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Nucleotide sequence and transcriptional analysis of a third function (Flm) involved in F-plasmid maintenance
S M Loh1, D S Cram, R A Skurray
1Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Abstract:
The leading region of the conjugative F plasmid encodes for a function, Flm, capable of extending the maintenance of normally unstable plasmids. Nucleotide sequencing and functional studies of flm locus have shown that it consists of at least two genes, flmA and flmB, which are physically and functionally homologous to hok and sok of parB in plasmid R1. The 52-amino acid flmA-coded polypeptide is almost identical to the hok product which has been shown to be a membrane-associated lethal protein [Gerdes et al., EMBO J. 5 (1986) 2023-2029]. Gene flmB codes for a 100 nucleotide, non-translated, complementary RNA which overlaps the 5' leader sequence of the flmA RNA. The flmA RNA also encodes an open reading frame (ORF70) which overlaps the flmA-coding sequence and may be a third gene involved in the Flm function. S1 analysis and functional studies suggest that the antisense flmB RNA binds to the flmA RNA and suppresses the expression of the lethal product, presumably by blocking coupled translation of ORF70 and flmA. Secondary structure analysis predicts that the flmA RNA is extremely stable compared to the regulatory flmB RNA. We suggest that when these RNA species are retained by cells which have lost the F plasmid, the more stable flmA RNA will eventually be translated thus leading to cell death. This phenomenon provides a third mechanism, additional to ParFIA and Ccd functions, to ensure maintenance of the F plasmid in a growing bacterial population.
Insights
The F plasmid uses a novel Flm system, involving lethal protein production from the stable flmA RNA, to ensure its maintenance in bacterial populations. This system acts as a safeguard against plasmid loss.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The F plasmid, a conjugative plasmid, possesses a function termed Flm (Factor for plasmid maintenance) that enhances the stability of plasmids.
- The flm locus shares homology with the hok/sok system of plasmid R1, suggesting a conserved mechanism for plasmid stability.
Purpose of the Study:
- To elucidate the genetic organization and functional mechanism of the Flm system in F plasmid maintenance.
- To investigate the roles of flmA, flmB, and ORF70 in plasmid stability and cell death.
Main Methods:
- Nucleotide sequencing and functional studies of the flm locus.
- S1 analysis to study RNA interactions.
- Secondary structure analysis of RNA molecules.
- Comparative analysis with known plasmid stabilization systems (ParFIA, Ccd).
Main Results:
- The flm locus comprises at least two genes, flmA and flmB, homologous to hok and sok.
- flmA encodes a 52-amino acid lethal protein, similar to Hok.
- flmB encodes a 100-nucleotide antisense RNA that regulates flmA expression.
- A potential third gene, ORF70, overlaps the flmA coding sequence.
- Antisense flmB RNA binding to flmA RNA suppresses lethal protein production by inhibiting coupled translation.
- flmA RNA is significantly more stable than flmB RNA.
Conclusions:
- The Flm system utilizes a post-segregational killing mechanism involving stable flmA RNA and regulatory flmB RNA.
- Upon F plasmid loss, the stable flmA RNA is translated, leading to cell death and ensuring plasmid inheritance.
- This represents a novel, third mechanism for F plasmid maintenance, complementing ParFIA and Ccd functions.