Date of Award

4-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Ocean Engineering and Marine Sciences

First Advisor

Alan C. Leonard

Second Advisor

Julia Grimwade

Third Advisor

David J. Carroll

Fourth Advisor

Shaohua Xu

Abstract

To ensure that all cells replicate their genomes faithfully before they divide, the initiation step of chromosome replication must be tightly regulated during the cell cycle. In bacteria, new rounds of replication are triggered by the assembly of a higher order protein complex, termed the orisome, that unwinds a well-defined origin (oriC) region on the circular chromosome, and prepares it for new replication forks. Orisomes comprise multiple copies of a highly conserved initiator protein, DnaA, and instructions for ordered orisome assembly are encoded into the oriC nucleotide sequence of nearly all bacteria as specifically-spaced clusters of 9 base pair (bp) DnaA recognition sites. Since orisomes are required for bacterial viability, and all bacteria share the DnaA initiator, the stages of orisome assembly should be excellent targets for antibiotic inhibitors. Unfortunately, for reasons that remain unclear, bacterial types build orisomes in different ways with high diversity in the number, spacing, and orientation of DnaA recognition sites in their copies of oriC. Little progress towards rational antibiotic design is expected until orisome assembly can be studied in many different bacterial types and shared stages of orisome assembly are identified. This can be a daunting task, especially when culture methods are lacking, and genetic tools are not available for many pathogenic strains. To avoid the problems associated with comparative orisome analysis, a heterologous chromosomal origin transplant system was developed using the well-understood and genetically tractable Escherichia coli (E. coli) bacterium. The transplant recipient allows oriC DNA from any bacterial donor to be seamlessly swapped into the native oriC position while the host chromosome is replicated from an integrated plasmid replication origin. Heterologous oriC function can then be tested by shutting off the plasmid origin although partial/non-functional orisomes can still be studied. Initial testing of the E. coli system was performed using a previously uncharacterized donor oriC from a distant Gammaproteobacteria relative; the soil microbe Acinetobacter baylyi (ADP1). Unexpectedly, the oriC of ADP1 functioned as the sole chromosomal replication origin in E. coli. Further examination revealed little similarity between E. coli and ADP1 origins and orisome assembly. A minimal functional ADP1 oriC (221 bp) was identified that required a centrally-located AT-rich DNA unwinding element (DUE) that was flanked by high and low DnaA recognition sites, a feature not commonly found in Gammaproteobacteria. Several important E. coli oriC features, including arrays of low affinity DnaA-ATP recognition sites, DeoxyAdenosine Methylation (DAM) sites (GATC), and 13 mer repeat motifs in the DUE were missing from ADP1 oriC. Based on these observations, ADP1 and related Acinetobacter species, some of which are important human pathogens, appear to initiate chromosome replication using a new version of orisomes that differ from those previously studied in other cell types and are worthy of further study. Furthermore, assembly of functional orisomes on ADP1 oriC was not coupled to the E. coli cell division cycle and DNA replication did not initiate synchronously from all origin copies as is the case for E. coli oriC. We propose that two sets of instructions are encoded into every oriC: one that performs the mechanical function of origin activation and will likely to be shared among bacterial types, and species-specific instructions that are used to regulate cell cycle timing. The ADP1 oriC must carry functional instructions, but lacks the sequences needed to monitor cell cycle availability of DnaA-ATP in E. coli, raising questions about cell cycle regulation of DNA synthesis in ADP1 cells. The newly found relationship between E. coli DnaA and ADP1 oriC provides a unique opportunity to study how one initiator can be assembled into two different functional orisomes. It is also likely that the transplant system described here will uncover additional functioning heterologous oriCs whose dissection will help to identify new targets for antibiotic development.

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