10 copies in M. bovis, depending on the strain (Fig. 5). Six isolates presented unique profiles. Strains 86p and 39G and isolate 9585 (passage 98) presented a same EcoRV band pattern, as it was the case also between the two isolates of strain 0435 (passage 7 and passage 80). Due to the very high similarity between IS1634 and ISMbov3, the M. mycoides subsp. mycoides SC strain PG1 also reacted with the ISMbov3 specific probe.
Three IS elements, ISMbov1, ISMbov2 and ISMbov3, have been sequenced from M. bovis. Different copies of all three IS elements were sequenced from isolate 2610 (passage 7) and shown to be well conserved at the nucleotide sequence level. Minor variations were observed, with a range of 0.5–11.25 differences per 1000 bp, and only half of them are able to affect the amino acid composition of the three putative transposases. Based on the criteria adopted by Mahillon and Chandler [ review on IS elements, ISMag1 and ISMbov1 may be considered isoforms (less than 10% nucleotide divergence). The relationships between ISMmy1 and ISMbov2, and between IS1634 and ISMbov3 are even stricter. All the M. bovis strains tested in this study contained several copies of each IS. It has been previously described that M. bovis contained a further IS element designated IS30-like [. This IS element is present in all the M. bovis strains tested in this study and Southern blotting profiles were not the same as those obtained for ISMbov1, ISMbov2 and ISMbov3. No correlation has been found between IS profiles and the clinical symptoms of the animals from which the M. bovis strains were isolated (bronchopneumonia, arthritis, mastitis, cattle, rabbit), the number of passages, or adherence rates of the strains (Table 1). However, it is evident that the genome of M. bovis contains a large number of IS elements and this feature could be associated with a rapid gene rearrangement, as observed for Mycoplasma fermentans [.
A clear picture can be observed from the different Southern blot analyses, where the frequency of IS elements and the variation in IS profiles in M. bovis contrast, for instance, with the situation in M. mycoides subsp. mycoides SC, where the profiles for the three different IS elements are rather homogeneous among the strains [[1,[2,. Additionally, in M. bovis strains 2610, 0435 and 9585 subjected to more than 80 passages in vitro, further transposition or recombination events could be detected on the Southern blots reacted with the probes for all four IS elements, if compared to the strains after only 7 passages. This indicates that genetic variants of M. bovis may arise upon extended growth in vitro. An in vitro passaging effect was also observed in M. fermentans, whereby analysis of insertion element sequences revealed inter- and intra-strain polymorphisms [.
The presence of ISMbov1 (homologous to ISMag1) and of IS30-like in M. bovis and M. agalactiae indicates horizontal gene transfer between the two species or suggests that both mycoplasmas had a common ancestor. Note however, that the M. agalactiae strain 3990 shows only a single copy of IS30-like, while it shows about 25 copies of the ISMbov1-homologue ISMag1. The association of M. bovis and M. agalactiae was also underlined by the previous findings that both mycoplasmas show a very close phylogenetic relationship, as shown by 16S rRNA and uvrC sequence similarities [[9,[7,. In this respect, it is interesting to note that ISMbov1 and ISMag1 are more closely related to each other than are the housekeeping genes uvrC from M. bovis and M. agalactiae (83% identity at the nucleic acid level) [. Previous experiments based on uvrC analysis [ suggested that M. bovis might have evolved more recently than M. agalactiae. Data as to whether M. agalactiae and M. bovis have ever been isolated from a common host are, however, not available.
M. bovis can be differentiated from M. agalactiae by the presence of multiple copies of ISMbov2 and ISMbov3 only in M. bovis. Interestingly, M. bovis shares ISMbov2 (homologous to ISMmy1) and ISMbov3 (homologous to IS1634) with M. mycoides subsp. mycoides SC. Since these two mycoplasmas both infect bovines, it is possible that a horizontal transfer of the two IS elements might have occurred during an ancient co-infection of a cow. The lower number of polymorphisms found between ISMmy1 and ISMbov2, and between IS1634 and ISMbov3 (97% identity between M. mycoides subsp. mycoides SC and M. bovis) if compared to those found between ISMag1 and ISMbov1 (92% between M. agalactiae and M. bovis) may imply that the horizontal transfer events between the two bovine mycoplasmas are more recent than the divergent evolution of M. agalactiae and M. bovis.
In conclusion, M. bovis, like M. mycoides subsp. mycoides SC, is striking in its exceptionally high number of insertion elements (6–12 copies of ISMbov1, 11–15 copies of ISMbov2, 4–10 copies of ISMbov3 and 4–8 copies of IS30-like), representing approximately 60 kb or 6% of total genomic DNA, whose length was established to be 961 ± 18.9 kb for M. bovis [. This insertion sequence variability can be associated with rapid gene rearrangements that may confer improved genetic fitness of M. bovis towards specific host tissues.
The authors are grateful to Isabelle Dizier for technical help, to Dr. Sachse (BGVV, Jena, Germany), Dr. Ball (DARDNI, Belfast, UK), Dr. Blanchard (INRA, Bordeaux, France), Dr. Kempf (AFSSA, Ploufragan, France) and Dr. Boucher (Les Herbiers, France) for providing M. bovis isolates. This study was possible thanks to the grant from the Belgian ‘Ministère de l'Agriculture’ (convention 6039) and to the European COST Action 826.