Molecular analysis of RepHI1B, a replicon specific to IncHI1 plasmids

RepHI1B is one of the replicons that is specific to IncHI1 multireplicon plasmids. Its general organization resembles that of several replicons 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 kDa replication protein (RepA), sharing significant similarity with the initiator proteins of other replicons belonging to various incompatibility (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 repA. By complementation testing, we identified the replication origin (ori) of RepHI1B in a 223 bp locus upstream from repA. By primer extension we mapped two promoters of repA (Pr1 and Pr2) in the ori sequence. We used repA::lacZ transcriptional fusions to study regulation of the repA gene. This analysis showed that repA is transcriptionally autoregulated. Gel mobility shift assays demonstrated that RepA binds specifically to the origin and to iterons overlapping the Pr1 and Pr2 promoters. A G to A transition at nucleotide position 13 of the iteron located in Pr2 (repeat 5) drastically decreases autoregulation of repA by inhibiting binding of RepA.