ORGANIZATION OF THE LEADING REGION OF INCN PLASMID PKM101 (R46) - A REGULON CONTROLLED BY CUP SEQUENCE ELEMENTS

Analysis of the nucleotide sequence of the 13.8 kb leading region of t he IncN plasmid pKM101 (a deletion derivative of R46) revealed eight c opies of highly conserved repetitive elements, CUP (Conserved UPstream ), and at least nine novel open reading frames (ORFs). Appropriate pro tein products were identified for eight ORFs and the analysis of their deduced amino acid sequences revealed similarities with some well-kno wn proteins (KorA of RK2/RP4, RecX and PsiB) that may play a role in t he adaptation of promiscuous plasmids to the new host. Comparison of C UP elements revealed that the CUP core is 417 nucleotides long and con sists of two portions that markedly differ in GC content. The larger p ortion (307 nucleotides) of the core is about 74% GC and contains at l east one NotI site, while the other (110 nucleotides) is only about 40 % GC. The remarkable feature 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 ATC; start codons of these ORFs are slightly different in length (16 to 18 bp), highly divergent in sequence but in all cases contain the c onserved 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 a ppears to function as a genetic switch that coordinately controls the expression of CUP-fused genes during the conjugal transfer. These find ings suggest that seven plasmid genes fused to the CUP elements includ ing repA and two ard genes encoding positively acting replication prot ein and antirestriction proteins, respectively, may be members of one regulatory network based on the CUP elements and two plasmid-encoded r egulatory proteins ArdK and ArdR. At least, the ArdK protein may act a s a typical repressor by binding to the promoter region of the CUP seq uence. 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 engineeri ng process of organizing various functionally related genes in one reg ulon. (C) 1997 Academic Press Limited.