Endogenous mutagenesis in recombinant sulfolobus plasmids.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Extreme thermoacidophiles (Sulfolobus spp.) typically exhibit high genome integrity despite harsh environments (high temperature, low pH).
• A discrepancy was observed between chromosomal stability and high mutation rates in a specific gene (lacS) on a shuttle plasmid (pJlacS) in Sulfolobus acidocaldarius.

Purpose of the Study:

• To investigate the unusually frequent mutations in the beta-D-glycosidase gene (lacS) on a shuttle plasmid (pJlacS) in Sulfolobus acidocaldarius.
• To quantify the mutation rate and characterize the types of mutations occurring in the plasmid-borne lacS gene.
• To explore the factors influencing lacS mutation frequency, particularly gene expression levels.

Main Methods:

• Development of a mutant accumulation assay adapted for the Sulfolobus system.
• Implementation of corrections for plasmid copy number and differential growth rates of mutants.
• Analysis of mutation types (transitions, slipped-strand events, deletions) through sequencing of independent lacS mutants.
• Comparison of mutation rates under varying lacS expression levels (high vs. low) and in chromosomal vs. plasmid contexts.

Main Results:

• A corrected mutation rate of 5.1 × 10⁻⁴ events per replication at the lacS gene was determined for the shuttle plasmid.
• Identified mutation types include G·C-to-A·T transitions, slipped-strand events, and deletions.
• High expression of the plasmid-borne lacS gene resulted in a 175-fold increase in spontaneous mutation rate compared to low-expression conditions.
• Nearly all substitution mutations created nonsense codons, and were concentrated at specific DNA primase priming sites.

Conclusions:

• High expression of the lacS gene on the pJlacS shuttle plasmid leads to significantly elevated mutation rates in Sulfolobus acidocaldarius.
• The findings suggest that DNA repair or replication fidelity mechanisms are overwhelmed or impaired under high-expression conditions on the plasmid.
• These results are analogous to transcription-associated mutagenesis observed in other organisms, highlighting potential conserved mechanisms of DNA damage and repair under specific cellular conditions.