PcsR2 Is a LuxR-Type Regulator That Is Upregulated on Wheat Roots and Is Unique to Pseudomonas chlororaphis.

Huiqiao Pan,Leland S Pierson,Elizabeth A Pierson

doi:10.3389/fmicb.2020.560124

Abstract

LuxR solos are common in plant-associated bacteria and increasingly recognized for playing important roles in plant-microbe interkingdom signaling. Unlike the LuxR-type transcriptional regulators of prototype LuxR/LuxI quorum sensing systems, luxR solos do not have a LuxI-type autoinducer synthase gene associated with them. LuxR solos in plant-pathogenic bacteria are important for virulence and in plant endosymbionts contribute to symbiosis. In the present study, we characterized an atypical LuxR solo, PcsR2, in the biological control species Pseudomonas chlororaphis 30–84 that is highly conserved among sequenced P. chlororaphis strains. Unlike most LuxR solos in the plant-associated bacteria characterized to date, pcsR2 is not associated with a proline iminopeptidase gene and the protein has an atypical N-terminal binding domain. We created a pcsR2 deletion mutant and used quantitative RT-PCR to show that the expression of pcsR2 and genes in the operon immediately downstream was upregulated ∼10-fold when the wild type strain was grown on wheat roots compared to planktonic culture. PcsR2 was involved in upregulation. Using a GFP transcriptional reporter, we found that expression of pcsR2 responded specifically to root-derived substrates as compared to leaf-derived substrates but not to endogenous AHLs. Compared to the wild type, the mutant was impaired in the ability to utilize root carbon and nitrogen sources in wheat root macerate and to colonize wheat roots. Phenazine production and most biofilm traits previously shown to be correlated with phenazine production also were diminished in the mutant. Gene expression of several of the proteins in the phenazine regulatory network including PhzR, Pip (phenazine inducing protein) and RpeA/RpeB were reduced in the mutant, and overexpression of these genes in trans restored phenazine production in the mutant to wild-type levels, indicating PcsR2 affects the activity of the these regulatory genes upstream of RpeA/RpeB via an undetermined mechanism. Our results indicate PcsR2 upregulates the expression of the adjacent operon in response to unknown wheat root-derived signals and belongs to a novel subfamily of LuxR-type transcriptional regulators found in sequenced P. chlororaphis strains.

Highlights

Albeit bacteria are unicellular organisms, they communicate with each other via small diffusible molecules to orchestrate their behaviors in a population density-dependent manner, which facilitates rapid adaptation to the environment (Fuqua et al, 1994; Mukherjee and Bassler, 2019)
P. chlororaphis was grown at 28◦C in Luria-Bertani (LB) medium (Fisher BioReagentsTM, Hampton, NH), pigment production medium D (PPMD), AB minimal media (AB), AB amended with 0.4% glucose (AB + G), or AB + G amended 2% casamino acids (AB + CAA) media (CAA is from BD Bacto, San Jose, CA), as described previously (Yu et al, 2018)
Bacterial quorum sensing (QS) regulation via the production of acyl homoserine lactones (AHLs), the prototypical QS signal, has been shown to be important for regulating key bacterial traits for bacteria-plant interactions, and the production of AHL mimics by plants highlights the importance of LuxR-based signal-response regulators as contributing to the recognition of the chemical language involved in interkingdom communication (Loh et al, 2002; Kan et al, 2017)

Summary

Introduction

Albeit bacteria are unicellular organisms, they communicate with each other via small diffusible molecules to orchestrate their behaviors in a population density-dependent manner, which facilitates rapid adaptation to the environment (Fuqua et al, 1994; Mukherjee and Bassler, 2019) This cell-cell communication mechanism, known as quorum sensing (QS), enables populations to solve problems that single cells cannot, such as colonization, conjugation, secondary metabolites biosynthesis, biofilm formation, and effective invasion of host organisms (Piper et al, 1993; Fuqua and Winans, 1994; Pierson et al, 1994; Wood et al, 1997; Maddula et al, 2006; Schuster and Greenberg, 2006). Quorum sensing was first identified in Vibrio fischeri, where LuxR binds the signal and directly activates transcription of the luxICDABE operon, resulting in an exponential increase in the production of both the signal and bioluminescence (Nealson and Hastings, 1979; Engebrecht et al, 1983; Fuqua and Greenberg, 2002)

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