Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons

Abstract

The so-called Mycoplasma mycoides cluster consists of six species or subspecies of mycoplasmas (Mollicutes). These species are pathogenic for ruminants and some of them are of great concern in veterinary medicine. The members of the M. mycoides cluster have two rRNA operons (rrnA and rrnB). The nucleotide sequences of the 16S rRNA genes of 10 strains, representing all of the known species and subspecies of the M. mycoides cluster, were determined by direct automated solid-phase DNA sequencing. The sequences of both rRNA operons were determined by a novel strategy involving in vitro amplification by PCR with one operon-specific primer pair and one general primer pair. Interestingly, sequence differences (polymorphisms) between the two operons were observed for all strains. Two strains of M. capricolum subsp. capripneumoniae were sequenced, and 15 polymorphisms were found in the type strain (F38) and 17 polymorphisms were found in the other strain (4/2LC). Eight polymorphisms were found in the 16S rRNA genes of the M. mycoides subsp. mycoides smallcolony type, and sequence length variations in a poly(A) region were observed in the 16S rRNA genes of the two operons of this species. Secondary-structure analysis showed that polymorphisms were present in both stem and loop regions. The nucleotide substitutions in the polymorphic sites of the stem regions often resulted in a change from a canonical to a noncanonical base pairing or vice versa. A compensatory mutation was never observed in the other nucleotide of the base pair. Phylogenetic analysis based on the 16S rRNA sequences indicated that Mycoplasma sp. strain PG50 should be included in the M. capricolum species group. Furthermore, the 16S rRNA sequences of M. mycoides subsp. capri and the M. mycoides subsp. mycoides large-colony type were 99.9% identical. We therefore suggest that these species be reclassified in a common species group (for instance, "Mycoplasma capri") distinct from the M. mycoides subsp. mycoides small-colony type, which formed an intermediate branch between the M. capricolum species group and the M. capri species group.Many species belonging to the class Mollicutes (trivial name, mycoplasmas) are pathogenic and of great economic concern in livestock production. There is one interesting group of six closely related mycoplasmas named the Mycoplasma mycoides cluster, consisting of several ruminant pathogens (13,49,50). This group comprises the following species, subspecies, or strains: M. capricolum subsp. capricolum (formerly M. capricolum), M. capricolum subsp. capripneumoniae (formerly Mycoplasma sp. strain F38), M. mycoides subsp. capri, the M. mycoides subsp. mycoides large-colony (LC) and small-colony (SC) types, and Mycoplasma sp. strain PG50.The best-known species in the M. mycoides cluster is perhaps the M. mycoides subsp. mycoides SC type, which is the causative agent of contagious bovine pleuropneumonia. This disease has been known since the 18th century, and the organism was first isolated in 1898 by Nocard and Roux (38). The M. myco...

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

“…It has been reported that MmmLC and Mmc are antigenically very similar, as assessed by the numerical analysis of one-dimensional SDS-PAGE protein patterns [18]. Phylogenetic studies based on sequence analysis of 16S rRNA genes have shown that it is obviously impossible to genetically distinguish between the MmmLC biotype and the subspecies Mmc (99.9% similarity) [26]. Therefore, MmmLC and Mmc may represent two different phenotypes of a same and unique subspecies of the M. mycoides species [21].…”

“…Among the field strains tested in this study, five strains were proven to be strictly identical to Msp7 strain PG50 by MF dot, by PCR and on the basis that these strains shared some strictly similar IS 1296 and vmm Southern blot profiles. Most of the phylogenetic analysis [14,25,33], particularly based on the 16S rRNA sequences [26], place Msp7 strain PG50 as a sub-species of the M. capricolum species. However, other genetic particularities place Msp7 strain PG50 phylogenetically closer to the M. mycoides species.…”

“…But classifying the members of M. mycoides cluster has always been problematic: (i) Extensive and complex cross-reactions occur between subspecies or types, and acute antigenic identification of CBPP and CCPP agents has been achieved only through the use of specific monoclonal antibodies [4,32]; (ii) High antigenic heterogeneity within some types or subspecies (MmmLC, Mcc) hampers identification [6,18,27]; (iii) The definitive taxonomy of the M. mycoides cluster has not yet been established and discrepancies have recently appeared between their taxonomy and phylogeny. Msp7 strain PG50 produces significant cross-reactions with Mccp and, to some extent, with MmmSC [13,27], and should be phylogenetically included as a subspecies of the M. capricolum species [1,14,26,33]. The 16S rRNA genes of Mmc and MmmLC are 99.9% similar, suggesting they should be considered as two phenotypes of the same species, distinct from MmmSC [21,26]; (iv) Animal host specificity, previously thought as very specific and used as a clue to identification, has proven to be unreliable [23].…”

“…Msp7 strain PG50 produces significant cross-reactions with Mccp and, to some extent, with MmmSC [13,27], and should be phylogenetically included as a subspecies of the M. capricolum species [1,14,26,33]. The 16S rRNA genes of Mmc and MmmLC are 99.9% similar, suggesting they should be considered as two phenotypes of the same species, distinct from MmmSC [21,26]; (iv) Animal host specificity, previously thought as very specific and used as a clue to identification, has proven to be unreliable [23].…”

Mycoplasma mycoidessubsp.capriandMycoplasma mycoidessubsp.mycoidesLC can be grouped into a single subspecies

“…capri and M. mycoides subsp. mycoides LC [24]. Since phylogenetic analysis based upon the 16S rRNA gene sequences alone provided only a limited understanding of species relationships and evolutionary history, and 16S rRNA operons show high intraspecific variation, sequences of protein-encoding genes have been suggested and used.…”