RepAM of the Amycolatopsis methanolica integrative element pMEA300 belongs to a novel class of replication initiator proteins

1 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands 2 Laboratory of Microbial Ecology, Centre for Ecological and Evolutionary Studies (CEES), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Correspondence Lubbert Dijkhuizen L.Dijkhuizen{at}rug.nl Accessory genetic elements, such as plasmids and integrative elements, are widespread amongst actinomycetes, but little is known about their functions and mode of replication. The conjugative element pMEA300 from Amycolatopsis methanolica is present mostly in an integrated state at a single specific site in the chromosome, but it can also replicate autonomously. Complete nucleotide sequencing, in combination with deletion studies, has revealed that o rfB of pMEA300 is essential for autonomous replication in its host. In this study, it was shown that purified OrfB protein binds specifically to the 3' end of its own coding sequence. Within this short sequence, a putative hairpin structure is located, which contains several direct and inverted repeats, and a nucleotide stretch that resembles the nicking site of the pC194 family of rolling circle replicating plasmids. Additional binding studies revealed that OrfB binds to an 8 bp inverted repeat that occurs three times within the hairpin structure. The data presented show that OrfB is the replication initiator (Rep) protein of pMEA300, and is therefore termed RepAM. Surprisingly, RepAM lacks significant sequence similarity with known prokaryotic Rep proteins, but it is highly similar to a number of yet uncharacterized ORFs that are located on integrative and conjugative elements of other actinomycetes. It is concluded that RepAM and its homologues are members of a novel class of Rep proteins. Abbreviations: DSO, double-stranded origin; ICE, integrative and conjugative element; RCR, rolling circle replication