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Determination of the single strand origin of Shigella sonnei plasmid pKYM
1Molecular Biology Group, Faculty of Engineering, Toyama University, Japan.
Abstract:
The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechanism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cloned the fragment into a -ori defective M13 vector phage, M13 delta lac183. The chimera phage, MDKY5, showed a larger plaque size, and increased phage yield and rate of progeny replicative form DNA (RF) synthesis. Rifampicin reduced rate of conversion of the single- to double-stranded RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hairpin loops and formed smaller plaques. The in vivo analyses revealed that a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single strand origin, referred to as SSO) and harbors a recognition site(s) by host E. coli RNA polymerase, for primer RNA synthesis. Moreover, we found a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RSA) which functions in plasmid sitespecific recombination and/or transfer.
Insights
The Shigella sonnei plasmid pKYM origin of replication was identified. This single-strand origin (SSO) contains hairpin loops essential for complementary strand synthesis and primer RNA formation by E. coli RNA polymerase.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The Shigella sonnei plasmid pKYM utilizes a rolling-circle replication mechanism.
- Understanding plasmid replication origins is crucial for genetic manipulation and understanding bacterial conjugation.
Purpose of the Study:
- To identify and characterize the origin of replication for the Shigella sonnei plasmid pKYM.
- To elucidate the functional role of specific DNA sequences and structures within the pKYM replication origin.
Main Methods:
- Cloning of a pKYM DNA fragment into an M13 vector to create a chimera phage.
- Site-directed mutagenesis using Bal31 nuclease to create deletion mutants.
- Analysis of phage plaque formation, DNA synthesis rates, and effect of rifampicin.
- In vivo characterization of the single-strand origin (SSO) and its interaction with E. coli RNA polymerase.
Main Results:
- A chimera phage (MDKY5) exhibited enhanced replication characteristics compared to the wild-type vector.
- Deletion mutants lacking hairpin loops resulted in smaller plaques, indicating impaired replication.
- A 136-nucleotide sequence containing two hairpin loops was identified as the pKYM single-strand origin (SSO).
- The SSO contains recognition sites for E. coli RNA polymerase, essential for primer RNA synthesis.
- A sequence upstream of the SSO showed homology to recombination site A (RSA), suggesting a role in recombination or transfer.
Conclusions:
- The identified 136-nucleotide sequence is the functional single-strand origin (SSO) of the pKYM plasmid.
- The SSO's hairpin structures and interaction with E. coli RNA polymerase are critical for initiating complementary strand synthesis.
- The upstream sequence may play a role in plasmid site-specific recombination or transfer.