Chloramphenicol acetyltransferase expression in marineRhodobacter sp. NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid

A vector, pUK318, was constructed to allow the expression of foreign genes in the marine photosynthetic bacteriumRhodobacter sp. NKPB 0021. This strain has been cured of its two endogenous plasmids pUK318 consists of a 2.3-kbPstI-BamHI restriction fragment, containing a marineRhodobacter plasmid replication region, cloned into pUC18. This fragment was derived from plasmid pRD31, a 3.1-kb endogenous plasmid purified from the marine strainRhodobacter sp. NKPB 043402. A kanamycin resistance gene from Tn903 was cloned into thePstI restriction site to provide antibiotic selection. pUK318 was transferred toRhodobacter sp. NKPB 0021 by transformation, and efficiencies of 7.2 × 10^-5 were obtained. Furthermore, pUK318 was stably maintained when transformants were grown either heterotrophically or photosynthetically in the absence of antibiotics. pUK318 was used to express theEscherichia coli chloramphenicol acetyl transferase (CAT) gene inRb. NKPB 0021. Transformants expressed a maximum CAT activity of 1.12 mmol/min/g dry cells. In addition, the DNA region essential for pUK318 replication inRb. NKPB 0021 was localized to a 1.36-kbHincII-PstI fragment. This is the first report of a plasmid vector containing a marineRhodobacter-specific replicon that allows stable expression of foreign genes in the absence of antibiotic selection.