Abstract
Mycoplasma sp. (strain F38) is the causative agent of contagious caprine pleuropneumonia, which is a goat disease of great global concern. Strain F38 belongs to the so-called "Mycoplasma mycoides cluster," and the members of this cluster have many biochemical and serological properties in common, which makes it difficult to differentiate between them by conventional methods. Their phylogenetic interrelationships are thus uncertain. The 16S rRNA gene of the rrnB operon from strain F38 was cloned and sequenced. The sequence was compared with the 16S rRNA sequences of related mycoplasmas, and phylogenetic trees were constructed by parsimony analysis. A three-way ambiguity among strain F38, Mycoplasma capricolum, and Mycoplasma sp.
Abstract
strain PG50 was observed in the trees. This observation is in agreement with a recent proposal to reclassify strain F38 and M. capricolum. A primer set was designed for in vitro amplification by PCR of a fragment of the 16S rRNA genes from the M. mycoides cluster. The amplimers of strain F38 could be distinguished easily from the corresponding amplimers from other members of the M. mycoides cluster by restriction enzyme analysis with PstI. This observation was utilized to design an identification system for strain F38. Part of the 16S rRNA gene of the rrn4 operon from strain F38 was also cloned, and several sequence differences between the two rRNA operons were discovered, revealing microheterogeneity between the two 16S rRNA genes of this organism.
Abstract
Mycoplasmas are the smallest free-living microorganisms found so far, and they have a genome size only one-sixth to one-third of that of Eschenichia coli. They lack cell walls and belong to the class Mollicutes (44). However, mycoplasmas are phylogenetically related to gram-positive bacteria with low G+C content (32). The mycoplasmas cause great economic losses, since they are widely distributed and many of them are pathogenic in humans, animals, and plants (33). Furthermore, they often contaminate cell cultures (36).
Abstract
Mycoplasmas are to a greater or lesser extent host specific. Goats harbor a number of species (12), and one of the most important is Mycoplasma sp. (strain F38), which is the etiological agent of contagious caprine pleuropneumonia (CCPP). This disease occurs mainly in Asia and Africa. It is considered to be the major economic obstacle to goat breeding in several countries in those parts of the world and thus represents a potential threat to neighboring countries (42). Mycoplasma mycoides subsp. capn was for many years designated as the causative agent of CCPP, but it is now generally accepted that strain F38 is solely responsible for classical CCPP (31). Strain F38 has been isolated from several outbreaks of CCPP, and it has proved possible to induce the natural disease by inoculating the agent into susceptible animals (30 Sequence comparison of 16S rRNA from bacteria is one of the most powerful and precise methods for determining phylogenetic interrelationships (40, 58). Mycoplasmas have recently been classified into five groups (the hominis group, the pneumoniae group, the spiroplasma group, the anaeroplasma group, and the asteroleplasma group) on the basis of rRNA sequences, and the M. mycoides cluster (including M. putrefaciens) constitutes one cluster of the spiroplasma group (57). This classification has resulted in a revised taxonomy of the Mollicutes (55), and the class now contains eight genera: Mycoplasma, Ureaplasma, Spiroplasma, Acholeplasma, Anaeroplasma, Asteroleplasma, Entomoplasma, and Mesoplasma. The last two genera are new, and some former Mycoplasma species and Acholeplasma species, associated with insects and plants, have been transferred to these genera, in which also some species from the spiroplasma group were included. The generic distinctions are based on molecular data as well as on differences in morphology, nutritional requirement, and ecological habitat.
Abstract
rRNA has also been used to develop identification systems for mycoplasmas (21, 25, 34, 35, 56) . Therefore, we have determined the sequence of the 16S rRNA gene from the rmB operon of strain F38 and have used it for phylogenetic studies and for the development of an identification system. The identification system was based on in vitro amplification by PCR (47) and restriction enzyme analysis of the amplimers. We also revealed microheterogeneity in the two different 16S rRNA genes of strain F38.
Abstract
on November 7, 2017 by guest

Mycoplasmas are the smallest free-living microorganisms found so far, and they have a genome size only one-sixth to one-third of that of Eschenichia coli. They lack cell walls and belong to the class Mollicutes (44) . However, mycoplasmas are phylogenetically related to gram-positive bacteria with low G+C content (32) . The mycoplasmas cause great economic losses, since they are widely distributed and many of them are pathogenic in humans, animals, and plants (33) . Furthermore, they often contaminate cell cultures (36) .

Mycoplasmas are to a greater or lesser extent host specific. Goats harbor a number of species (12) , and one of the most important is Mycoplasma sp. (strain F38), which is the etiological agent of contagious caprine pleuropneumonia (CCPP). This disease occurs mainly in Asia and Africa. It is considered to be the major economic obstacle to goat breeding in several countries in those parts of the world and thus represents a potential threat to neighboring countries (42) . Mycoplasma mycoides subsp. capn was for many years designated as the causative agent of CCPP, but it is now generally accepted that strain F38 is solely responsible for classical CCPP (31) . Strain F38 has been isolated from several outbreaks of CCPP, and it has proved possible to induce the natural disease by inoculating the agent into susceptible animals (30) . Strain F38 belongs to the so-called "Mycoplasma mycoides cluster," which includes the following mycoplasmas: Mycoplasma capnicolum, M. mycoides subsp. mycoides (large-and small-colony type [LC and SC, respectively]), M. mycoides subsp. capni, and Mycoplasma sp. strain PG50 (11, 15) . Mycoplasma putrefaciens was included in the M. mycoides cluster when the clustering was based only on 16S rRNA sequences (57) . All of the above species are The F38-like strains were grown in WJ medium (29) . The other mycoplasmas were grown in F medium or HA medium (3) . The amounts of mycoplasmas used in the PCR experiments were estimated by CFU or color-changing units as described by Rodwell and Whitcomb (46) . Nucleic acid extraction. DNA extraction and precipitation were carried out essentially as described by Sambrook et al. (48) . Briefly, a 200-ml culture from strain F38 in a lateexponential phase was centrifuged for 30 min at 25,000 x g. The pellet was washed twice with phosphate-buffered saline (PBS), resuspended in 5 ml of ice-cold cell lysis buffer (10 mM Tris-HCl [pH 7 .4]-10 mM NaCl-10 mM EDTA), and incubated for 1 (20) . The sizeselected DNA fragments were then cloned into pUC19 and transformed into E. coli DHSa. Preparation of competent cells and transformation were carried out by the one-step procedure described by Chung et al. (10) . Clones from the transformation were screened by colony hybridization (48) with the oligonucleotide P-F38 as probe (5'-AGC TCC CCT TCA CAG GAT TGC-3'). P-F38 was complementary to the V9 region and designed on the basis of sequence information obtained by direct sequencing of 16S rRNA with the U7 oligonucleotide as sequencing primer (27) . For a definition of different sequence regions in 16S rRNA, see Gray et al. (22) .

Positive colonies were grown overnight, and small-scale preparations of plasmid DNA were sequenced (50) . A largescale preparation of plasmid DNA was made from a clone containing the whole 16S rRNA gene by precipitation with polyethylene glycol (48 (27) . The nucleotide analog dITP was used to resolve band compressions. The products from the sequencing reactions were separated by electrophoresis on 8% polyacrylamide gels with 7 M urea.

Sequence analysis. The sequences were analyzed by programs developed by the Genetics Computer Group at the University of Wisconsin (Madison) (13) . Phylogenetic analysis was conducted with the phylogenetic inference program package PHYLIP (17) .

In vitro amplification by PCR. One milliliter of broth culture of each bacterium tested by PCR was centrifuged, washed in PBS, resuspended in water, and lysed by being heated at 100°C for 10 min. DNA from 5-,ul samples of undiluted and 10-folddiluted lysed cells was used as PCR templates. A fragment of the 16S rRNA genes from the members of the M. mycoides cluster was amplified by PCR with the forward primer MmF (5'-CGA AAG CGG Cll ACT GGC TTG TT-3') and the reverse primer MmR (5'-TIG AGA TTA GCT CCC CiT CAC AG-3') (see Fig. 1 ). The amplification was performed in a 50-,u reaction mixture containing 0.2 mM (each) deoxynucleotide, 20 pmol of each primer, 50 mM KCI, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, and 1.5 mM MgCl2. Two droplets of mineral oil were layered on reaction mixtures to prevent evaporation. To enhance the specificity, a manual hot start was used to prevent initial nonspecific annealing of primers (8 Restriction enzyme analysis of amplimers. Without further purification, the amplimers were digested with PstI in OnePhor-All buffer PLUS (Pharmacia LKB Biotechnology) at 37°C for 2 h, and the resulting products were analyzed by agarose gel (2%) electrophoresis.

Analysis of rRNA operons. The strain F38 amplimers were digested with PstI, and the fragments were separated by electrophoresis in a 2% low-temperature melting agarose gel (FMC BioProducts, Rockland, Maine). The large PstI fragment, representing part of the 16S rRNA gene of the rnA operon, was excised from the gel and purified with the Magic PCR Preps (Promega). The fragment was then cloned into pUC19 and sequenced as described above. Five different clones were chosen to compensate for any nucleotide incorporation error introduced by the Taq DNA polymerase.

Nucleotide sequence accession numbers. The sequences of 16S rRNA from strain F38 have been deposited in GenBank under the accession numbers M94728 and L14607.
RESULTS
The nucleotide sequence of 16S rRNA from strain F38. A 2.0-kb HindlIl fragment from Mycoplasma sp. strain F38 was cloned into pUC19. The insert included the 16S rRNA gene of the rmB operon, flanking regions, and part of the 5' end of the 23S rRNA gene of the same operon. Figure 1 shows the complete sequence of the 16S rRNA from strain F38 aligned with the corresponding sequences from related mycoplasmas deposited in the EMBL data bank for nucleotide sequences. The locations of the 5' and 3' termini of the 16S rRNA have not been determined experimentally, but are based on the established mature ends of the 16S rRNA from other bacteria (39) . The length of the 16S rRNA gene from the rmB operon of strain F38 was tentatively estimated at 1,525 nucleotides. A partial sequence of 425 nucleotides from the 16S rRNA of the nrA operon of strain F38 was also determined (Fig. 2) .
RESULTS
The 16S-23S rRNA-spacer region from the rnB operon of strain F38 was nearly identical to that from M. capricolum (24) , since only four nucleotide differences out of 225 nucleotides were found (data not shown). In agreement with data from most other mycoplasmas, no tRNA genes were identified in the spacer region (24, 52) . However, a recent report describes two spacer tRNA genes in one of the two operons from another member of the class Mollicutes, namely, Acholeplasma laidlawii (38) .
RESULTS
Sequence analysis. The 16S rRNA sequences from several members of the spiroplasma group (57) and strain F38 were aligned by the PileUp program of the sequence analysis software package from Genetics Computer Group (13) . Given the degree of similarity among the sequences, only minor adjustments of the computer-aligned sequences were made by hand before the phylogenies were estimated. The evolutionary trees shown in Fig. 3 were inferred by parsimony analysis with the DNAPARS program from PHYLIP (17) . Three equally parsimonious trees were found, in which M. capricolum, strain F38, and strain PG50 formed a group with unresolved branching order. Each of the trees required a total of 512 changes. The data were also subjected to bootstrapping (16) 
RESULTS
cluster was therefore constructed. A fragment of 548 bp stretching from nucleotide 722 to 1269 in the 16S rRNA of Mycoplasma sp. strain F38 (strain F38 numbering) was amplified with this primer pair (Fig. 1) . The restriction enzyme PstI has a recognition site at positions 845 to 850 (Fig. 1) in both operons for all of the members of the M. mycoides cluster except strain F38, which has that recognition site only in the nrnA operon (Fig. 2) . Two fragments of 420 and 128 bp were thus generated by restriction enzyme analysis with PstI of all of these mycoplasmas except for strain F38. Three fragments of 548, 420, and 128 bp were obtained from strain F38. The 548-bp fragment corresponded to undigested amplimer of the rnB operon. Samples from a goat experimentally infected with an F38-like strain were also analyzed to test the diagnostic potential of the system. Pleura fluid from the diseased goat was centrifuged, washed in PBS, resuspended in water, and lysed as described above. The expected amplimer of 548 bp was Table 1 were analyzed with the identification system for strain F38 with the result shown in Fig. 4 and summarized in Table 1 . All members of the M. mycoides cluster were amplified by the PCR system used, but only F38-like strains gave three bands after restriction enzyme analysis (Fig. 4) . In addition, we have analyzed 5 F38-like strains, 9 M. capnicolum strains, and 10 M. mycoides strains from different parts of the world, and they all gave consistent electrophoresis patterns after PCR (and restriction enzyme analysis) (4).
RESULTS
Number of operons. It has been shown earlier that many mycoplasmas of the M. mycoides cluster (including strain F38) have two operons for rRNA (9, 43 Table 1 . In panel B, strain F38 was applied before and after digestion (lanes 1 and 2, respectively) . The amplimer from M. capricolum was also applied before and after digestion with PstI ( lanes 3 and 4, respectively) . BglI-cleaved pBR 328 DNA and HinfI-cleaved pBR 328 DNA from Boehringer Mannheim (Mannheim, Germany) were used as molecular size markers in lanes M.
RESULTS
blots (data not shown) and by cloning and sequence analysis of parts of both 16S rRNA genes. The rmB operon lacks a recognition site for PstI in the 16S rRNA gene, whereas the rrnA operon has a recognition site for PstI located at a position identical to the PstI sites present in the other species from the M. mycoides cluster. This microheterogeneity explains the presence of three bands for the digested amplimers from strain F38. Furthermore, several variant positions were found in the part of the 16S rRNA genes that was sequenced from both operons (Fig. 2) . These differences were either unique for the second gene (see positions 1064 and 1155) or similar to 16S rRNA sequences from the other mycoplasmas in the M. mycoides cluster (see positions 848, 875, 1083, 1084, and 1150).
DISCUSSION
The complete 16S rRNA sequence of the rmB operon from Mycoplasma sp. strain F38 has been determined in this work. The strain F38 sequence was then compared with complete 16S rRNA sequences of M. capricolum (24) and Mycoplasma sp. strain PG50 (19) as well as the partial 16S rRNA sequences of M. mycoides subsp. mycoides (LC) and other related mycoplasmas (57) . The sequences of M. capricolum and Mycoplasma sp. strain PG50 have been determined from the rmB operon and the rmnA operon, respectively. Approximately 120 nucleotides (about 70 in the U5 region and about 50 in the 3' end) are unknown in the sequences of the latter species, since they were determined by direct RNA sequencing with reverse transcriptase. The partial sequences of Mycoplasma agalactiae and M. putrefaciens (57) were included in the alignment as representatives for goat mycoplasmas which are not members of the classical M. mycoides cluster.
DISCUSSION
M. capricolum has long been regarded as the closest relative to strain F38, a notion that has been supported by DNA-DNA hybridization data, serological data, and protein fingerprints (44, 45) . The taxonomic position of F38 with respect to M. capricolum has recently been thoroughly investigated by DNA-DNA hybridization experiments on several isolates (6) . The DNA relatedness values were about 70% between the F38 and M capricolum groups, whereas the values were about 90 and 85%, respectively, for the strains within each group. Consequently, strain F38 and F38-like species have been proposed to be classified as M capnicolum subsp. capripneumoniae and M capricolum as M capricolum subsp. capricolum (28) . This is also, in principle, supported by the 16S rRNA sequence data presented in the present work. However, a three-way ambiguity (F38 with M capricolum, F38 with PG50, and M capnicolum with PG50) was observed. M capricolum is usually regarded as a rather inhomogeneous species (12) , and the type strain of M capricolum, California kid, from which the 16S rRNA was analyzed (24) , is not a typical representative of the species. Usually, it is necessary to use antisera towards several isolates to identify strains of M capricolum (12) . It (18) . Instead, they suggest that species with effectively identical 16S rRNA sequences should be regarded as belonging to the same rRNA species complex. In this work, different trees are supported in the parsimony analysis depending upon how a single nucleotide position (no. 71 in Fig. 1 ) in the PG50 sequence is treated in the calculations. A single most parsimonious tree can be drawn with F38 and M. capricolum as the closest relatives if the position is treated as a gap. This is also supported in a bootstrap analysis (data not shown). However, three equally parsimonious trees, shown in Fig. 3 , can be drawn if the position is treated as a missing datum. Analysis of the secondary structure in that region suggests that the position should be treated as a missing datum, since it is on the second side of a known hairpin. In all, these results call for a further investigation on the taxonomy of PG50 and strains with similar characteristics.
DISCUSSION
Since rRNA is subject to comparatively slow changes in primary structure, alternative molecules are required to resolve close phylogenetic relationships. In an attempt to resolve the phylogenetic relationships within the M. mycoides cluster, a DNA fragment from M. capricolum was cloned and sequenced along with the corresponding PCR products from the other members (54) . The result indicates that strain F38 is most closely related to M. capricolum, although nothing is known about the biological function of the cloned DNA fragment. Surprisingly, upon analyzing open reading frames, the authors interpreted TGA as a stop codon (54), although TGA is read as tryptophan in mycoplasmas (41) .
DISCUSSION
The use of rRNA for measuring phylogenetic distances has several advantages, among which are the functional constancy of the molecules, the wide phylogenetic range, and the fact that results from different researchers can be utilized easily by others (40) . An interesting alternative to rRNA might be the RNA subunit of RNase P, the enzyme responsible for 5'-end processing of precursor tRNA (1). This enzyme is essential, and it is the RNA subunit that constitutes the catalytic core in bacteria. Furthermore, the molecule seems to be less conserved among closely related species than are rRNA sequences (51) , and the list of published sequences is growing rapidly (7) . Mycoplasmas in general show a more rapid evolutionary pace than other bacteria, which is reflected in variations of otherwise conserved nucleotides in mycoplasma 16S rRNA (57) as well as RNase P RNA sequences (51) . However, because of less pronounced functional constraints on RNase P RNA, compared with rRNA, this molecule is suitable only to solve close phylogenetic relationships (40) .
DISCUSSION
The biological significance of the microheterogeneity in the 16S rRNA genes is unclear, and little is known about sequence variations in bacteria (14) . Usually, there is only a low level of heterogeneity at about 0.1% of the nucleotide positions (37) . An exception to this observation is a protozoan parasite, Plasmodium berghei, which expresses structurally stage-specific rRNA sequences (23 (53) . Operon microheterogeneity does not pose a problem in dealing with phylogenetically distant organisms, but complicates phylogenetic inferences between closely related species (14) . For strain F38, one of the 16S rRNA genes (from the rnB operon) seems to be more distant from the other mycoplasmas, whereas the other gene (from the rnA operon) is almost identical in sequence to the other members of the cluster.
DISCUSSION
The identification system for Mycoplasma sp. strain F38 involved two steps. In the first, which was based on PCR, a 548-bp segment of the 16S rRNA genes from the members of the M. mycoides group was amplified. So far, all strains and isolates from the M. mycoides cluster have been positive in the PCR, whereas all other species tested, including several caprine mycoplasmas, were negative. In the second step, the amplimers were cleaved with the restriction enzyme PstI. This resulted in a unique restriction pattern for F38, differentiating it from the other members of the cluster. Three bands were generated for all F38-like strains, and two bands were observed with the other species. The presence of a PstI site in only one of the 16S rRNA genes from strain F38 distinguishes it from the other members of the M. mycoides cluster. Furthermore, this single site was also used as an internal control for the activity of the restriction enzyme. All amplimers, not only from non-F38 strains, were cleaved. A single band in any of the lanes would suggest that the enzyme does not function properly. The strains that were tested included all of the named mycoplasma species isolated from goats and some incompletely characterized strains from goats. The Swedish strain G5 of M. capricolum was included because of its extensive cross-reactions with an antiserum against strain F38 in the immunofluorescence and growth inhibition tests, which are the tests regularly used for mycoplasma identification. M. equigenitalium and Mycoplasma primatum were also analyzed for the same reason, although they have never been isolated from goats (29) . In conclusion, these results are very promising, and the system can thus be used for rapid and convenient identification of Mycoplasma sp. strain F38. The diagnostic potential of the system is currently under further evaluation with clinical samples from goats suffering from CCPP.

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