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Mycoplasma mycoides subsp. mycoides small-colony type (SC) is the infectious agent of contagious bovine pleuropneumonia (CBPP), a severe, highly contagious disease of cattle causing important economic losses and large socioeconomic problems in many countries worldwide. CBPP has reemerged in a few European countries during the last few years, thus threatening the industrialized countries in which the disease was successfully eradicated during the late 1950s (5, 12, 21). Substantial efforts in research on M. mycoides subsp. mycoides SC are required to elaborate efficient methods for diagnosis, prevention, and treatment of CBPP, which are necessary to eradicate this epizootic and prevent it from further spread. Although M. mycoides subsp. mycoides SC is distinguished by its very high pathogenicity from the other members of closely related mycoplasmas of the M. mycoides cluster, which are mostly of only low epidemiological importance, it is phenotypically and antigenically very similar to these. This fact is a major hindrance to the serodiagnosis of CBPP and to detection and identification of M. mycoides subsp. mycoides SC. It has also been shown that, phylogenetically, M. mycoides subsp. mycoides SC can hardly be distinguished from most other members of the M. mycoides cluster, as revealed by sequence analysis of the rrn (16S rRNA) genes and physical genome mapping (14, 16). Moreover, all genes or DNA fragments from M. mycoides subsp. mycoides SC characterized so far have also been found with a variable degree of similarity in other members of the M. mycoides cluster (6-8, 20). A detailed genetic analysis is therefore necessary to discover the main factors that differentiate M. mycoides subsp. mycoides SC from the close relatives of the M. mycoides cluster.

Insertion sequences are transposable elements of about 800 to 2,500 bp (10) which are often present in multiple copies in bacterial genomes. They are able to induce high-frequency chromosomal rearrangements causing phenotypic changes and are commonly used as a genetic tool for the identification of bacterial pathogens. Recently, an insertion element, IS1296, was discovered in M. mycoides subsp. mycoides SC (6). Its insertion profile allowed differentiation between strains isolated from the reemerging outbreaks in Europe since 1980 and strains from the African and Australian continents collected over the last 45 years (4). IS1296 was, however, found at low copy numbers also in a few strains of M. mycoides subsp. mycoides LC and Mycoplasma sp. bovine group 7. We now report the discovery of a novel insertion sequence, named IS1634, which is highly specific for M. mycoides subsp. mycoides SC and produces unusually long and variable-length direct repeats.

Strains, growth conditions, and DNA extraction. The mycoplasma isolates used in this study are listed in Table 1. Mycoplasmal cultures were made in a standard PPLO broth medium enriched with 20% horse serum, 2.5% yeast extract, and 1% glucose at 37°C until stationary growth phase (1). DNA extractions using guanidium thiocyanate were performed as previously described (4).

Cloning, detection, and analysis of IS1634. IS1634 was detected while studying the DNA segments proximal to copies of IS1296 of M. mycoides subsp. mycoides SC strains L2 and Afadé. Genomic HindIII fragments were cloned into the HindIII site of pBluescriptII SK, and clones were screened with a specific IS1296 probe prepared as described previously (4). Fragments neighboring IS1296 were sequenced by using the oligonucleotide primers IS1296S1 (5'-CAACTGAAATATCATATTTATTTGC-3') and IS1296S2 (5'-GGATTGTCTCCTGTACAATTCAG-3') matching the ends of IS1296 and facing outward from the insertion element. In one clone, plasmid pJFFev7.0-L2 containing a 7.0-kb-long insert from M. mycoides subsp. mycoides SC strain L2, we detected a new insertion element only 17 bp away from IS1296. This novel insertion element has been designated IS1634 by the Plasmid Reference Center, Stanford University School of Medicine, Stanford, Calif. Determination of the entire nucleotide sequence of IS1634 was done by creation of deletion subclones by exonuclease III digestion and sequencing with oligonucleotide primers complementary to the T3 and T7 promoters flanking the cloning site of pBluescriptII SK. This copy of IS1634 has a size of 1,872 bp, contains characteristic 13-bp inverted repeats at its ends, and includes a single open reading frame (ORF) spanning nearly the entire sequence between the two inverted repeats. There are several putative mycoplasmal start codons for this ORF, including GUG at nucleotides (nt) 110 and 194 and UUA at nt 182, but no AUG. Although UUA is most rarely used as a start codon in eubacterial genes, it must be noted that UUA at nt 182 is preceded by a canonical sequence for a ribosome binding site (AAAGG) 8 bp upstream (11, 13) and could be a potential start codon of this transposase, as was also observed for the closely related Mycoplasma capricolum (2). The resulting peptide of 533 amino acids has 28% identical and 45% similar positions in a 266-amino-acid overlap of the central region with the corresponding part of the putative transposase from insertion element IS1549 of Mycobacterium smegmatis (15). This indicates that the ORF represents the transposase of IS1634. There are 5 UGA_Trp codons in this transposase gene. UGA encodes tryptophan in the genus Mycoplasma but is generally used as a stop codon in prokaryotes. The A+T content of IS1634 is 73.9%, reflecting the A+T-rich genome characteristic of mycoplasmas.

Identification of direct repeats. To verify the correct ends of IS1634 and to study its sites of transposition, we have cloned and analyzed independent copies of this insertion element. Primers IS1634(in)L (5'-ACTTAGTCGCGAAAACACTC-3') and IS1634(in)R (5'-AGCATAAAACAAGAAAACATAC-3'), matching the ends of IS1634 and facing inward, were designed to produce a probe for IS1634 from plasmid pJFFev7.0-L2. This probe was used to screen the two gene banks from M. mycoides subsp. mycoides SC strains L2 and Afadé. In both libraries, 30 to 50 of 300 colonies hybridized to the IS1634 probe, indicating a relatively high copy number of this insertion element. Four clones from the L2 library (plasmids pJFFev7.0-L2, pJFFev9.0-L2, pJFFev2.0-L2, and pJFFev6.0-L2) and five clones from the Afadé library (plasmids pJFFev3.9-Af, pJFFevAM5802, pJFFevAM5830, pJFFevAM2520, and pJFFevAM2516) were retained to be sequenced across the junctions of IS1634 integration, using primers ISnew(out)L (5'-TATAGCCGCTGAAAACTGAG-3') and ISnew(out)R (5'-AAAAAACATAACTTATGAGATC-3') facing outward from IS1634. Long stretches and segments which could not be sequenced accurately by this strategy were sequenced by the use of primers derived from sequenced segments. Analysis of the sequences obtained confirmed the ends of IS1634 and revealed that the different copies of IS1634 were flanked by direct repeats of variable length, ranging from 17 to 478 bp (Fig. 1). This sequence strategy was not successful with plasmids pJFFev6.0-L2 and pJFFevAM2520 since they both presented overlapping sequences, suggesting the presence of two (or more) IS1634 copies in the same clone.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Structure of IS1634 insertion sites. The map for the IS1634 clones (open boxes) that were analyzed shows the long, variable-length direct repeats at the site of insertion as black boxes and black arrows beneath (A to F). The plasmid pJFFevAM2516 contains one copy of IS1634 with no apparent flanking direct repeats (G). Hatched boxes represent all or part of IS1296. Grey triangles represent the inverted repeats IR-L and IR-R of IS1634 or IS1296.

Distribution of IS1634 in M. mycoides subsp. mycoides SC and related species. To analyze the distribution of IS1634, we have performed Southern blot hybridization analysis with HindIII-digested genomic DNA of a large number of M. mycoides subsp. mycoides SC strains as well as many strains from the other members of the M. mycoides cluster by using the IS1634 probe. As shown in Fig. 2, all strains of M. mycoides subsp. mycoides SC originating from different countries and continents and collected over a period of more than 60 years contain about 30 copies of IS1634 and show a constant picture with only minor differences. These observations indicate that IS1634, similarly to the previously discovered IS1296 (4, 6), has a relatively low transposition frequency or that M. mycoides subsp. mycoides SC has acquired an optimal constellation of IS1634 copies on its genome for survival in its ecological niche, the infected tissues of the animal host. Since IS1634 insertions show a lower percentage of variations than IS1296 insertions do, typing of strains with this new IS element gives fewer different profiles than the previously reported typing with IS1296 (4).

View larger version (94K): [in this window] [in a new window]   FIG. 2.   Southern blot hybridization of HindIII-digested DNA of a selection of M. mycoides subsp. mycoides SC isolates. DNA digests of different M. mycoides subsp. mycoides SC strains were separated electrophoretically on a 0.5% agarose gel, transferred to a positively charged nylon membrane, and hybridized to the IS1634 probe. The strains are described in Table 1. Abbreviations: Eu, Europe; Af, Africa; Au, Australia; T, type strain; superscripts v, vaccine strain; superscript c, caprine strain; superscript o, ovine strain. Continent designations with no superscript indicate strains from cattle. Std, molecular mass standards: 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. Arrows indicate particular IS1634 copies. (A) Blot results for strains from Europe in comparison to PG1. A thin arrow indicates the region of the blot at about 4.0 kb where the IS1634 pattern of strain O326 differed from the others. The 7.-kb fragment characteristic of all European strains is indicated by a horizontal dotted arrow. (B) Blot results for strains from Africa and Australia in comparison to PG1. A horizontal arrow indicates the bands at about 3.0 to 3.5 kb where African strains showed heterogeneity in the IS1634 pattern.

Concluding remarks. M. mycoides subsp. mycoides SC, by far the most pathogenic mycoplasma in the M. mycoides cluster, is striking in its exceptionally high number of insertion elements, about 20 copies of IS1296 and about 30 copies of IS1634, representing 7% of total genomic DNA (approximately 90 kb). In view of the capacity of insertion elements to modulate gene expression (10, 19), one might speculate that M. mycoides subsp. mycoides SC has used these elements to regulate the expression of its genetic potential, resulting in a phenotype for invasion ability into host tissue being conferred to this microorganism. The fact that IS1634 is specific to M. mycoides subsp. mycoides SC, where it is found at a high copy number, makes it an optimal target for identification of M. mycoides subsp. mycoides SC and for the development of highly sensitive detection methods.

Nucleotide sequence accession number. The EMBL/GenBank accession numbers for the nucleotide sequences of five of the copies of insertion element IS1634 analyzed are AF062493, AF101410, AF101411, AF101412, and AF101413.