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Generation of deletions in pneumococcal mal genes cloned in Bacillus subtilis
Abstract:
The pneumococcal recombinant plasmid pLS70, which contains two strong promoters for transcription of the malM and malX genes, is unstable when transferred to Bacillus subtilis, and it gives rise to deleted derivatives. Analysis of proteins produced by the deleted plasmids and restriction mapping of 29 different deletions showed that stabilization in B. subtilis was accompanied by deletions affecting both promoters. Plasmids containing even a single strong promoter were at a selective disadvantage. Nucleotide sequences surrounding the deletions in 10 plasmids were determined. Six different deletions occurred between directly repeated sequences of 3-13 base pairs in length, presumably by a recombination mechanism involving short homologies. Four deletions occurred between sites not contained within repeated sequences. A weak but significant similarity of an 11-base sequence was found surrounding these deletions and the corresponding points of junction in the progenitor plasmids. It is suggested that this sequence may be the recognition site for a topoisomerase-like enzyme that can produce deletions.
Insights
The pneumococcal plasmid pLS70 shows instability in Bacillus subtilis, leading to deletions that affect its promoters. This suggests a mechanism involving DNA recombination and potentially a topoisomerase-like enzyme for plasmid stabilization.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Recombinant plasmids are crucial tools in molecular biology.
- Plasmid stability is essential for maintaining genetic constructs in host organisms.
- The pneumococcal plasmid pLS70 contains strong promoters for malM and malX genes.
Purpose of the Study:
- To investigate the instability of the pneumococcal recombinant plasmid pLS70 in Bacillus subtilis.
- To identify the mechanisms responsible for deletions in the plasmid.
- To understand the role of promoters and DNA sequences in plasmid stabilization.
Main Methods:
- Transfer of plasmid pLS70 to Bacillus subtilis.
- Analysis of proteins produced by deleted plasmids.
- Restriction mapping of 29 different plasmid deletions.
- Nucleotide sequencing of deletion junctions in 10 plasmids.
Main Results:
- Plasmid pLS70 exhibited instability in B. subtilis, resulting in deleted derivatives.
- Stabilization was associated with deletions affecting both strong promoters.
- Plasmids with a single strong promoter were at a selective disadvantage.
- Six deletions occurred within directly repeated sequences, suggesting homologous recombination.
- Four deletions occurred at non-repeated sites, associated with an 11-base sequence similarity.
Conclusions:
- Plasmid instability in B. subtilis is linked to deletions impacting strong promoters.
- Short direct repeats facilitate deletions via recombination.
- A conserved 11-base sequence may be a recognition site for a topoisomerase-like enzyme involved in deletion formation.
- Understanding these mechanisms is vital for stable plasmid-based genetic engineering in bacteria.