Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata

The basic replicon of the narrow-host-range plasmid pHE1 from the moderatel y halophilic bacterium Halomonas elongata ATCC 33174 has been identified an d characterized. The replicon consists of a 1.7-kb DNA fragment, which cont ains the genetic information required for autonomous replication and stable maintenance. Analysis of its sequence revealed the presence of two ORFs wh ich seem to form one transcription unit. ORF1 encodes a replication initiat or protein (RepA), which has a high degree of homology to the theta-replica se (RepA) protein of ColE2 plasmid and to the RepA proteins of a family of replicons from gram-positive and gram-negative bacteria, also related to Co lE2. The product encoded by ORF2 showed a certain similarity to the RepB pr oteins of the same family of replicons and perhaps represents the pHE1 RepB function. Deletion analysis suggests that the pHE1 origin of replication ( ori) is located in an 800-bp region upstream of repA. A third putative gene , incA, was found on the complementary strand to the leader region of the r epA mRNA. This, together with the presence in the 5' untranslated region of the repA mRNA of inverted repeats that could form stable stem-loop structu res, suggests that the incA gene encodes a small antisense RNA. A possible control mechanism for pHE1 replication is proposed, involving an RNA molecu le which sequesters the translational initiation region of the replication protein RepA. The basic replicon characterized here shows very interesting properties that should allow it to be used in the construction of cloning a nd expression vectors for moderate halophiles.