PLASMID 24,90-99 ( 1990) Chloramphenicol Acetyltransferase Expression in Marine Rhodobacter sp. NKPB 0021 by Use of Shuttle Vectors Containing the Minimal Replicon of an Endogenous Plasmid TADASHI MATSUNAGA,’ KAZUFUMI TSUBAKI, HIDEAKI MIYASHITA, AND J. GRANT BURGESS Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan Received April 19, 1990; revised August 6, 1990 A vector, pUK3 18, was constructed to allow the expression of foreign genes in the marine photosynthetic bacterium Rhodobacter sp. NKPB 002 1. This strain has been cured of its two endogenous plasmids. pUK3 I8 consists of a 2.3-kb P.vtI-BamHI restriction fragment, contain- ing a marine Rhodobactar plasmid replication region, cloned into pUCl8. This fragment was derived from plasmid pRD3 1, a 3.1-kb endogenous plasmid purified from the marine strain Rhodobacter sp. NKPB 043402. A kanamycin resistance gene from Tn903 was cloned into the Pstl restriction site to provide antibiotic selection. pUK3 18 was transferred to Rhodobacter sp. NKPB 002 I by transformation, and efficiencies of 7.2 X 10m5 were obtained. Furthermore, pUK3 I8 was stably maintained when transformants were grown either heterotrophically or photosynthetically in the absence of antibiotics. pUK3 18 was used to express the Escherichia coli chloramphenicol acetyl transferase (CAT) gene in Rb. NKPB 0021. Transformants ex- pressed a maximum CAT activity of 1.12 mmol/min/g dry cells. In addition, the DNA region essential for pUK318 replication in Rb. NKPB 002 1 was localized to a 1.36-kb HincII-PstI fragment. This is the first report of a plasmid vector containing a marine Rhodobacter-specific replicon that allows stable expression of foreign genes in the absence of antibiotic selection. 0 1990 Academic Prrsq. Inc Despite many reports of efficient gene transfer systems for freshwater photosynthe- tic bacteria, in particular, Rhodobacter sphaeroides and Rb. capsulatus (for a review see Scolnik and Marrs, 1987), no such sys- tems have been reported for marine photo- synthetic bacteria despite their biotechno- logical and environmental importance (Ma- tsunaga and Mitsui, 1982; Miura et al., 1989; Tolbert, 1989). The development of a useful gene cloning system for marine strains of purple photosynthetic bacteria is therefore an essential prerequisite to their biotechnologi- cal utilization and to environmental studies of important processes such as CO, fixation in the oceans at the molecular level. Plasmid vectors currently in use for gene transfer in freshwater phototrophic bacteria are derivatives of broad host range vectors I To whom correspondence should be addressed 0147-619X/90 $3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction I” any form reserved. such as RSFlO 10 (Guerry et al., 1974) and RK2 (Meyer et al., 1977). In addition, deriva- tives of pBR322 are also used for Rb. capsu- latus (Pollock et al., 1988) and Rhodopseudo- monas viridis (Lang and Oesterhelt, 1989). However, until now, there have been no re- ports of the use of expression vectors that contain Rhodobacter-specific replicons. A vector constructed from an endogenous plas- mid of a marine Rhodobacter strain (Matsu- naga et al., 1986) was found to be unstable due to deletion of the Rhodohacter-specific fragment from the hybrid plasmid (Matsu- naga et al., unpublished observations). Barb& et al. ( 1986) have also reported the construc- tion of a chimeric plasmid bearing a 5.6-kb replication region isolated from an endoge- nous plasmid of Rb. sphaeroides; however, this work has not been continued. Johnson et al. ( 1986) reported the expression of cellulase genes in Rb. capsulatus using expression vec- 90