Genetic evidence for a periplasmic protein as a component for a subset of NtrYX two-component systems

PlrSR, a member of the NtrYX family of two-component regulatory systems (TCSs), is required for the classical bordetellae, including the causative agent of whooping cough, Bordetella pertussis, to persist in the lower respiratory tract. The plrSR genes are in the middle of a six-gene cluster whose regulation and roles during infection were unknown. rsmB and plrP are often found 5' to plrSR homologs in β- and γ-proteobacteria, while trkAH are often found 3' to plrSR homologs in α-proteobacteria. We investigated these genes to determine if they have a functional link to plrSR. We found that this gene cluster does not function as an operon. Rather, it contains two internal promoters: a weaker promoter in the 3' end of rsmB and a stronger promoter in the 3' end of plrS. Additionally, our results indicate that PlrP functions as a third component of the PlrSR TCS. Genetic manipulations of plrP, plrS, and plrR indicate that PlrP is essential in vitro and inhibits PlrS phosphatase activity, likely through PlrS's PDC domain. Since our results indicate that PlrR can be phosphorylated by another unknown phosphodonor in vitro, limiting PlrS phosphatase activity ensures PlrR~P is not dephosphorylated to lethally low levels. Using natural-host models, we determined that high levels of PlrR~P are required for in vivo survival, and PlrP affects PlrS activity in vivo. Given that plrP homologs always colocalize with ntrYX homologs, we propose that PlrP may fulfill similar functions in other β- and γ-proteobacteria that encode NtrYX-family TCSs, including nonpathogens.