Two Related Rolling Circle Replication Plasmids from Salt-Tolerant Bacteria

Abstract

In a range of salt-tolerant bacteria isolated from soil, rhizosphere, and phyloplane, cross-hybridization tests revealed a group of seven plasmids which were chosen for further study. Two plasmids (pSH1418 and pSH1451) were picked as representative examples of the two related subgroups. 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-copy-number vectors but this was possible with low-copy-number IncP vectors. InEscherichia coli,cloned plasmids pSH1418 and pSH1451 conferred salt tolerance but the phenotype was unstable, with the loss of salt tolerance apparently being correlated with structural instability of the plasmid DNA. Plasmid pSH1451 was sequenced and shown to be closely related to RCR plasmids of Gram-positive bacteria. The host of this plasmid was classified asBacillus pumilusby rDNA typing and lipid profiling by gas chromatography. A number of open reading frames (orfs) which could code for salt tolerance or other functions were identified in the plasmid sequence. Sequence similarity to previously sequenced genes suggested that the products of orf4 and orf5 may work together to transport a molecule such as aspartate ion that may promote osmotolerance.