COMPARISON OF MYCOBACTERIUM 23S RIBOSOMAL-RNA SEQUENCES BY HIGH-TEMPERATURE REVERSE TRANSCRIPTION AND PCR

We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium, We combined the sensitivity of the reverse t ranscriptase PCR for amplifying nanogram amounts of template rRNA mate rial with the elevated extension temperatures used for the thermostabl e DNA polymerase from Thermus thermophilus, A 70 degrees C reverse tra nscription extension step permitted improved read-through of highly st ructured rRNA templates from members of the genus Mycobacterium, which have G+C-contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle s equencing with alpha-P-33-labeled primers, again with extension at 70 degrees C. Nonspecifically terminated bands were chased by using termi nal deoxynucleotidyl transferase. Our method had a template requiremen t of nanogram amounts or less of purified RNA or 2,000 CFU of intact c ells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Se quences from a 250-nucleotide stretch of the 23S rRNA were aligned, an d phylogenetic trees were evaluated by using the De Soete distance tre eing algorithm and Rhodococcus bronchialis as the outgroup. Our 23S rR NA trees were compared with previously published 16S rRNA trees, inclu ding the comprehensive trees developed by the University of Illinois R ibosomal Database Project, and included 15 species not evaluated previ ously. Most of the groups were in general agreement and were consisten t with relationships determined on the basis of biochemical characteri stics, but some new relationships were also observed.