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Published on: August 25, 2020
Characterization of pMa025, a plasmid from the cyanobacterium Microcystis aeruginosa UV025
Margaret M Wallace1, David W Miller, Shirley Raps
1Department of Biological Sciences, Hunter College, CUNY, New York, NY 10021, USA.
Abstract:
The characterization of pMa025, a plasmid isolated from the unicellular, toxin-producing cyanobacterium Microcystis aeruginosa UV025, is described. A recombinant plasmid, pMaL [pMa025-pBluescript II SK(-)] was constructed for mapping, sequencing, and development of shuttle vectors capable of transforming both Escherichia coli and M. aeruginosa. pMa025 is 8,018 bp in length and has a G+C content of 62.3 mol%. Nineteen presumptive ORFs, ORF A - ORF S were identified using ATG or GTG as initiation codons. Fifteen different ORFs, ORF a - ORF o were identified using TGA as a degenerate codon for tryptophan. GTG was the start codon in two-thirds of the putative ORFs when TGA was the termination codon. GTG was the start codon in one-third of the putative ORFs when TGA was used as a codon for tryptophan. The deduced amino acid sequence from ORF j (3,114 bp) was significantly similar to that of a putative plasmid replication protein, RepA, from plasmid pUH24 of Synecho coccus sp. strain PCC7942. M. aeruginosa UV027 and E. coli were transformed to carbenicillin resistance with pMaL-D7, a 6.4-kb hybrid plasmid (3.46 kb pMa025, 2.95 kb pBluescript II) generated from the nested deletion strategy. pMaL-D7 will be used as a shuttle vector.
Insights
This study characterizes plasmid pMa025 from Microcystis aeruginosa, identifying 19 open reading frames (ORFs) and a replication protein. A shuttle vector, pMaL-D7, was developed for transforming E. coli and M. aeruginosa.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Microcystis aeruginosa is a unicellular, toxin-producing cyanobacterium.
- Plasmids play crucial roles in bacterial adaptation and virulence.
- Characterizing plasmids from cyanobacteria can reveal novel genetic elements and functions.
Purpose of the Study:
- To characterize the plasmid pMa025 from Microcystis aeruginosa UV025.
- To construct a recombinant plasmid for genetic manipulation and shuttle vector development.
- To enable transformation of both Escherichia coli and M. aeruginosa.
Main Methods:
- Plasmid isolation and characterization (size, G+C content).
- Open reading frame (ORF) identification using sequence analysis.
- Construction of recombinant plasmids (pMaL) and nested deletions (pMaL-D7).
- Transformation assays in E. coli and M. aeruginosa.
Main Results:
- Plasmid pMa025 is 8,018 bp with 62.3 mol% G+C content.
- Nineteen presumptive ORFs (A-S) and fifteen different ORFs (a-o) were identified.
- ORF j showed similarity to a plasmid replication protein (RepA).
- A shuttle vector, pMaL-D7, was successfully constructed and used for transformation.
Conclusions:
- Plasmid pMa025 harbors genes potentially involved in replication and other functions.
- The developed shuttle vector pMaL-D7 is functional for both E. coli and M. aeruginosa.
- This work facilitates further genetic studies of M. aeruginosa.
