Pettersson B, Leitner T, Ronaghi M, Bölske G, Uhlen M, Johansson K E
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
J Bacteriol. 1996 Jul;178(14):4131-42. doi: 10.1128/jb.178.14.4131-4142.1996.
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 small-colony 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.
所谓的丝状支原体簇由六种支原体（柔膜菌纲）物种或亚种组成。这些物种对反刍动物具有致病性，其中一些在兽医学中备受关注。丝状支原体簇的成员有两个rRNA操纵子（rrnA和rrnB）。通过直接自动化固相DNA测序，测定了代表丝状支原体簇所有已知物种和亚种的10个菌株的16S rRNA基因的核苷酸序列。两个rRNA操纵子的序列通过一种新策略测定，该策略涉及用一对操纵子特异性引物和一对通用引物通过PCR进行体外扩增。有趣的是，在所有菌株中均观察到两个操纵子之间的序列差异（多态性）。对两株山羊支原体山羊肺炎亚种进行了测序，在模式菌株（F38）中发现了15个多态性，在另一菌株（4/2LC）中发现了17个多态性。在丝状支原体丝状亚种小菌落型的16S rRNA基因中发现了8个多态性，并且在该物种两个操纵子的16S rRNA基因中观察到一个聚（A）区域的序列长度变异。二级结构分析表明，茎区和环区均存在多态性。茎区多态性位点的核苷酸替换常常导致从典型碱基对变为非典型碱基对，反之亦然。在碱基对的另一个核苷酸中从未观察到补偿性突变。基于16S rRNA序列的系统发育分析表明，支原体菌株PG50应归入山羊支原体物种组。此外，丝状支原体山羊亚种和丝状支原体丝状亚种大菌落型的16S rRNA序列有99.9%的同一性。因此，我们建议将这些物种重新分类到一个与丝状支原体丝状亚种小菌落型不同的共同物种组（例如，“山羊支原体”）中，丝状支原体丝状亚种小菌落型在山羊支原体物种组和山羊支原体物种组之间形成一个中间分支。
