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

## TL;DR

This study shows that a periplasmic protein, PlrP, acts as a third component in a bacterial signaling system, helping pathogens like Bordetella survive in the lungs.

## Contribution

The paper identifies PlrP as a novel third component in a subset of NtrYX family two-component systems.

## Key findings

- PlrP prevents PlrS from acting as a strong phosphatase, maintaining PlrR phosphorylation levels.
- PlrP is essential for bacterial survival in the lower respiratory tract.
- PlrP homologs are found with NtrYX-family TCSs in other bacteria, suggesting a conserved function.

## Abstract

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 is often found 3′ to plrSR homologs in ⍺-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 strongly suggest that it inhibits PlrS phosphatase activity, likely through PlrS’s PhoQ-DcuS-CitA (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 bacterial survival in the lower respiratory tract, and that 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.

Bordetella species, including B. pertussis, cause respiratory infections in humans and other animals. Their PlrSR two-component regulatory systems are required for survival in the lower respiratory tract. We characterized the six-gene cluster that includes plrS and plrR, identifying two internal promoters that drive expression of plrP and plrS separately from plrR. Our data indicate that the plrP gene product is an essential third component of the PlrSR two-component regulatory system (TCS), functioning to prevent PlrS from acting as a strong phosphatase in vitro. PlrP homologs are present, and may function similarly, in NtrYX-family TCSs in other β- and γ-proteobacteria. Our results are important because they provide insight into how bacteria, including pathogens, sense and respond to their environment.