2 RELATED ROLLING CIRCLE REPLICATION PLASMIDS FROM SALT-TOLERANT BACTERIA

In a range of salt-tolerant bacteria isolated from soil, rhizosphere, and phyloplane, cross-hybridization tests revealed a group of seven pl asmids which were chosen for further study. Two plasmids (pSH1418 and pSH1451) were picked as representative examples of the two related sub groups. Restriction mapping and Southern blotting identified a region common to the two plasmids which later was identified as encoding the replication functions. We were unable to join these plasmids to high-c opy-number vectors but this was possible with low-copy-number IncP vec tors. In Escherichia coli, cloned plasmids pSH1418 and pSH1451 conferr ed salt tolerance but the phenotype was unstable, with the loss of sal t tolerance apparently being correlated with structural instability of the plasmid DNA. Plasmid pSH1451 was sequenced and shown to be closel y related to RCR plasmids of Gram-positive bacteria The host of this p lasmid was classified as Bacillus pumilus by rDNA typing and lipid pro filing by gas chromatography. A number of open reading frames (orfs) w hich could code for salt tolerance or other functions were identified in the plasmid sequence. Sequence similarity to previously sequenced g enes suggested that the products of orf4 and orf5 may work together to transport a molecule such as aspartate ion that may promote osmotoler ance. (C) 1996 Academic Press.