SEQUENCE-ANALYSIS OF 16S RIBOSOMAL-RNA FROM MYCOPLASMAS BY DIRECT SOLID-PHASE DNA-SEQUENCING

Automated solid-phase DNA sequencing was used for determination of par tial 16S ribosomal DNA sequences of mycoplasmas. The sequence informat ion was used to establish phylogenetic relationships of 11 different m ycoplasmas whose 16S rRNA; sequences had not been determined earlier. A biotinylated fragment corresponding to positions 344 to 939 in the E scherichia coli sequence was generated by PCR. The PCR product was imm obilized onto streptavidin-coated paramagnetic beads, and direct seque ncing was performed in both directions. One previously unclassified av ian mycoplasma was found to belong to the Mycoplasma lipophilum cluste r of the hominis group. Microheterogeneities mere discovered in the rR NA operons of Mycoplasma mycoides subsp. mycoides (SC type), confirmin g the existence of two different rRNA operons. The 16S rRNA sequence o f M. mycoides subsp. capri was identical to that of M. mycoides subsp. mycoides (type SC), except that no microheterogeneities were revealed . Furthermore, automated solid-phase DNA sequencing was used to identi fy a mycoplasmal contamination of a cell culture as Mycoplasma hyorhin is, which proved to be very difficult by conventional methods. The res ults suggest that the direct solid-phase DNA sequencing procedure is a powerful tool for identification of mycoplasmas and is also useful in taxonomic studies.