PLASMID 34, 157–164 (1995) Construction of Novel Shuttle Vectors for Use between Moderately Halophilic Bacteria and Escherichia coli ENCARNACIO ´ N MELLADO,JOAQUIN J. NIETO, AND ANTONIO VENTOSA 1 Faculty of Pharmacy, Department of Microbiology and Parasitology, University of Sevilla, 41012 Seville, Spain Received April 21, 1995; revised June 25, 1995 Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed. These vectors are based on the minimal replicon of pCM1, a cryptic plasmid from Chromohalobacter marismortui, combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites, lacZ fragment), as well as with the trimethoprim resistance gene as a selection marker for moderate halophiles. These vectors can be efficiently transferred by RP4-mediated conjugation from Escherichia coli to the moderate halophiles Chromohalobacter marismortui, Deleya halophila, Halomonas elongata, Halomonas subglaciescola, and Volcaniella eurihalina.  1995 Academic Press, Inc. Besides the extremely halophilic aerobic The genetic manipulation of this group of extremophiles has been classically hampered Archaea (halobacteria), moderately halophilic bacteria are the most abundant prokaryotes by the lack of studies on genetic transfer mechanisms existing among them and other that inhabit hypersaline environments (Rodrı ´- guez-Valera, 1988). Moderate halophiles are nonhalophilic bacteria such as Escherichia coli. So far, transformation, electroporation, defined as those microorganisms that grow op- timally in media containing 3 to 15% NaCl or transduction procedures have not been re- ported for these halophiles. A few autochtho- (Kushner and Kamekura, 1988). In contrast with halobacteria, moderately halophilic bac- nous plasmids from moderately halophilic bacteria have been described (Ferna ´ndez-Cas- teria have been less attractive for research al- though in recent years they are considered tillo et al., 1992; Ventosa et al., 1994; Vargas of great interest, especially due to their bio- et al., 1995). The cryptic plasmid pHE1, iso- technological potential (Galinski and Tin- lated from Halomonas elongata, was used for dall, 1992; Ventosa and Nieto, 1995). They the construction of a shuttle vector, which was produce enzymes (amylases, proteases, mobilized by a RK2 derivative of several Ha- nucleases) of industrial interest (Kamekura, lomonas strains. However, this vector is only 1986; Ventosa and Nieto, 1995) and accumu- useful for cloning purposes in several species late a variety of organic compounds, known of the genus Halomonas, and on the other as compatible solutes, that could be used as hand, it lacks several features generally found enzymes or cells stabilizing agents (Galinski in other cloning vectors. and Tindall, 1992; Galinski, 1993). Since the Our recent studies on plasmid pCM1 from commercial production of compounds from Chromohalobacter marismortui have permit- halophiles would be limited by the problems ted the location and molecular characteriza- associated with the high-salt concentrations tion of the minimal replicon region of this required for growing them, genetic studies that plasmid (Mellado et al., 1995). To facilitate would permit the manipulation of halophiles the genetic engineering of moderate halophilic and the exploitation of their properties are bacteria, we have combined the oriV fragment strictly necessary. of pCM1 and the trimethoprim resistance gene, which we have found is a good genetic 1 To whom correspondence should be addressed. marker for these bacteria, with the cloning 157 0147-619X/95 $12.00 Copyright  1995 by Academic Press, Inc. All rights of reproduction in any form reserved.