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ABSTRACT: Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.
Journal of Microbiological Methods 08/2002; 50(2):215-23. · 2.09 Impact Factor
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ABSTRACT: 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, rrnI-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 114 bp 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.
Molecular Microbiology 09/2001; 42(1):101 - 109. · 5.01 Impact Factor
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ABSTRACT: 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.
Journal of Microbiological Methods.
