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This file contains all supplementary figures of this study (S1-S5).

Iron is important for bacterial growth and survival, as it is a common co-factor in essential enzymes. Although iron is very abundant in the earth crust, its bioavailability is low in most habitats because ferric iron is largely insoluble under aerobic conditions and at neutral pH. Consequently, bacteria have evolved a plethora of mechanisms to solubilize and acquire iron from environmental and host stocks. In this review, I focus on Pseudomonas spp. and first present the main iron uptake mechanisms of this taxa, which involve the direct uptake of ferrous iron via importers, the production of iron-chelating siderophores, the exploitation of siderophores produced by other microbial species, and the use of iron-chelating compounds produced by plants and animals. In the second part of this review, I elaborate on how these mechanisms affect interactions between bacteria in microbial communities, and between bacteria and their hosts. This is important because Pseudomonas spp. live in diverse communities and certain iron-uptake strategies might have evolved not only to acquire this essential nutrient, but also to gain relative advantages over competitors in the race for iron. Thus, an integrative understanding of the mechanisms of iron acquisition and the eco-evolutionary dynamics they drive at the community level might prove most useful to understand why Pseudomonas spp., in particular, and many other bacterial species, in general, have evolved such diverse iron uptake repertoires.

Bacterial infections are often polymicrobial, leading to intricate pathogen-pathogen and pathogen-host interactions. There is increasing interest in studying the molecular basis of pathogen interactions and how such mechanisms impact host morbidity. However, much less is known about the ecological dynamics between pathogens and how they affect virulence and host survival. Here we address these open issues by co-infecting larvae of the insect model hostGalleria mellonellawith one, two, three or four bacterial species, all of which are opportunistic human pathogens. We found that host mortality was always determined by the most virulent species regardless of the number of species and pathogen combinations injected. In certain combinations, the more virulent pathogen simply outgrew the less virulent pathogen. In other combinations, we found evidence for negative interactions between pathogens inside the host, whereby the more virulent pathogen typically won a competition. Taken together, our findings reveal positive associations between a pathogen’s growth inside the host, its competitiveness towards other pathogens, and its virulence. Beyond being generalizable across species combinations, our findings predict that treatments against polymicrobial infections should target the most virulent species to reduce host morbidity, a prediction we validated experimentally.ImportanceThere is increasing evidence that polymicrobial infections are common and that host morbidity is impacted by interactions between the co-infecting pathogens. In this study, we infected the larvae of the insect hostGalleria mellonellawith four opportunistic human pathogens as mono or multispecies bacterial infections in all possible combinations and followed host survival and pathogen interactions over time. We discovered that species followed the exact same rank order in terms of their virulence, their growth inside the host, and their competitive ability against co-infecting pathogens. As consequence of this consistent rank order, we found that the most virulent species determines host survival dynamics in mixed infections. Beyond revealing a generalizable predictive pattern for virulence outcomes, our findings also predict that treatments should target the most virulent species in polymicrobial infections, a prediction we validated experimentally.

Iron is an essential trace element for most organisms. A common way for bacteria to acquire this nutrient is through the secretion of siderophores, which are secondary metabolites that scavenge iron from environmental stocks and deliver it to cells via specific receptors. While there has been tremendous interest in understanding the molecular basis of siderophore synthesis, uptake and regulation, questions about the ecological and evolutionary consequences of siderophore secretion have only recently received increasing attention. In this Review, we outline how eco-evolutionary questions can complement the mechanistic perspective and help to obtain a more integrated view of siderophores. In particular, we explain how secreted diffusible siderophores can affect other community members, leading to cooperative, exploitative and competitive interactions between individuals. These social interactions in turn can spur co-evolutionary arms races between strains and species, lead to ecological dependencies between them and potentially contribute to the formation of stable communities. In brief, this Review shows that siderophores are much more than just iron carriers: they are important mediators of interactions between members of microbial assemblies and the eukaryotic hosts they inhabit.

Pseudomonas aeruginosa populations evolving in cystic fibrosis (CF) lungs, animal infection models, natural environments or in vitro undergo extensive genetic adaption and diversification. A common mutational target is the quorum sensing (QS) regulon, a three-unit regulatory system that controls the expression of a suite of virulence factors and secreted public goods. Three scenarios have been advocated to explain selection for QS mutants, which include (I) disuse of the regulon, (II) cheating on public goods, or (III) modulation of the network. Here, we test these scenarios by examining a set of 61 QS mutants from an experimental evolution study. We observed non-synonymous mutations in all three QS systems – Las, Rhl and PQS. Most Las mutants carried large deletions, resulting in loss of QS function, and the inability to produce QS-regulated traits (scenario I or II). Conversely, phenotypic and gene expression analyses of Rhl mutants support network modulation (scenario III), as these mutants overexpressed the Las and Rhl regulators and showed an altered QS-regulated trait production portfolio. PQS mutants also showed patterns of network modulation (scenario III), spurring strain diversification and phenotypic trade-offs, where the upregulation of certain QS traits is associated with the downregulation of others. Overall, our results indicate that mutations in different QS systems lead to diverging effects on the social portfolio of bacterial populations. These mutations might not only affect the plasticity and diversity of evolved populations but could also impact bacterial fitness and virulence in infections.ImportancePseudomonas aeruginosa uses quorum sensing (QS), a three-unit multi-layered network, to coordinate expression of traits for growth and virulence in the context of infections. Despite its importance for bacterial fitness, the QS regulon appears to be a common mutational target during long-term adaptation of P. aeruginosa in the host, natural environments and experimental evolutions. This raises the questions why such an important regulatory system is under selection and how mutations change the portfolio of QS-regulated traits. Here, we examine a set of 61 naturally evolved mutants to address these questions. We found that mutations involving the master regulator, LasR, resulted in an almost complete breakdown of QS, while mutations in RhlR and PqsR resulted in modulations of the QS regulon, where both the QS regulon structure and the QS-regulated trait portfolio changed. Our work reveals that natural selection drives diversification in QS activity patterns in evolving populations.