Transporter Gene-Mediated Typing for Detection and Genome Mining of Lipopeptide-Producing Pseudomonas

Pseudomonas lipopeptides (LPs) are involved in diverse ecological functions and have biotechnological application potential associated with their antimicrobial and/or antiproliferative activities. They are synthesized by multimodular nonribosomal peptide synthetases which, together with transport and regulatory proteins, are encoded by large biosynthetic gene clusters (BGCs). These secondary metabolites are classified in distinct families based on the sequence and length of the oligopeptide and size of the macrocycle, if present. The phylogeny of PleB, the MacB-like transporter that is part of a dedicated ATP-dependent tripartite efflux system driving export of Pseudomonas LPs, revealed a strong correlation with LP chemical diversity. As each LP BGC carries its cognate pleB, PleB is suitable as a diagnostic sequence for genome mining, allowing assignment of the putative metabolite to a particular LP family. In addition, pleB proved to be a suitable target gene for an alternative PCR method for detecting LP-producing Pseudomonas sp. and did not rely on amplification of catalytic domains of the biosynthetic enzymes. Combined with amplicon sequencing, this approach enabled typing of Pseudomonas strains as potential producers of a LP belonging to one of the known LP families, underscoring its value for strain prioritization. This finding was validated by chemical characterization of known LPs from three different families secreted by novel producers isolated from the rice or maize rhizosphere, namely, the type strains of Pseudomonas fulva (putisolvin), Pseudomonas zeae (tensin), and Pseudomonas xantholysinigenes (xantholysin). In addition, a new member of the Bananamide family, prosekin, was discovered in the type strain of Pseudomonas prosekii, which is an Antarctic isolate. IMPORTANCE Pseudomonas spp. are ubiquitous bacteria able to thrive in a wide range of ecological niches, and lipopeptides often support their lifestyle but also their interaction with other micro- and macro-organisms. Therefore, the production of lipopeptides is widespread among Pseudomonas strains. Consequently, Pseudomonas lipopeptide research not only affects chemists and microbiologists but also touches a much broader audience, including biochemists, ecologists, and plant biologists. In this study, we present a reliable transporter gene-guided approach for the detection and/or typing of Pseudomonas lipopeptide producers. Indeed, it allows us to readily assess the lipopeptide diversity among sets of Pseudomonas isolates and differentiate strains likely to produce known lipopeptides from producers of potentially novel lipopeptides. This work provides a valuable tool that can also be integrated in a genome mining strategy and adapted for the typing of other specialized metabolites.