Genome‐based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species

Abstract

SummaryAnalysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.

Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca

“…An additional chromatographic peak from the T. fusca preparation, eluting just before fuscachelin B, exhibited NMR spectra very similar to fuscachelin B. 15 N gHSQC, are consistent with an ␣-amide structure (termed fuscachelin C) as shown in Fig. 2B (Fig.…”

“…S3 and S4). Unexpectedly, 13 C and 15 N gHMBC experiments to confirm the amino acid connectivity revealed a heterodimeric peptide with the sequence Dhb-Arg-Gly-Gly-Ser-HOOrn-Gly-Gly-Arg-Dhb (Table 1 (Fig. S6).…”

“…NMR solvents were purchased from Cambridge Isotope Laboratories. 1 H, 13 C, 15 N, and 1 H-13 C correlation spectra were acquired on a Varian INOVA 500 MHz ( 1 H) NMR spectrometer with VNMR 6.1C software. Spectra were processed by using MestReC version 4.9.9.8 software.…”

“…13 C spectra were referenced to CD3OD, which was used as an internal standard. 15 N spectra were externally referenced to 2.9 M 15 NH4Cl/1 M DCl in D2O (Cambridge Isotope Laboratories). 1 H-15 N correlation spectra (gHSQC, 256 increments, 4 transients; and gHMBC, 128 increments, 16 transients) were acquired on a Varian VNMRS 600-MHz ( 1 H) NMR spectrometer equipped with a 5-mm HCN AutoX inverse probe.…”

“…NRP synthetases are large, multidomain enzymes catalyzing the assembly of peptides by a thioester-templated mechanism (10). Identification of amino acid building blocks is possible from analysis of the sequence of the NRP synthetase adenylation domains (12)(13)(14)(15). By using this information in combination with the frequently observed colinearity of the biosynthetic genes with the product, prediction of the peptide structure is possible.…”