Figure 3

The nuclease activity of NurA and its interaction with HerA are essential for cell viability. (A) A schematic map depicting the procedures of the complementation analysis. pMID-nurA-T cells were transformed with pSSRA-NurA-C-His plasmids or empty vector. Three genotypes of the cells grown up after counter-selection were indicated as ➀, ➁, and ➂. The star indicates a putative spontaneous mutation in pyrEF. (B) Genotype analysis of the cells grown up by PCR using nurA flanking primers and sequencing of the nurA on the isolated plasmid. “+”, the designed plasmid maintained; “R”, plasmid maintained, but nurA gene had reverted to wild-type. (C) Gel filtration analysis of the interaction between wild type HerA and NurA mutants I295L, I295E, F300Y, and F300E by a Superdex™ 200 10/300 GL column. A total of 500 μg HerA, 500 μg NurA, and the mixture were heated incubated at 60°C for 20 min before gel filtration. The sample fractions were analyzed by SDS-PAGE. A small amount of NurA formed dimers in gels when NurA concentration was high. The elution peaks of the molecular markers are indicated by arrows at the bottom.