Insertional mutation of the rpoS gene contributes to alteration in biosynthesis of antifungal agents in Pseudomonas sp. M18

Abstract

As a plant-beneficial rhizobacterium, Pseudomonas sp. M18 was isolated from the rhizosphere of watermelon in a Shanghai suburb. Two kinds of antifungal compounds, phenazine-1-carboxylic acid and pyoluteorin produced by Pseudomonas sp. M18, contribute to suppression of pathogenic fungi in agricultural soil. In order to examine how production of these antifungal agents is regulated, an rpoS gene with its flanking fragments was first cloned in strain M18. With the insertional inactivation of rpoS, the rpoS null mutant strain M18S was constructed, and it showed improved production of antifungal agents. In King’s medium B, phenazine-1-carboxylic acid reduced to nondetectable level, while pyoluteorin increased approximately 10-fold. In PPM medium, strain M18S produced less phenazine-1-carboxylic acid and much more pyoluteorin in comparison with wild-type strain M18. Complementation with the wild type ropS gene in trans restored wild-type phenotypes. Expression of the translational fusions phzA‘-’lacZ and pltA‘-’lacZ confirmed the effect of rpoS on phenazine-1-carboxylic acid and pyoluteorin biosynthetic operons. In in vitro plate assays, the mutant strain M18S inhibiting mycelial growth of Pythium aphanidermatum more than wild-type strain M18. Altogether, these results suggest that phenazine-1-carboxylic acid biosynthesis is positively regulated and pyoluteorin production negatively regulated by alternative sigma factor S in Pseudomonas sp. M18.

Introduction

Some strains of fluorescent pseudomonads, which usually inhabit plant surfaces including roots, leaves, and floral parts, have been proved to act as biological control agents because of their ability to protect plants against a range of agricultural plant-pathogenic fungi (Weller and Cook, 1983). A set of antifungal metabolites produced in these strains contribute to their biocontrol capabilities, including phenazine compounds, hydrogen cyanide (HCN), 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt), pyrrolnitrin (Prn), and their relevant derivatives (Fenton et al., 1992, Keel et al., 1992, Loper, 1988, Maurhofer et al., 1994, Stutz et al., 1986, Thomashow and Weller, 1988, Voisard et al., 1989).

In Escherichia coli, the alternative sigma factor S (σ^S, RpoS) functions as a global regulator and is responsible for the activation of many genes expressed mainly during the stationary phase and under various stress conditions (Loewen and Hengge-Aronis, 1994). Genome-wide analysis in E. coli revealed that 10% of genes were controlled directly or indirectly by RpoS (Weber et al., 2005). In Pseudomonas spp., sigma factor S may be involved in regulating the production of many antifungal agents (Haas and Keel, 2003). RpoS positively regulates production of exotoxin A and alginate and inhibits production of pyocyanin and the siderophore pyoverdine by P. aeruginosa (Suh et al., 1999). In an rpoS null mutant of P. fluorescens Pf-5, the lack of σ^S increased Plt, HCN and Phl expression several fold, and completely blocked Prn biosynthesis (Sarniguet et al., 1995, Whistler et al., 1998). In an rpoS mutant of P. fluorescens CHA0, the consequences of rpoS disruption, however, enhanced expression of the plt genes and the hcnABC genes (encoding HCN synthase), relative to that in the wild-type, but apparently did not over-express the phl genes (encoding Phl synthase) (Haas and Keel, 2003). In P. chlororaphis PCL1391, phenazine-1-carboxamide (PCN), a derivative of phenazine-1-carboxylic acid (PCA), was repressed when the rpoS gene was knocked out (Girard et al., 2006). Thus, it can be deduced that biosynthesis of some antifungal agents requires RpoS in Pseudomonas spp., while biosynthesis of other antifungal agents does not.

Pseudomonas sp. M18 is a strain of plant growth promoting rhizobacteria (PGPR), which can produce both PCA and Plt (Hu et al., 2005). In the last two decades, PCA, which exhibits broad-spectrum activity against various species of fungi, has well been shown to be an effective factor for suppression of growth of Gaeumannomyces gramminis var. tritici which causes take-all disease of wheat (Thomashow and Weller, 1988, Thomashow, 1996). Plt shows its antifungal properties by inhibiting growth of Pythium ultimum which induces damping-off of cotton seedling and cress (Howell and Stipanovic, 1980, Maurhofer et al., 1994). Up to the present time, M18 has been the only reported strain which can produce these two antifungal compounds, although PCA or Plt has been found in many other P. fluorescens strains, respectively. In previous study, GacA, a global regulator, showed differential regulation on production of PCA and Plt in strain M18, suggesting that there seems to be a specific regulation mechanism for antifungal metabolites in strain M18 (Ge et al., 2004). Although the effects on PCA or Plt biosynthesis made by σ^S have been investigated in many strains, respectively, it is not clear how RpoS regulates PCA and Plt production at one time in a single strain. For this, an rpoS null mutant strain M18S was first constructed and the production of antifungal secondary metabolites was determined by high performance liquid chromatography (HPLC) in strain M18S or the wild type strain M18, respectively. Here, we present data that provide answers to this question.