Unique and Universal Features of Epsilonproteobacterial Origins of Chromosome Replication and DnaA-DnaA Box Interactions

In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts ( ). While DnaA proteins share significant homology regardless of phylogeny, regions exhibit more variable structures. The general architecture of s is universal, i.e., they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of s are shared by related species while being significantly different in unrelated bacteria. In this work, we characterized Epsilonproteobacterial regions. was the only species of the class for which was characterized. A few unique features were found such as bipartite structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual features raised questions of whether structure and DnaA-DNA interactions are unique to this bacterium or whether they are common to related species. By and analyses we identified putative s in three Epsilonproteobacterial species: pathogenic , symbiotic , and free-living . We propose that s typically co-localize with in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream ( ) and downstream ( ) of , and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in . Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial s, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5'-TTCAC-3' (4-8 nt), which, together with the significant changes in the DNA-binding motif of corresponding DnaAs, determines the unique molecular mechanism of DnaA-DNA interaction. Our results will facilitate identification of s and subsequent identification of factors which regulate chromosome replication in other Epsilonproteobacteria. Since replication is controlled at the initiation step, it will help to better characterize life cycles of these species, many of which are considered as emerging pathogens.