Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species

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 Pseudornonas 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 Pseudornonas 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

Antimicrobial and Insecticidal: Cyclic Lipopeptides and Hydrogen Cyanide Produced by Plant-Beneficial Pseudomonas Strains CHA0, CMR12a, and PCL1391 Contribute to Insect Killing.

Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes, and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains Pseudomonas protegens CHA0, Pseudomonas chlororaphis PCL1391, and Pseudomonas sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e., 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host

Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A

Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225 mg L-1) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated beta-hydroxy fatty acid chain residue (from C-13 to C-15), differed each other from peptide sequence in the position of Leu(7) or Val(7). All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115 A degrees C) and sodium chloride concentration (from 2.5 to 20.0 %, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent

Production and characterization of iturinic lipopeptides as antifungal agents and biosurfactants produced by a marine Pinctada martensii-derived Bacillus mojavensis B0621A

Bacillus mojavensis B0621A was isolated from a pearl oyster Pinctada martensii collected from South China Sea. While screening for cyclic lipopeptides potentially useful as lead compounds for biological control against soil-bone fungal plant pathogens, three lipopeptides were isolated and purified from the fermentation broth of B. mojavensis B0621A via vacuum flash chromatography coupled with reversed-phase high performance liquid chromatography (RP-HPLC). The structural characterization and identification of these cyclic lipopeptides were performed by tandem mass spectrometry (MS/MS) combined with gas chromatography-mass spectrometry (GC-MS) analysis as well as chemical degradation. These lipopeptides were finally characterized as homologues of mojavensins, which contained identical amino acids back bones of asparagine1, tyrosine2, asparagine3, glutamine4, proline5, asparagine6, and asparagine7 and differed from each other by their saturated β-amino fatty acid chain residues, namely, iso-C14 mojavensin, iso-C16 mojavensin, and anteiso-C17 mojavensin, respectively. All lipopeptide isomers, especially iso-C16 mojavensin and anteiso-C17 mojavensin, displayed moderate antagonism and dose-dependent activity against several formae speciales of Fusarium oxysporum and presented surface tension activities. These properties demonstrated that the lipopeptides produced by B. mojavensis B0621A may be useful as biological control agent to fungal plant pathogens

The cyclic lipopeptide orfamide induces systemic resistance in rice to Cochliobolus miyabeanus but not to Magnaporthe oryzae

The Pseudomonas- derived cyclic lipopeptide orfamide can induce resistance to Cochliobolus miyabeanus but not to Magnaporthe oryzae in rice. Abscisic acid signaling is involved in the induced systemic resistance response triggered by orfamide. Diverse natural products produced by beneficial Pseudomonas species have the potential to trigger induced systemic resistance (ISR) in plants, and thus may contribute to control of diseases in crops. Some beneficial Pseudomonas spp. can produce cyclic lipopeptides (CLPs), amphiphilic molecules composed of a fatty acid tail linked to an oligopeptide which is cyclized. CLPs can have versatile biological functions, but the capacity of Pseudomonas-derived CLPs in triggering ISR responses has barely been studied. Pseudomonas protegens and related species can produce orfamide-type CLPs. Here we show that in rice, orfamides can act as ISR elicitors against the necrotrophic fungus Cochliobolus miyabeanus, the causal agent of brown spot disease, but are not active against the blast fungus Magnaporthe oryzae. Orfamide A can trigger early defensive events and activate transcripts of defense-related genes in rice cell suspension cultures, but does not cause cell death. Further testing in rice cell suspension cultures and rice plants showed that abscisic acid signaling, the transcriptional activator OsWRKY4 and pathogenesis-related protein PR1b are triggered by orfamide A and may play a role in the ISR response against C. miyabeanus

Pseudophomins A–D Produced from Pseudomonas sp. HN8‑3 Using an OSMAC Approach and Their Roles in Biocontrol of Phytophthora capsici in Cucumbers

In this study, two new cyclic lipopeptides (CLPs) pseudophomins C (3) and D (4) and two known CLPs pseudophomins A (1) and B (2) were produced and characterized from the bacterial supernatant of Pseudomonas sp. HN8-3 by an OSMAC (one strain-many compounds) approach. OSMAC is a strategy that involves feeding of a single microorganism with divergent substrates to stimulate the production of new secondary metabolites. These pseudophomins were purified and identified via chromatographic methods, droplet collapse assay, genome mining, spectroscopic and spectrometric analyses, and single-crystal X-ray diffraction (XRD). Moreover, bioactivity tests showed that pseudophomins could lyse the zoospores of Phytophthora capsici in vitro, and coapplication of pseudophomins with zoospores of P. capsici further reduced the incidence of P. capsici on cucumber leaves. Collectively, these results indicated that pseudophomins have the potential to be developed as biopesticides for controlling P. capsici in cucumber

Pseudophomins A–D Produced from Pseudomonas sp. HN8‑3 Using an OSMAC Approach and Their Roles in Biocontrol of Phytophthora capsici in Cucumbers

In this study, two new cyclic lipopeptides (CLPs) pseudophomins C (3) and D (4) and two known CLPs pseudophomins A (1) and B (2) were produced and characterized from the bacterial supernatant of Pseudomonas sp. HN8-3 by an OSMAC (one strain-many compounds) approach. OSMAC is a strategy that involves feeding of a single microorganism with divergent substrates to stimulate the production of new secondary metabolites. These pseudophomins were purified and identified via chromatographic methods, droplet collapse assay, genome mining, spectroscopic and spectrometric analyses, and single-crystal X-ray diffraction (XRD). Moreover, bioactivity tests showed that pseudophomins could lyse the zoospores of Phytophthora capsici in vitro, and coapplication of pseudophomins with zoospores of P. capsici further reduced the incidence of P. capsici on cucumber leaves. Collectively, these results indicated that pseudophomins have the potential to be developed as biopesticides for controlling P. capsici in cucumber

Isolation and characterization of a new iturinic lipopeptide, mojavensin A produced by a marine-derived bacterium Bacillus mojavensis B0621A

Three lipopeptides were isolated by bioactivity-guided fractionation from the fermentation broth of Bacillus mojavensis B0621A. A new iturinic lipopeptide, named mojavensin A, was tentatively characterized by 1D, 2D NMR and MS spectroscopy, Marfey's method containing a novel peptide backbone of L-Asn(1), D-Tyr(2), D-Asn(3), L-Gln(4), L-Pro(5), D-Asn(6), L-Asn(7) and an anteiso-type of the saturated beta-fatty acid side chain. Compound 2 and 3 were tentatively identified as iso-C16 fengycin B and anteiso-C17 fengycin B, respectively. These lipopeptides displayed dose-dependent antifungal activity against a broad spectra of phytopathogens and were weakly antagonistic to Staphylococcus aureus. Moreover, they all revealed cytotoxic activities against the human leukemia (HL-60) cell line. Mojavensin A, iso-C16 fengycin B, and anteiso-C17 fengycin B inhibited the growth of HL-60 with IC(5)(0) of 100, 100 and 1.6 muM, respectively