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Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri
L Radnedge1, M A Davis, B Youngren
1Gene Regulation and Chromosome Biology Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
Abstract:
The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.
Insights
The Shigella flexneri virulence plasmid harbors a stability determinant (Stb) with novel genes. These genes, STBORF1 and STBORF2, resemble postsegregational killing systems and ensure plasmid stability.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The Shigella flexneri virulence plasmid pMYSH6000 possesses a stability determinant (Stb).
- Understanding plasmid maintenance mechanisms is crucial for bacterial pathogenesis research.
Purpose of the Study:
- To elucidate the genetic basis and mechanism of the Stb determinant on the pMYSH6000 plasmid.
- To investigate the role of novel open reading frames in plasmid stability and incompatibility.
Main Methods:
- Plasmid DNA sequencing and fragment analysis (SalI digestion).
- Functional analysis of gene homologs in Escherichia coli, including stability assays.
- Deletion analysis to identify essential regions for stability.
Main Results:
- A RepFIIA replicon and the Stb determinant were localized on adjacent SalI fragments.
- A 1.1-kb region homologous to the F plasmid's trbH gene conferred stability.
- Two overlapping open reading frames (STBORF1 and STBORF2) were identified as crucial for stability, showing homology to Salmonella vagC/D genes.
- The Stb system functions as a plasmid incompatibility determinant, potentially acting as an active partition or postsegregational killing system.
Conclusions:
- The Stb system on pMYSH6000 represents a novel plasmid stability mechanism.
- STBORF1 and STBORF2 are key components of the Stb system, likely functioning via postsegregational killing.
- The genetic organization of Stb shares similarities with other bacterial plasmid and chromosomal stability systems.