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Published on: December 8, 2023
Plasmid pWW115, a cloning vector for use with Moraxella catarrhalis
1Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9048, USA.
Abstract:
The plasmid shuttle vector pWW102B is able to replicate in only a modest number of Moraxella catarrhalis strains. Plasmid pWW115, a spontaneous deletion mutant of pWW102B, was shown to lack both the pACYC184-derived origin of replication and the associated chloramphenicol-resistance gene but was able to replicate in every M. catarrhalis strain tested in this study, including one strain that had been previously refractory to all types of genetic manipulations. To test the utility of this plasmid, a M. catarrhalis gene encoding the UspA2 serum-resistance factor was cloned into pWW115 and the resultant recombinant plasmid was shown to confer serum-resistance on a serum-sensitive M. catarrhalis uspA2 mutant.
Insights
A new plasmid, pWW115, replicates in all tested Moraxella catarrhalis strains, overcoming limitations of previous vectors. This enables successful cloning and expression of M. catarrhalis genes, like the UspA2 serum-resistance factor.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The plasmid shuttle vector pWW102B exhibits limited replication in Moraxella catarrhalis strains.
- Genetic manipulation of M. catarrhalis has been challenging due to vector limitations.
Purpose of the Study:
- To develop a novel plasmid vector for efficient replication in diverse Moraxella catarrhalis strains.
- To demonstrate the utility of the new vector for gene cloning and functional studies in M. catarrhalis.
Main Methods:
- Construction of pWW115 as a deletion mutant of pWW102B, removing the pACYC184 origin and chloramphenicol-resistance gene.
- Testing replication efficiency of pWW115 across various M. catarrhalis strains.
- Cloning the M. catarrhalis UspA2 gene into pWW115 and assessing its function in a mutant strain.
Main Results:
- Plasmid pWW115 demonstrated replication in all tested M. catarrhalis strains, including previously refractory ones.
- The UspA2 serum-resistance factor gene was successfully cloned into pWW115.
- The recombinant plasmid conferred serum resistance to a serum-sensitive M. catarrhalis uspA2 mutant.
Conclusions:
- The novel plasmid pWW115 is a versatile tool for genetic studies in Moraxella catarrhalis.
- pWW115 facilitates the functional analysis of M. catarrhalis genes, such as UspA2.
- This advancement improves genetic manipulation capabilities for this important bacterial pathogen.
