<i>Pseudomonas savastanoi</i> Two-Component System RhpRS Switches between Virulence and Metabolism by Tuning Phosphorylation State and Sensing Nutritional Conditions

Abstract

<i>Pseudomonas savastanoi</i> uses a type III secretion system (T3SS) to invade host plants. Our previous studies have demonstrated that a two-component system (TCS), RhpRS, enables <i>P. savastanoi</i> to coordinate the T3SS gene expression, which depends on the phosphorylation state of RhpR under different environmental conditions. Orthologues of RhpRS are distributed in a wide range of bacterial species, indicating a general regulatory mechanism. How RhpRS uses external signals and the phosphorylation state to exercise its regulatory functions remains unknown. We performed chromatin immunoprecipitation sequencing (ChIP-seq) assays to identify the specific binding sites of RhpR and RhpR^D70A in either King's B medium (KB [a T3SS-inhibiting medium]) or minimal medium (MM [a T3SS-inducing medium]). We identified 125 KB-dependent binding sites and 188 phosphorylation-dependent binding sites of RhpR. In KB, RhpR directly and positively regulated cytochrome <i>c</i>₅₅₀ production (via <i>ccmA</i>) and alcohol dehydrogenase activity (via <i>adhB</i>) but negatively regulated anthranilate synthase activity (via <i>trpG</i>) and protease activity (via <i>hemB</i>). In addition, phosphorylated RhpR (RhpR-P) directly and negatively regulated the T3SS (via <i>hrpR</i> and <i>hopR1</i>), swimming motility (via <i>flhA</i>), c-di-GMP levels (via PSPPH_2590), and biofilm formation (via <i>algD</i>). It positively regulated twitching motility (via <i>fimA</i>) and lipopolysaccharide production (via PSPPH_2653). Our transcriptome sequencing (RNA-seq) analyses identified 474 and 840 new genes that were regulated by RhpR in KB and MM, respectively. We showed nutrient-rich conditions allowed RhpR to directly regulate multiple metabolic pathways of <i>P. savastanoi</i> and phosphorylation enabled RhpR to specifically control virulence and the cell envelope. The action of RhpRS switched between virulence and regulation of multiple metabolic pathways by tuning its phosphorylation and sensing environmental signals in KB, respectively.<b>IMPORTANCE</b> The plant pathogen <i>Pseudomonas savastanoi</i> invades host plants through a type III secretion system, which is strictly regulated by a two-component system called RhpRS. The orthologues of RhpRS are widely distributed in the bacterial kingdom. The master regulator RhpR specifically depends on the phosphorylation state to regulate the majority of the virulence-related genes. Under nutrient-rich conditions, it modulates many important metabolic pathways, which consist of one-fifth of the genome. We propose that RhpRS uses phosphorylation- and nutrition-dependent mechanisms to switch between regulating virulence and metabolism, and this functionality is widely conserved among bacterial species.