2 DIFFERENT 16S RIBOSOMAL-RNA GENES IN A MYCOBACTERIAL STRAIN

Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-establ ished method used to identify bacteria, particularly mycobacteria, Uni que sequences allow identification of a particular genus and species, If more than one 16S rDNA is present on one mycobacterial genome, thei r sequences are assumed to be strictly or almost identical, We have is olated a slowly growing Mycobacterium strain, ''X'', identified by con ventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous res ults in two variable regions, suggesting the presence of different cop ies of the sequenced gene, Total DNA was digested by restriction enzym es and hybridized after Southern blotting to a probe representing abou t two-thirds of the 16S rDNA. Two copies of 16S rDNA mere identified a nd cloned, By sequencing, the clones were of two different types, A an d B, differing in 18 positions, Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two gen es observed in the Southern blot, We conclude that Mycobacterium strai n ''X'' has two different copies of 16S rDNA. Variations in the sequen ce between two copies of 16S rDNA gene have been described in archaeob acteria, but not in mycobacteria, When placed in a phylogenetic tree t ogether with other slowly growing mycobacteria, gene A shows a common root with M. terrae, whereas gene B is placed separately.