Siderophores are soluble or membrane-embedded molecules that bind the oxidized form of iron, Fe(III), and play roles in iron acquisition by microorganisms. Fe(III)-bound siderophores bind to specific receptors that allow microbes to acquire iron. However, certain soil microbes release a compound (pulcherriminic acid, PA) that, upon binding to Fe(III), forms a precipitate (pulcherrimin) that apparently functions by reducing iron availability rather than contributing to iron acquisition. Here, we use Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model to show that PA is involved in a peculiar iron-managing system. The presence of the competitor induces PA production, leading to precipitation of Fe(III) as pulcherrimin, which prevents oxidative stress in B. subtilis by restricting the Fenton reaction and deleterious ROS formation. In addition, B. subtilis uses its known siderophore bacillibactin to retrieve Fe(III) from pulcherrimin. Our findings indicate that PA plays multiple roles by modulating iron availability and conferring protection against oxidative stress during inter-species competition.

Plant growth-promoting rhizobacteria (PGPR) such as the root colonizers Bacillus spp. may be ideal alternatives to chemical crop treatments. This work sought to extend the application of the broadly active PGPR UD1022 to Medicago sativa (alfalfa). Alfalfa is susceptible to many phytopathogens resulting in losses of crop yield and nutrient value. UD1022 was cocultured with four alfalfa pathogen strains to test antagonism. We found UD1022 to be directly antagonistic toward Collectotrichum trifolii, Ascochyta medicaginicola (formerly Phoma medicaginis), and Phytophthora medicaginis, and not toward Fusarium oxysporum f. sp. medicaginis. Using mutant UD1022 strains lacking genes in the nonribosomal peptide (NRP) and biofilm pathways, we tested antagonism against A. medicaginicola StC 306-5 and P. medicaginis A2A1. The NRP surfactin may have a role in the antagonism toward the ascomycete StC 306-5. Antagonism toward A2A1 may be influenced by B. subtilis biofilm pathway components. The B. subtilis central regulator of both surfactin and biofilm pathways Spo0A was required for the antagonism of both phytopathogens. The results of this study indicate that the PGPR UD1022 would be a good candidate for further investigations into its antagonistic activities against C. trifolii, A. medicaginicola, and P. medicaginis in plant and field studies.

Siderophores are soluble or membrane-embedded molecules that play a major role in Fe acquisition by microorganisms. Pulcherriminic acid (PA) is a compound produced by different microbes that sequesters Fe in the precipitated pulcherrimin, but which role in Fe homeostasis remains elusive. Using Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model, we demonstrated that PA is involved in a yet undescribed Fe-managing system. When challenged by a competitor, PA production creates a local Fe(III) source, which can be retrieved via the bacillibactin siderophore produced by B. subtilis. Furthermore, precipitation of Fe(III) as pulcherrimin prevents oxidative stress in bacterial competition by restricting the Fenton reaction and deleterious ROS formation. Together, our findings uncover that PA is at the core of a counterintuitive Fe management strategy that capitalizes on controlled Fe precipitation when challenged by a competitor. This makes PA a unique and multifunction tool in the iron war.

Root-associated microorganisms play an important role in plant health, such as plant growth-promoting rhizobacteria from the Bacillus and Pseudomonas genera. Although bacterial consortia including these two genera would represent a promising avenue to efficient biofertilizer formulation, we observed that B. subtilis root colonization is decreased by the presence of P. fluorescens and P. protegens. To determine if B. subtilis can adapt to the inhibitory effect of Pseudomonas on roots, we conducted adaptative laboratory evolution experiments with B. subtilis in mono-association or co-cultured with P. fluorescens on tomato plant roots. Evolved isolates with various colony morphology and stronger colonization capacity of both tomato plant and A. thaliana roots emerged rapidly from the two evolution experiments. Certain evolved isolates had also a better fitness on root in presence of other Pseudomonas species. Whole genome sequencing revealed that single nucleotide polymorphism (SNPs) in negative biofilm regulator genes ywcC or sinR were found in all independent lineages, suggesting their involvement in enhanced root colonization. These findings provide insights into the molecular mechanisms underlying B. subtilis adaptation to root colonization and highlight the potential of directed evolution to enhance beneficial traits of PGPRs.