Biosynthesis, Chemical Structure, and Structure-Activity Relationship of Orfamide Lipopeptides Produced by Pseudomonas protegens and Related Species

Abstract

Orfamide-type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudomonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudomonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P. protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens.

Root‐associated microbes in sustainable agriculture: models, metabolites and mechanisms

“…Sequencing revealed three additional ‘orphan’ gene clusters subsequently shown to encode for the synthesis of a novel cyclic lipopeptide (CLP) designated orfamide A, the macrolide polyketide rhizoxin, and the previously unrecognized siderophore enantio‐pyochelin. Like other cyclic lipopeptides, orfamide A is a surfactant with antifungal activity, influencing bacterial motility and lysing zoospores of oomycete plant pathogens . Rhizoxin and its structural analogs, encoded by a hybrid NRPS/PKS cluster, also are active against fungi and oomycetes but in addition, they exhibit broad, differential phytotoxic, cytotoxic and antitumor activities by binding to β‐tubulin, thereby interfering with microtubule dynamics during mitosis in eukaryotes .…”

Coregulation of the cyclic lipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a

“…This strain produces two classes of CLPs namely orfamides and sessilins together with two types of phenazines, phenazine‐1‐carboxylate (PCA) and phenazine‐1‐carboxamide (PCN) (D'aes et al., ; Perneel et al., ). Orfamides are also produced by biocontrol agents belonging to the P. protegens group (Gross et al., ; Jang et al., ; Ma, Geudens, et al., ; Takeuchi, Noda, & Someya, ), while sessilins are structurally related to the tolaasins produced by the mushroom pathogen, P. tolaasii . Sessilins are important for biofilm formation, while orfamides are crucial for the swarming motility of CMR12a (D'aes et al., ) and both CLPs are important for biocontrol (D'aes et al., ; Hua & Höfte, ; Ma, Hua, Ongena, & Höfte, 2016; Olorunleke, Hua, Kieu, Ma, & Höfte, 2015).…”

“…Intriguingly, there is no nodT ‐like gene in the orfamide gene cluster of Pseudomonas sp. CMR12a, while this gene is present in the orfamide gene clusters of P. protegens isolates (Ma, Geudens, et al., ). In addition, a LuxR‐type regulatory gene, ofaR1 , is located upstream of the orfamide biosynthesis cluster and a second one, ofaR2 , is situated downstream of the macA2B2 genes, whereas a single LuxR‐type regulatory gene, sesR , is located upstream of the sessilin biosynthesis cluster next to the sesT gene (D'aes et al., ).…”

Role of phenazines and cyclic lipopeptides produced by pseudomonas sp. CMR12a in induced systemic resistance on rice and bean

“…CMR12a produces phenazine‐1‐carboxylate (PCA) and phenazine‐1‐carboxamide (PCN), and two different classes of CLPs. The strain produces orfamide‐type CLPs, which are commonly produced by P. protegens strains (Ma et al ., ) and sessilins, which are related to tolaasins, CLPs produced by the mushroom pathogen P. tolaasii (D'aes et al ., ). Phenazines are nitrogen‐containing heterocyclic compounds that can be produced by Brevibacterium , Burkholderia , Pseudomonas , Pectobacterium and Streptomyces (Budzikiewicz, ; Mavrodi et al ., ) Phenazines play important roles in antibiosis, antitumor, antimalaria, antiparasitic activities, biofilm formation, and can even act as virulence factors (Laursen and Nielsen, ; Ramos et al ., ).…”