Utility of 16S-23S rRNA spacer region methodology: how similar are interspace regions within a genome and between strains for closely related organisms?

Use of 16S-23S interspace region sequence variability, as a relatively new method, is becoming an important supplement to 16S rRNA sequencing as the standard for differentiating bacterial species. If interspace regions are as variable within a genome as between strains for closely related organisms, this limits the utility of the technique. Strains W23 and 168 represent two distinct genetic clusters within the species Bacillus subtilis. B. atrophaeus var. niger was selected as a member of a group of species closely related to B. subtilis. Comparison of the 10 rDNA operons, available from Genbank, for B. subtilis 168 shows three distinct types of interspace (ISR) regions. Two of the ten 16S-23S ISRs contain the sequences for isoleucine and alanine tRNA and are identical in sequence. The remaining eight ISRs lack tRNA sequences and have two distinct sizes. Variability among non-tRNA operons ranged from 97-100%. Counting the tRNA insert as one change, variability between tRNA and non-tRNA containing sequences ranged from 95.3-97%. The sequences of equivalent 16S-23S ribosomal operon interspace regions (ISRs) are highly conserved between W23 and 168 (99.9-100%). Thus the sequence differences between strains 168 and W23 are less than between multiple operons within 168. However, the sequence of an ISR from B. atrophaeus var, niger is quite distinct from any of the ISRs found in B. subtilis (range 88.2-91.6%). These observations are consistent with the previous suggestion that B. atrophaeus is distinct genetically from the B. subtilis sub-groups represented by W23 and 168 respectively. This is the first study to make sequence comparisons at the genome, strain and species level for the rRNA interspace region. Considerations of this issue will be important in using ISR methodology to differentiate other closely related bacterial species. (C) 1998 Elsevier Science B.V. All rights reserved.