Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates

The genome of the Bacillus subtilis 168-type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnl-H-G and rrnJ-W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168-type strain and B. subtilis 168-like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168-type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.