Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides

Abstract

Pseudomonads are cosmopolitan microbes able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density, and enhance or inhibit growth of competing microbes. However, these valuable metabolites are typically characterized one-molecule-one-microbe at a time instead of inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates discovery of new molecules. Herein, through indexing the Pseudomonas specialized metabolome, we report the molecular networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analog, and a molecular sub-family of cyclic lipopeptides, the bananamides 1, 2, and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies akin to the way newly generated genomic information can be compared to data from public databases.

Increased diversity of peptidic natural products revealed by modification-tolerant database search of mass spectra

“…The +113 Da offset corresponds to insertion of Leucine or Isoleucine residue and matches the recently identified poaeamide B with sequence TISLLSLI*EL and mass 1252.8 Da. Note that a single run of VarQuest instantly achieved the same goal as the time-consuming semi-manual discovery of poaeamide B 25 .…”

“…VarQuest also rediscovered bananamides, a family of PNPs discovered in the same study 25 . Bananamide (referred to as Bananamide-1093) was identified with P -value 4.3 · 10 −10 as a variant of massetolide A (sequence TIS −46 LSLI*EL and mass 1093.7 Da) using a spectrum from P. fluorescens .…”

“…These offsets may correspond to the combination of the amino acid loss (−113 Da for Leucine/Isoleucine and −99 Da for Valine) and hydration (+18 Da). The abundance of such offsets suggests that the recently described phenomenon of amino acid deletions/insertions in NRPs 25 (due to A-domain stuttering and skipping in NRPSs 32 ) may be more prevalent than previously thought. Genome mining efforts typically rule out such events due to the consecutive arrangements of A-domains in NRP synthetases.…”

“…We benchmarked VarQuest on five high-resolution GNPS datasets: Spectra PSEUD (≈400,000 spectra from 260 Pseudomonas isolates 25 ), Spectra STREP 1 (≈200,000 spectra from Streptomyces 7 ), Spectra STREP 2 (≈500,000 spectra from Streptomyces 11, 26 ), Spectra CYANO (≈11 million spectra from Cyanobacteria 27 ), and Spectra GNPS (≈130 million spectra from GNPS 11 ). Details on these datasets are provided in Supplementary Table 1.…”

“…Bananamide (referred to as Bananamide-1093) was identified with P -value 4.3 · 10 −10 as a variant of massetolide A (sequence TIS −46 LSLI*EL and mass 1093.7 Da) using a spectrum from P. fluorescens . While the recent study 25 did not derive the amino acid sequence from this spectrum, it purified and sequenced a related PNP (named bananamide 2) with sequence TL LQ LI* D L (along with C12 3-hydroxy unsaturated acid lipid tail of mass 197 Da) and mass 1105.7 Da (amino acids differing from massetolide A are highlighted except for a change between amino acids I and L with identical masses). While amino acid sequences TIS −46 LSLI*EL and TLLQLI*DL appear to be rather different, note that S −46 LS has the same mass as LQ suggesting that TIS −46 LSLI*EL may actually correspond to TILQLI*EL with a single deleted amino acid as compared to massetolide A.…”