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

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• The Bacillus subtilis 168 genome possesses 10 ribosomal RNA (rRNA) operons.
• Five rRNA operons (rrnI-H-G and rrnJ-W) in the intergenic spacer region (ISR) between 16S and 23S rRNA genes lack a trinucleotide signature region.

Purpose of the Study:

• To investigate genetic variability within Bacillus subtilis 168 isolates.
• To characterize the presence or absence of a trinucleotide signature region in rRNA operons.
• To understand variations in rRNA operon number and chromosomal organization.

Main Methods:

• Polymerase chain reaction (PCR) to amplify ISR segments.
• Electrospray mass spectrometry (MS) for precise molecular weight determination of PCR products.
• Restriction fragment length polymorphism (RFLP) analysis of rRNA operons.
• Southern blot analysis.
• DNA sequencing of specific rRNA operons.

Main Results:

• Mass spectrometry and PCR revealed variations in PCR product size (114 bp vs. 111 bp) due to the presence or absence of the trinucleotide insert.
• Five of seven B. subtilis 168 isolates exclusively generated 114 bp PCR products, indicating the presence of the trinucleotide signature in all rRNA operons.
• RFLP and Southern blot analysis identified three distinct patterns among the isolates, confirming genetic variability beyond operon deletion.
• Restriction analysis and sequencing confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates.

Conclusions:

• Significant genetic variability exists within Bacillus subtilis 168 subgroup isolates concerning rRNA operons.
• The trinucleotide insert's presence or absence influences PCR product size and suggests potential roles in rRNA maturation and protein synthesis.