Construction of a high-efficiency shuttle vector for Histophilus somni
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Published on: May 6, 2015
Indra Sandal1, Mohamed N Seleem, Shaadi F Elswaifi
1Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, United States.
Abstract:
The genetic manipulation of Histophilus somni is limited due to its high-fidelity restriction-modification system. The broad host-range shuttle plasmid pLS88 is capable of transforming some strains of H. somni, but is an inefficient vector. We have constructed an improved version of pLS88, pNS3K, that transforms H. somni strain 2336 100-fold more efficiently than its predecessor. The transformation efficiency was further increased when pNS3K was isolated from H. somni and retransformed into the same strain. As proof of principle, the lipooligosaccharide biosynthesis gene lob-2A was cloned into pNS3K and expressed in H. somni strain 129Pt, which lacks this gene. Thus, pNS3K is a useful shuttle vector for H. somni and a potential vector for genetic manipulation of this bacterium.
Insights
Researchers developed a new plasmid, pNS3K, to improve genetic manipulation of Histophilus somni. This enhanced vector transforms bacteria 100-fold more efficiently, aiding in understanding and modifying this important pathogen.
Area of Science:
- Bacteriology
- Molecular Biology
- Genetics
Background:
- Histophilus somni presents challenges for genetic manipulation due to its robust restriction-modification system.
- Existing vectors, like the broad host-range shuttle plasmid pLS88, exhibit limited transformation efficiency in H. somni.
Purpose of the Study:
- To engineer an improved shuttle vector for enhanced genetic manipulation of Histophilus somni.
- To increase the transformation efficiency of H. somni compared to previously available vectors.
Main Methods:
- Construction of an improved pLS88 derivative, designated pNS3K.
- Transformation efficiency assays of pNS3K in H. somni strain 2336.
- Cloning and expression of the lipooligosaccharide biosynthesis gene lob-2A using pNS3K.
Main Results:
- The novel pNS3K vector demonstrated a 100-fold increase in transformation efficiency in H. somni strain 2336 compared to pLS88.
- Further enhancement of transformation efficiency was observed when pNS3K was isolated from H. somni and retransformed.
- Successful cloning and expression of the lob-2A gene in H. somni strain 129Pt, demonstrating vector functionality.
Conclusions:
- The pNS3K shuttle vector significantly enhances the genetic manipulation capabilities of Histophilus somni.
- pNS3K represents a valuable tool for future research involving genetic modification of H. somni, including functional gene studies.
