Abstract
Bacterial chromosome replication is triggered by the initiator protein, DnaA, which assembles into a multimeric complex (orisome) that unwinds the replication origin, oriC, and helps to load the replicative DNA helicase. DnaA is highly conserved and the oriCs from nearly all bacteria carry clusters of oriC‐encoded DnaA recognition sites that guide step‐wise and properly timed orisome assembly. However, the arrangement of these sites is widely divergent among bacterial types and prior studies show that heterologous oriCs are only functional in extremely close relatives with nearly identical origin nucleotide sequence. To investigate the basis for DnaA recognition site diversity and possibly identify shared steps in orisome assembly, we replaced E. coli's chromosomal oriC with heterologous donor oriCs from its distant Gammaproteobacterial relatives and measured replication activity in vivo. Despite carrying little sequence similarity to the host, oriCs from Acinetobacter baylyi (ADP1) and Moraxella catarrhalis (ATCC 25240) were active in E. coli and used alternative pathways to assemble E. coli DnaA into functional orisomes. Mapping studies revealed the transplanted oriCs lacked the distinctive DNA unwinding element comprising 13mer repeat motifs as well as the arrayed low affinity DnaA recognition sites that are both hallmarks of E. coli oriC. Using flow cytometry analysis, we found that ADP1 and M. catarrhalis oriCs were also unable to initiate DNA synthesis at the proper cell cycle time in their E. coli hosts. Thus, the mechanisms for initiation timing must be distinct from those that permit replication fork assembly on these origins. We propose that E. coli and ADP1‐type orisomes share a sub‐architecture required for mechanical function (unwinding and helicase loading), but also carry type‐specific variations in the arrangement and affinity of DnaA recognition sites that are required for proper initiation timing using their cognate DnaAs. We propose that replication origin diversity reflects the many varieties of cell cycle‐coupled regulatory mechanisms used to control the availability/accessibility of DnaA at the oriCs of different bacterial types. Further studies of DnaA‐oriC interactions on heterologous origins may also reveal novel targets for broad spectrum bacterial growth inhibitors as well as those for type‐specific inhibition.
Support or Funding Information
National Institutes of Health (GM54042 to AL) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to JG).
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.