Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF.

Rosanna C Hennessy,Kristian F Nielsen,Peter Stougaard,Christopher B W Phippen,Stefan Olsson

doi:10.1002/mbo3.516

Abstract

Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun–nup regulon. Organization of the regulon is similar to clusters found in other CLP‐producing pseudomonads except for the border regions where putative LuxR‐type regulators are located. This study focuses on understanding the regulatory role of the LuxR‐type‐encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real‐time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun–nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.

Highlights

Pseudomonas fluorescens strain In5 originally isolated from an agricultural suppressive soil in southern Greenland is a promising biocontrol agent capable of suppressing Rhizoctonia solani infection of tomato seedlings and inhibiting the growth of diverse phytopathogens (Michelsen, Watrous, Glaring, Kersten, Koyama, et al, 2015)
Nunamycin and nunapeptin biosynthesis is directed by nonribosomal peptide synthetase (NRPS) genes located on a large genomic island spanning over 100 kb
LuxR-like regulators are involved in the production of viscosin by P. fluorescens SBW25 mediated by ViscAR and ViscBCR and the massetolide biosynthesis genes of P. fluorescens SS101 have been shown to be regulated by a LuxR-type transcriptional regulator

Summary

| INTRODUCTION

Pseudomonas fluorescens strain In5 originally isolated from an agricultural suppressive soil in southern Greenland is a promising biocontrol agent capable of suppressing Rhizoctonia solani infection of tomato seedlings and inhibiting the growth of diverse phytopathogens (Michelsen, Watrous, Glaring, Kersten, Koyama, et al, 2015). The cyclic lipopeptides (CLP) nunamycin and nunapeptin are a distinctive feature of the Greenlandic strain P. fluorescens In5 and have recently been shown to play a key role in the suppressiveness of soilborne pathogens by. If the bacterium is specialized in using nunF-regulated genes to induce hyphal leakages, it is hypothesized that the fungal-associated carbon sources should result in a higher upregulation of nunF

| EXPERIMENTAL PROCEDURES

Findings

| DISCUSSION