MOLECULAR ANALYSIS OF REPHI1B, A REPLICON SPECIFIC TO INCHI1 PLASMIDS

RepHI1B is one of the replicons that is specific to IncHI1 multireplic on plasmids. Its general organization resembles that of several replic ons that control their copy number by an iteron mechanism. The RepHI1B replicon (2.4 kb) contains: (i) an 882 bp repA gene coding for a 32 k Da replication protein (RepA), sharing significant similarity with the initiator proteins of other replicons belonging to various ii incompa tibility (Inc) groups, including P1 (IncY), Rts1 (IncT), RepFIB (IncFI ), and RepHI1A (IncHI1); (ii) two sets of 17 bp DNA repeats (iterons), one upstream and one downstream from reyA. By complementation testing , we identified the replication origin (oi i) of RepHI1B in a 223 bp l ocus upstream from repA. By primer extension we mapped two promoters o f repA (Prl and Pr2) in the ori sequence. We used repA::lacZ transcrip tional fusions to study regulation of the repA gene. This analysis sho wed that repA is transcriptionally autoregulated. Gel mobility shift a ssays demonstrated that RepA binds specifically to the origin and to i terons overlapping the Prl and Pr2 promoters. A G to A transition at n ucleotide position 13 of the iteron located in Pr2 (repeat 5) drastica lly decreases autoregulation of repA by inhibiting binding of RepA.