THE POLYYNE PROTEGENCIN FROM Pseudomonas protegens HAS ANTIBACTERIAL ACTIVITY AGAINST Azospirillum AND Bacillus, AND IS REQUIRED FOR NORMAL WHEAT ROOT COLONIZATION

SOBRERO, P; PAGNUSSAT, LA; VALVERDE, CLAUDIO; CREUS, C.; MARONICHE GM

Congreso; SAMIGE 2022; 2022

Bacteria belonging to Azospirillum and Pseudomonas genera are ubiquitous members of the plantrhizomicrobiome. When inoculated exogenously, they are able to act as probiotics by improving thegrowth and health of plants. Pseudomonads are particularly interesting as biological control agentsbecause they produce a wide array of natural compounds with antibiotic activity. While studying theinteraction between Azospirillum and Pseudomonas species in vitro, we detected that A. brasilenseSp7 colonies are unable to grow near biofilms developed by P. protegens strains Pf-5 and CHA0. Theresulting strong exclusion halos were produced by a Gac/Rsm-regulated diffusible metabolite. But,unlike what has been previously reported for P. fluorescens F113, we found that neither the antibiotic2,4-diacetylphloroglucinol nor any other of the well-characterized Gac/Rsm-regulated exoproducts ofP. protegens were responsible for the inhibition. Random insertional mutagenesis allowed us toidentify the biosynthetic gene cluster (BGC) of protegencin (PGN), a newly characterized polyyne-typeantibiotic with reported antifungal and antialgal activity, as the putative genetic determinant requiredfor the inhibitory phenotype. P. protegens knock-in mutants in both the BGC pgnD-K and the flankingtranscription factor-coding gene pgnC were unable to inhibit A. brasilense growth. In accordance,extraction of P. protegens culture supernatant with dichloromethane, or ethyl acetate, allowed us torecover the Azospirillum-inhibitory compound from the wild type strain but not from a mutant strainimpaired in PGN production. We confirmed that the expression of a transcriptional pgnD reporterfusion in P. protegens requires a functional pgnC allele and is under the direct or indirect control ofthe Gac-Rsm pathway. Further testing of PGN antibacterial activity on other species indicated thatBacillus sp. are also highly sensitive to this compound. In addition, wheat inoculation assays showedthat P. protegens root colonization is negatively affected when the PGN BGC is non-functional. Weconclude that the P. protegens Gac/Rsm-controlled exoproduct PGN has a previously undescribednarrow-spectrum antibacterial activity, that its production through the pgnD-K BGC is dependent on the putative transcriptional activator PgnC, and that it is required for Azospirillum and Bacillus growthinhibition as well as for normal root colonization, possibly by counteracting other competingrhizobacteria