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 sigma factor (sigma(54)) 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 sigma(54)-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 similar to 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-RcNtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC similar to P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB similar to 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 similar to 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 similar to P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific RcNtrB phosphotransfer to the RcNtrC protein results in increased oligomerization at the enhancer but with subsequent activation of a sigma(54)-independent promoter.