Use of cobA and cysGA as Red Fluorescent Indicators

Abstract

This chapter is based on the observations (1–3) that Escherichia coli cells bearing the plasmid pISA417, for the overexpression of the cobA gene from the bacterium Propionibacterium freudenreichii, or the plasmid pEB1, for the overexpression of a truncated cysG (cysGA) gene of E. coli, exhibit bright red fluorescence Fig. 1) when cultured on Luria-Bertani (LB) growth medium and illuminated with ultraviolet (UV) light. The genes both encode uroporphyrinogen III (urogen III) methyltransferases (referred herein to as CobA or CysG^A) which catalyze the methylation of urogen III, an intermediate in heme biosynthesis, using S-adenosyl-L-methionine as the methyl donor. Plasmid pISA417 was constructed by insertion of a DNA fragment bearing the complete cobA gene into pUC19 Fig. 2) and was originally used for the characterization of urogen III methyltransferase (1). During this study, it was noticed that E. coli colonies harboring pISA417 are brightly red fluorescent when illuminated with UV light. However, E. coli cells harboring pISA417 bearing a DNA insert that deletes or knocks out the expression of cobA are not fluorescent, thus providing the basis for its first use as a fluorescent indicator in selecting recombinant plasmids (2).