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Most habitats on earth are colonized by diverse bacterial communities, offering ample opportunities for inter-species interactions. While competition for space and nutrients might often dominate such interactions, little is known about whether bacteria can sense competitors and mount specific responses to withstand their attacks. The competition-sensing hypothesis proposes that bacteria can do so through nutrient stress and cell damage cues. Here, we conducted a replicated RNAseq-transcriptomic study to test this hypothesis. We exposedPseudomonas aeruginosato either its own spent medium or to the supernatant of its competitorBurkholderia cenocepacia. Compared to controls, we detected significant changes in the transcriptome ofP. aeruginosathat entail both general responses to spent medium and specific responses to the competitor. Specifically, genes encoding various competitive traits, including the type-VI secretion system, the siderophore pyoverdine, and the toxins phenazines and hydrogen cyanide, were upregulated when exposed to the competitor supernatant. Similarly, several genes coding for stress response and quorum-sensing regulators were overexpressed. Moreover, we found transcriptional responses to vary as a function of iron availability, whereby metabolically more costly responses were launched under iron rich conditions. Altogether, our results reveal nuanced competitive responses ofP. aeruginosawhen exposed toB. cenocepaciasupernatant, integrating both environmental and social cues.

The ability for all organisms to acquire metals from their environment is essential for life. To overcome the metal restriction imposed by the host’s nutritional immunity, bacterial pathogens exploits the use of small high metal affinity molecules called metallophores. Metallophores are first synthetized in the cytoplasm, then secreted into the medium where they sequester the metal. The metal-metallophore complex is then imported into the bacterium following binding to dedicated cell surface receptors. Recently, a new family of metallophores has been discovered in pathogenic bacteria called staphylopine in Staphylococcus aureus and pseudopaline in Pseudomonas aeruginosa. Here, we are expending the molecular understanding of pseudopaline secretion/recovery cycle across the double-membraned envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first revealed that pseudopaline is secreted in a two-step process including export across the inner membrane by the CntI exporter followed by a specific transport across the outer membrane by the MexAB-OprM efflux pump. Such involvement of MexAB-OprM in pseudopaline secretion, reveal a new natural function that extends its spectrum of functions and therefore reasserts its interest as antibacterial target. We then addressed the fate of the recovered metal-loaded pseudopaline by combining in vitro reconstitution experiments using radio-labeled pseudopaline subjected to bacterial lysates, and in vivo phenotyping in absence of pseudopaline transporters. Our data support the existence of a pseudopaline degradation/modification mechanism, possibly involved in metal release following pseudopaline recovery. All together our data allowed us to provide an improved molecular model of secretion, recovery and fate of this important metallophore by P. aeruginosa.IMPORTANCEPseudopaline is a broad spectrum metallophore produced and used by Pseudomonas aeruginosa to supply the bacterium in metal in metal scarce environments. Here we are investigating the pseudopaline transport/recovery cycle across the bacterial envelope. We are first demonstrating that pseudopaline secretion in the medium is achieved by a specific efflux pump, usually dedicated to the release of toxic compounds such as antibiotics, thus revealing a new natural function for this efflux pump reasserting its interest as antibacterial target. Additional experiments also revealing the existence of an intracellular pseudopaline degradation mechanism providing new clues to another obscure step of the pseudopaline cycle which is the intracellular metal liberation from the imported metal-pseudopaline complex. All together our data allowed us to disclose important aspects of the secretion, recovery and fate of this essential molecule used by P. aeruginosa to survive during infections thus constituting new potential targets for antibacterial development.

Nicotianamine (NA) is a metabolite synthesized by all plants, in which it is involved in the homeostasis of different micronutrients such as iron, nickel or zinc. In some plants it also serves as a precursor of phytosiderophores, which are used for extracellular iron scavenging. Previous studies have also established the presence of NA in filamentous fungi and some mosses, whereas an analogue of NA was inferred in an archaeon. More recently, opine-type metallophores with homology to NA were uncovered in bacteria, especially in human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa or Yersinia pestis, synthesizing respectively staphylopine, pseudopaline and yersinopine. Here, we review the current state of knowledge regarding the discovery, biosynthesis, function and regulation of these metallophores. We also discuss the genomic environment of the cntL gene, which is homologous to the plant NA synthase (NAS) gene, and plays a central role in the synthesis of NA-like metallophores. This reveals a large diversity of biosynthetic, export and import pathways. Using sequence similarity networks, we uncovered that these metallophores are widespread in numerous bacteria thriving in very different environments, such as those living at the host–pathogen interface, but also in the soil. We additionally established a phylogeny of the NAS/cntL gene and, as a result, we propose that this gene is an ancient gene and NA, or its derivatives, is an ancient metallophore that played a prominent role in metal acquisition or metal resistance. Indeed, our phylogenetic analysis suggests an evolutionary model where the possibility to synthesize this metallophore was present early in the appearance of life, although it was later lost by most living microorganisms, unless facing metal starvation such as at the host–pathogen interface or in some soils. According to our model, NA then re-emerged as a central metabolite for metal homeostasis in fungi, mosses and all known higher plants.

International audience; Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a pi-pi interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Varroa destructor, an acarian parasite of the Western honey bee Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. The parasite lifecycle occurs in close synchrony with its host development. The females have to discriminate between different developmental stages of the host and trigger an appropriate behavioral response. Many studies have focused on these behavioral aspects, whether it is the choice of a precise host stage or the reproduction of female mites. Behavioral tests often require laboratory settings that are very different from the mite’s environment. Our first experiment was designed to study the impact of the surface of test arena on the mite behavior. We found that plastic from Petri dishes commonly used as test arenas disturbs the female mites and can cause death. We searched for a substrate that does not harm mites and found that gelatin-coated plastic Petri dishes responded to these expectations. We then investigated the host choice behavior of phoretic mites confronted with larval stages of the bee on gelatin-coated arenas to watch if the well-documented orientation towards 5th instar larva was observable in our conditions. Pupal stages were included in the host choice experiments, initially to act as neutral stimuli. As white-eyed pupae were revealed attractive to the mite, several pupal stages were then included in a series of host choice bioassays. These additional experiments tend to show that the positive response to the white-eyed pupa stage depends on cues only delivered by living pupae. Further investigation on the nature and impact of these cues are needed as they could shed light on key signals involved in the parasite lifecycle.