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Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1
K J Leenhouts1, B Tolner, S Bron
1Department of Genetics, University of Groningen, Haren, The Netherlands.
Plasmid
|July 1, 1991
Summary
The broad-host-range plasmid pWVO1 from Lactococcus lactis uses rolling-circle replication. Its replication proteins show similarity to streptococcal plasmids, with distinct host RNA polymerase involvement in DNA conversion.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The Lactococcus lactis plasmid pWVO1 is a broad-host-range replicon capable of replicating in both Gram-positive and Gram-negative bacteria.
- Understanding the replication mechanism of such plasmids is crucial for genetic engineering and biotechnology applications.
Purpose of the Study:
- To determine the nucleotide sequence of the pWVO1 plasmid.
- To identify genes and regulatory elements involved in its replication.
- To elucidate the replication mechanism and host interactions of pWVO1.
Main Methods:
- Nucleotide sequencing of the pWVO1 plasmid.
- Bioinformatic analysis to identify open reading frames (ORFs) and predict protein functions.
- Comparative sequence analysis with known plasmids (e.g., pLS1).
- Experimental analysis of single-stranded (ss) and double-stranded (ds) DNA production in different bacterial hosts (Bacillus subtilis and L. lactis) with and without rifampicin treatment.
Main Results:
- The complete nucleotide sequence of pWVO1 was determined, revealing four ORFs.
- ORF A encodes a replication protein (RepA) essential for replication, showing similarity to Rep proteins of streptococcal plasmid pLS1.
- The plus origin of replication was identified, also similar to pLS1.
- pWVO1 derivatives produced ssDNA in B. subtilis and L. lactis, indicating rolling-circle replication.
- Rifampicin treatment increased ssDNA levels in B. subtilis, suggesting host RNA polymerase involvement in ssDNA to dsDNA conversion, a process not requiring host RNA polymerase in L. lactis.
Conclusions:
- The pWVO1 plasmid utilizes a rolling-circle replication mechanism.
- Replication proteins (RepA) and origins share homology with streptococcal plasmids, suggesting a conserved replication strategy.
- The conversion of single-stranded to double-stranded DNA during replication involves host RNA polymerase in B. subtilis but not in L. lactis, highlighting host-specific differences in plasmid replication.