# Functional and Pangenomic Exploration of Roc Two‐Component Regulatory Systems Identifies Novel Players Across Pseudomonas Species

## TL;DR

This study expands the Roc regulatory system in Pseudomonas by identifying new components and their roles in virulence and biofilm formation.

## Contribution

The work identifies RocA3 and RocS4 as novel regulators in the Roc system and provides a pangenomic analysis of regulatory components across Pseudomonas species.

## Key findings

- PA4080, renamed RocA3, is a fourth response regulator activated by RocS1 and RocS2.
- A fourth branch of the Roc system includes RocS4 and HptA, which interact with other regulators.
- Pangenomic analysis reveals the plasticity of Roc regulators among Pseudomonas clades.

## Abstract

The opportunistic pathogen
Pseudomonas aeruginosa
relies on a large collection of two‐component regulatory systems (TCSs) to sense and adapt to changing environments. Among them, the Roc (regulation of
cup) system is a one‐of‐a‐kind network of branched TCSs, composed of two histidine kinases (HKs—RocS1 and RocS2) interacting with three response regulators (RRs—RocA1, RocR, and RocA2), which regulate virulence, antibiotic resistance, and biofilm formation. Based on extensive work on the Roc system, previous data suggested the existence of other key regulators yet to be discovered. In this work, we identified PA4080, renamed RocA3, as a fourth RR that is activated by RocS1 and RocS2 and that positively controls the expression of the cupB operon. Comparative genomic analysis of the locus identified a gene—rocR3—adjacent to rocA3 in a subpopulation of strains that encodes a protein with structural and functional similarity to the c‐di‐GMP phosphodiesterase RocR. Furthermore, we identified a fourth branch of the Roc system consisting of the PA2583 HK, renamed RocS4, and the Hpt protein HptA. Using a bacterial two‐hybrid system, we showed that RocS4 interacts with HptA, which in turn interacts with RocA1, RocA2, and RocR3. Finally, we mapped the pangenomic RRs repertoire, establishing a comprehensive view of the plasticity of such regulators among clades of the species. Overall, our work provides a comprehensive inter‐species definition of the Roc system, nearly doubling the number of proteins known to be involved in this interconnected network of TCSs controlling pathogenicity in Pseudomonas species.

The Roc system represents a highly interconnected but incomplete network of two‐component regulatory systems involved in the virulence of
Pseudomonas aeruginosa
. Our work has enabled us to identify the missing RocA3 regulator, to propose new players in the system and to delineate their conservation between clades of the species.