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The broad-host-range plasmid pTF-FC2 requires a primase-like protein for autonomous replication in Escherichia coli
R A Dorrington1, S Bardien, D E Rawlings
1Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
Abstract:
A 3202-bp fragment of plasmid pTF-FC2, cloned into PUC19, had previously been identified as the minimum region required for replication in either Pseudomonas aeruginosa or Escherichia coli polA- mutants. During the course of experiments to construct broad-host-range cloning vectors based on the pTF-FC2 replicon, it was found that the 3202-bp fragment had an absolute requirement for some function of the pUC19 vector. This requirement was eliminated in the presence of co-resident pTF-FC2 derivatives. An additional 1239-bp fragment from pTF-FC2, immediately adjacent to the 3202-bp fragment, was identified which restored the ability of the pTF-FC2 replicon to replicate autonomously. Sequence analysis of the region revealed a single open reading frame encoding a 40-kDa polypeptide, which was synthesised in an in vitro transcription/translation system. A comparison of the amino acid sequence of this protein with sequence data banks revealed limited homology with the RepB' primase of the IncQ plasmid, RSF1010. An M13 delta lac 110 replication-deficient phage system was used to demonstrate that the 40-kDa protein did function as a primase with respect to replication at the origin of replication (vegetative) of pTF-FC2.
Insights
A new 1239-bp fragment from plasmid pTF-FC2 enables autonomous replication. This fragment encodes a 40-kDa protein functioning as a primase, crucial for plasmid replication in various bacteria.
Area of Science:
- Molecular Biology
- Microbiology
- Plasmid Biology
Background:
- The minimum replication region of plasmid pTF-FC2 (3202-bp) cloned into pUC19 requires pUC19 vector functions for replication in Pseudomonas aeruginosa and Escherichia coli polA- mutants.
- This dependency suggests a need for additional factors or functions not inherent to the minimal replicon fragment alone.
Purpose of the Study:
- To identify the genetic element responsible for the autonomous replication of plasmid pTF-FC2.
- To characterize the function of the identified replication element and its encoded protein.
Main Methods:
- Plasmid construction and replication assays in bacterial hosts (Pseudomonas aeruginosa, Escherichia coli).
- DNA sequencing and in vitro transcription/translation to analyze the identified fragment.
- Homology searches against sequence databases.
- Functional characterization using an M13 replication-deficient phage system.
Main Results:
- An additional 1239-bp fragment from pTF-FC2 restored autonomous replication.
- Sequence analysis revealed a single open reading frame encoding a 40-kDa polypeptide.
- The 40-kDa protein exhibited limited homology to the RepB' primase of IncQ plasmid RSF1010.
- The 40-kDa protein was confirmed to function as a primase for pTF-FC2 replication.
Conclusions:
- The 1239-bp fragment contains the necessary genetic information for autonomous replication of pTF-FC2.
- The 40-kDa protein encoded by this fragment acts as a primase, essential for initiating replication at the vegetative origin of pTF-FC2.
- This finding contributes to understanding plasmid replication mechanisms and the development of novel cloning vectors.