RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris.

Kaihuai Li,Fengquan Liu,Yuling Liao,Quan Zeng,Haihong Wang,Guichun Wu

doi:10.1111/mpp.12938

Abstract

Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris. Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched‐chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues.

Highlights

Homologous regulatory factors are widely present in bacterial genomes
We describe for the first time the distinct roles of RpoN1 and RpoN2 and their different regulatory functions in basal metabolism, flagellar biosynthesis, extracellular polysaccharide (EPS) formation, biofilm formation, and virulence in X. campestris
RpoN has been shown to be involved in the regulation of many bacterial functions, such as nitrogen metabolism, flagellar biosynthesis, biofilm formation, motility, colonization, lipoprotein biosynthesis, and the activity of a type III secretion system

Summary

| INTRODUCTION

Homologous regulatory factors are widely present in bacterial genomes. These homologous regulators may display functional redundancy or different biological functions, and some homologues even have no biological function, for example the two homologous ArsR regulators of Pseudomonas putida KT2440 bind arsenite with similar affinities (Fernandez et al, 2016). TEM observation showed that the ΔrpoN1 mutant strain had a normal flagellar morphology compared with the wild-type strain (Figure 4c) These results indicate that RpoN2 is necessary for flagellar biogenesis and motility in X. campestris and suggest that the function of RpoN2 in flagellar synthesis cannot be performed by RpoN1. RT-qPCR showed that the expression of Xcc fliC in the ΔrpoN2 and ΔrpoN1N2 mutant strains was compromised compared with that in the wild-type X. campestris strain, indicating that RpoN2 positively regulates the expression of Xcc fliC (Figure 5b). The results showed that the ΔrpoN2 and ΔrpoN1N2 mutant strains exhibited 5.8-fold and 14.9-fold reductions, respectively, in biofilm formation on the polystyrene surface after staining with CV, compared with the wild type (Figure 6a); inactivation of rpoN1.

| DISCUSSION

Findings

| EXPERIMENTAL PROCEDURES