His-RcsB-P alone or mixed with excess MBP-RcsA could bind to the waaA promoter-proximal region in a dose-dependent manner.
The electrophoretic mobility shift assay (EMSA) (Fig. 1C) denoted that the purified His-PhoP protein was able to bind to the waaA promoter-proximal DNA in a dose-dependent manner; in contrast, His-PhoP did not bind the 16S rRNA gene at any concentration.
We created three strains to analyze the effect of RscA on binding of RscB to the waaA promoter: 1) rcsA-c containing endogenous RcsB and plasmid-borne RscA, 2) ΔrcsB lacking both RcsA and RscB, and 3) rcsA-c/ΔrcsB containing only plasmid-borne RscA.
To determine whether RcsA affects binding affinity of RcsB to the waaA promoter-proximal region, EMSAs were performed (Fig. 2C).
In addition to regulation of waaA, RcsAB is also known to bind to the hmsT promoter-proximal region to repress the transcription of hmsT, a gene encoding the diguanylate cyclase that is responsible for the biosynthesis of 3′,5′-cyclic diguanosine monophosphate (c-di-GMP, a second messenger promoting the production of biofilm matrix exopolysaccharide) (Sun et al., 2012).
Only the Δ5KA abolishes binding to PtdIns 3,4,5 P3.
Searches using the Chlamydia trachomatis WaaA revealed that this protein was much less similar to the neisserial WaaA, with homologies strongest in the middle of the protein.
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We used the WaaA sequence of E. coli or H. influenzae to search the available translated neisserial DNA sequences and found that the these proteins were very similar, with homology between the proteins seen across the entire protein sequence.
