ISOLATION OF CRYPTIC PLASMIDS FROM MODERATELY HALOPHILIC EUBACTERIA OF THE GENUS HALOMONAS - CHARACTERIZATION OF A SMALL PLASMID FROM H-ELONGATA AND ITS USE FOR SHUTTLE VECTOR CONSTRUCTION

Three cryptic plasmids have been isolated from moderately halophilic eubacteria belonging to three species of the genus Halomonas. These three plasmids were designated pHE1 (4.2 kb, isolated from H. elongata ATCC 33174), pHI1 (48 kb, isolated from ''H. israelensis'' ATCC 43985), and pHS1 (ca. 70 kb, isolated from H. subglaciescola UQM 2927). Because of its small size, the plasmid pHE1 was selected for further characterization and construction of a shuttle vector for Halomonas strains. pHE1 was cloned into pBluescript KS and a detailed restriction map was constructed. Hybridization experiments excluded the existence of sequences homologous to pHE1 in total DNA from other strains of the genus Halomonas. Moreover, no DNA homology with pMH1, the only plasmid described so far from moderate halophiles, was found. Since pHE1 appeared to be unable to replicate in Escherichia coli cells, a number of mobilizable pHE1-derived hybrid plasmids were constructed that could be selected and maintained both in E. coli and in H. elongata. Finally, an improved shuttle vector, pHS15, was generated. The vector pHS15 contains an origin of replication from E. coli as well as one from H. elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2. The vector pHS15 was readily mobilized by the RK2 derivative pRK2013 to all Halomonas strains tested so far. This is the first report on the development of a cloning vector useful for moderately halophilic eubacteria.