Organization of the leading region of IncN plasmid pKM101 (R46): a regulation controlled by CUP sequence elements

Analysis of the nucleotide sequence of the 13.8 kb leading region of the IncN plasmid pKM101 (a deletion derivative of R46) revealed eight copies of highly conserved repetitive elements, CUP (Conserved UPstream), and at least nine novel open reading frames (ORFs). Appropriate protein products were identified for eight ORFs and the analysis of their deduced amino acid sequences revealed similarities with some well-known proteins (KorA of RK2/RP4, RecX and PsiB) that may play a role in the adaptation of promiscuous plasmids to the new host. Comparison of CUP elements revealed that the CUP core is 417 nucleotides long and consists of two portions that markedly differ in GC content. The larger portion (307 nucleotides) of the core is about 74% GC and contains at least one NotI site, while the other (110 nucleotides) is only about 40% GC. The remarkable features of CUP elements is that five of them are oriented in the same direction and fused in a similar mode to the open reading frames (ORFs) that are able to encode unrelated proteins. The spacings between the right boundary of the CUP core and the potential ATG start codons of these ORFs are slightly different in length (16 to 18 bp), highly divergent in sequence but in all cases contain the conserved hexamer 5'-AGGAGT-3' at the position that is typical for the ribosome binding site of Escherichia coli. The A+T-rich portion of the CUP sequences contains the strong negatively regulated promoter and appears to function as a genetic switch that coordinately controls the expression of CUP-fused genes during the conjugal transfer. These findings suggest that seven plasmid genes fused to the CUP elements including repA and two ard genes encoding positively acting replication protein and antirestriction proteins, respectively, may be members of one regulatory network based on the CUP elements and two plasmid-encoded regulatory proteins ArdK and ArdR. At least, the ArdK protein may act as a typical repressor by binding to the promoter region of the CUP sequence. Most of the structural and functional features of organization of the CUP-controlled regulatory network are associated with the idea that the CUP elements may be involved in the natural genetic engineering process of organizing various functionally related genes in one regulon.