Hydrogen cyanide, which contributes to Pseudomonas chlororaphis strain PA23 biocontrol, is upregulated in the presence of glycine

Abstract

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the pathogenic fungus Sclerotinia sclerotiorum. This bacterium secretes the antibiotics pyrrolnitrin (PRN) and phenazine (PHZ), together with degradative enzymes and siderophores. Strain PA23 also produces hydrogen cyanide (HCN); however, the role of this compound in PA23 antifungal (AF) activity remains unknown. The aim of the current study was to characterize an hcn mutant and determine whether HCN contributes to biocontrol. Analysis of an hcn mutant revealed decreased AF activity both in vitro and in a newly established model of S. sclerotiorum root-rot infection in lettuce. When glycine (20mM) and ferric chloride (100µM) were included as media amendments, elevated AF activity was observed. Moreover glycine, but not FeCl3, increased HCN production and hcnA-lacZ transcriptional activity in the wild type. As the metabolic precursor for HCN biosynthesis, glycine is expected to enhance HCN production at the post-transcriptional level. We postulated that glycine might be indirectly affecting HCN transcription through the PhzRI quorum-sensing (QS) system. Analysis of phzR-lacZ and phzI-lacZ activity together with autoinducer levels revealed upregulation by glycine. This compound also increased expression of the QS-controlled prnA and phzA and their endproducts but, for reasons unknown, FeCl3 exerted a repressive effect. In a QS-deficient background the opposite occurred; FeCl3 increased hcnA-lacZ expression whereas glycine had no effect. HCN is under control of QS and the global regulator ANR. We present a model depicting how these regulators, in combination with media amendments, impact PA23 hcn expression.