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Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Published on: May 15, 2018

Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid.

Garima Jain¹, Helga Ertesvåg²

¹Department of Biotechnology and Food Science, NTNU - Norwegian University of Science and Technology, 7491, Trondheim, NO, Norway.

Applied Microbiology and Biotechnology

|October 4, 2022

Summary

This summary is machine-generated.

Researchers developed a new plasmid vector to improve genetic engineering in Rhodococcus opacus (R. opacus), a bacterium vital for bioremediation. This advancement also confirmed the essential role of mycolic acids for R. opacus survival.

Keywords:

Conjugative conditional suicide plasmid Homologous recombination Mycolic acid Rhodococcus opacus

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Area of Science:

Microbiology
Synthetic Biology
Biotechnology

Background:

Rhodococcus opacus PD630 is a versatile bacterium used in bioremediation, metal recovery, and triacylglycerol storage.
Genetic manipulation of R. opacus via homologous recombination is challenging due to low DNA transfer and recombination frequencies.

Purpose of the Study:

To develop an improved vector system for efficient homologous recombination in R. opacus.
To investigate the essentiality of mycolic acids for R. opacus viability using the developed vector.

Main Methods:

Construction and evaluation of a conjugative, conditional suicide plasmid based on the Corynebacterium glutamicum plasmid pGA1 replicon.
Utilized a dual inducible and repressible promoter system for replication control and sacB as a counterselection marker.
Employed the vector to attempt deletion of the accD3 gene, essential for mycolic acid synthesis.

Main Results:

The developed plasmid derivative enabled efficient homologous recombination in R. opacus, overcoming previous limitations.
Deletion of the accD3 gene was only possible in strains harboring a plasmid-encoded copy, indicating its essentiality.
Mycolic acid synthesis is crucial for the viability of R. opacus, similar to its role in Mycobacterium species.

Conclusions:

A novel conditional suicide plasmid vector significantly enhances homologous recombination in R. opacus.
Mycolic acids are essential for R. opacus survival, highlighting a conserved characteristic within certain bacterial genera.
The developed vector and approach are applicable for genetic engineering in Rhodococcus and related bacteria, and for plasmids requiring plasmid-encoded proteins for replication.