Construction of a Gordonia amarae shuttle vector using plasmid p1 replicon

Members of the genus Gordonia are Gram-positive mycolic acid producing Actinomycetota, with important bioremediation capabilities, including hydrocarbon and heavy metal degradation. However, their hydrophobic cell surface also contributes to stabilisation of foams, making Gordonia species especially G. amarae, major causes of foaming in wastewater treatment plants worldwide. Genetic studies of Gordonia have been hindered by the lack of suitable plasmid vectors. In this study, a native Gordonia pseudoamarae plasmid, was used to construct a shuttle vector capable of replicating in Gordonia and Escherichia coli. Thisvector was used for gene complementation inG. amaraeandtested successfully in additional Mycolata strains. However, because the native plasmid likely maintains a low copy number, the resulting shuttle vector exhibited instability. Incorporation of the native higAB plasmid maintenance system significantly improved plasmid stability producing plasmid pSPO3, which was successfully transformed, maintained, and functionally validated in G. amarae. This work expands the genetic toolkit available for Gordonia and the greater Mycolata and provides a strategy for developing stable vectors based on low copy number replication systems.