Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of Factors

Mycobacterial plasmid pAL5000 represents a family of plasmids foundmostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB.While BLAST searches indicate that RepA is a replicase family protein, the evolu-tionary connection of RepB cannot be established, as no significant homologous partner (E< 103) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was under-taken using homology modeling, phylogenetic, andmutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operonmay have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix do-main characteristic of region 4 of extracytoplasmic function (ECF) factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of factors. Mutational analysis identified two argi-nines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is impor-tant for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interactionmodules. The pAL5000 family (23) consists of a groupof plasmids that arefound mostly in bacteria belonging to the phylum Actinobac