DnaAE183Q mutant shows reduced ATPase activity and an underinitiating phenotype.
A) Coupled ATPase assay of purified wild type DnaA or of DnaAE183Q (10 μM). B) Surface plasmon resonance [SPR] experiments with DnaA and DnaAE183Q [2.5 μM] binding to oriC-DNA [0.25 pmol]. Black line: DnaAE183Q, grey line: wtDnaA. C) SPR in the presence of 2.5 mM ATP. D-E) Fluorescence microscopy analysis of cells growing exponentially in rich (LB) medium, D) expressing YFP-DnaAE183Q for 60 minutes, E) expressing YFP-DnaA for 60 minutes. Note that cells contain more oriC regions in rich medium compared with minimal medium as in Figs 1 and 2. The effect of induction of mutant DnaA is more apparent in rich medium. Septa are indicated by white lines, and are more difficult to see after induction of mutant DnaA. F)-I) Increased cell size and reduced number of origins in exponentially growing cells expressing DnaAE183Q for 60 min in minimal medium, white bars wild type cells, grey bars cells after induction of DnaAE183Q, F) Diagram showing the distribution of cell length, bars show percentage of cells with a certain size, white bars wild type cells, G) Diagram showing percentage of cells containing one or two nucleoids, or none, H) Diagram showing the percentage of cells containing a certain number of origins, I) Diagram showing the percentage of cells containing no YFP-DnaA foci, or different numbers of foci.