Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis

Abstract

DnaA is the bacterial replication initiator, which also functions as a transcription factor to regulate gene expression. In B. subtilis, DnaA has previously been shown to repress its own transcription and has also been implicated in directing part of the transcriptional response to replication stress. Because dnaA is essential, most of DnaA's potential effects on gene expression have been determined through indirect methods, which have implemented perturbations in replication and sequence analyses to predict direct effects of DnaA transcriptional regulation. Below, I take a more direct approach to assay DnaA's effect on gene expression and specific transcriptional regulatory networks by deleting dnaA in an oriN+ [delta]oriC strain background, which renders dnaA non-essential. Isogenic dnaA+ cells were constructed similarly and have dnaA constitutively expressed from an ectopic locus. In this background, DNA replication no longer depends on dnaA and is initiated instead by a plasmid replicon, oriN. The native origin of replication, oriC, is also deleted to eliminate differences in replication between [delta]dnaA and dnaA+ cells. Consequently, I can directly compare differences in gene expression due to the presence versus absence of dnaA. Deletion of dnaA results in approximately 463 significant differences in gene expression, most of which I show are due to DnaA direct activation of the gene sda. Many of these genes lie downstream of Sda activity and comprise several regulons, such as the Spo0A, AbrB, and SinR regulons. These regulons are known to become active during the transition from exponential growth to stationary phase. In addition to the many effects on gene expression, I show that deletion of dnaA results in lowered competence development. I also revisit the transcriptional response to replication stress and show that some of the previously predicted targets of DnaA respond to replication stress in a DnaA-dependent manner. Lastly, in collaboration with others, I have studied the relationship between a DnaA regulator, YabA and a nucleoid binding protein Rok. YabA and Rok associate at some of the same chromosomal regions, and at these regions YabA absolutely depends on Rok for its association. We are currently trying to understand the functional relationship between YabA, Rok, and DnaA.