Genetic and biochemical interactions between the DNA replication initiator and a chromosome architecture protein in Bacillus subtilis by

I described the co-association, in vitro interaction, and epistatic relationship between the DNA replication initiator DnaA and the nucleoid-associated protein Rok in Bacillus subtilis. Using ChIP-seq, I characterized the genome-wide association of DnaA and its regulator YabA. I found that DnaA and YabA associated with over 30 chromosomal regions that were bound by Rok, and association of DnaA and YabA with these regions was dependent on Rok. The DNAbinding domain of DnaA was dispensable for association of DnaA with these chromosomal regions. This indirect Rok-dependent association contrasted with the canonical sequencespecific binding of DnaA to eight chromosomal regions containing clusters of DnaA boxes. I found that association of YabA with Rok-dependent regions did not require DnaA, in contrast to the DnaA-dependent association of YabA with DnaA box cluster regions. Furthermore, I showed an in vitro interaction between DnaA and Rok using purified proteins in an electrophoretic mobility shift assay. DnaA depended on Rok for association with a DNA probe, recapitulating the dependence observed at this chromosomal region in vivo. DnaA and Rok are both transcription factors and regulate some of the same genes. I analyzed global gene expression to determine the genetic relationship between these two transcriptional regulators. In general, rok was epistatic to dnaA; that is, the gene expression effects of DnaA depended on Rok, consistent with the ChIP-seq dependence. I investigated a potential role for Rok in regulating replication initiation, but I did not detect a replication phenotype of a rok null mutant under various conditions. I found that a rok null mutation did not detectably affect DnaA or YabA protein levels, and I showed that the stationary phase growth defect of this strain was dependent on comK, a downstream target of Rok. Additionally, I determined that a putative regulator of replication initiation, the pyruvate dehydrogenase enzyme PdhC, affected replication via its metabolic function but was not a direct regulator of replication initation. Thesis Supervisor: Alan D. Grossman Title: Professor of Biology