Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D-radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radi odurans SARK plasmid pUE10 was determined, in order to direct the developme nt of small, versatile cloning systems for Deinococcus, Annotation of the s equence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with repli cation proteins and site-specific resolvases, respectively, The products of both genes were demonstrated using an overexpression system in Escherichia coli, RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysi s using purified His-tagged RepU identified putative binding sites and sugg ested that RepU may be involved in both replication initiation and autoregu lation of repU expression. In addition, a gene encoding a possible antirest riction protein was found, which was shown to be required for high transfor mation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is simil ar to that for plasmids found in Thermus but not to that for the 45.7-kb pl asmid found in D. radiodurans strain R1. The minimal region required for au tonomous replication in D. radiodurans was determined by sequential deletio n of segments from the 12-kb fragment. The resulting minimal replicon, whic h consists of approximately 2.6 kb, was used for the construction of a shut tle vector for E. coli and D. radiodurans. This vector, pRAD1, is a conveni ent general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus p romoter was shown to efficiently express LacZ.