In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system

Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative σ factor (σ⁵⁴) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of σ⁵⁴-independent promoters. To reconstitute this putative two-component system in vitro, the RcNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP- RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB P phosphorylates the S. typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC-P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB-P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC-P binds the upstream tandem binding sites of the glnB promoter 4-fold better than the unphosphorylated RcNtrC protein, presumably due to oligomerization of RcNtrC-P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc-NtrB phosphotransfer to the RcNtrC protein results in increased oligomerization at the enhancer but with subsequent activation of a σ⁵⁴-independent promoter.