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Recent advances in next-generation sequencing technologies (NGS) coupled with machine learning have demonstrated the potential of microbiome-based analyses in applied areas such as clinical diagnostics and forensic sciences. Particularly in forensics, microbial markers in biological stains left at a crime scene can provide valuable information for the reconstruction of crime scene cases, as they contain information on bodily origin, the time since deposition, and donor(s) of the stain. Importantly, microbiome-based analyses provide a complementary or an alternative approach to current methods when these are limited or not feasible. Despite the promising results from recent research, microbiome-based stain analyses are not yet employed in routine casework. In this review, we highlight the two main gaps that need to be addressed before we can successfully integrate microbiome-based analyses in applied areas with a special focus on forensic casework: one is a comprehensive assessment of the method's strengths and limitations, and the other is the establishment of a standard operating procedure. For the latter, we provide a roadmap highlighting key decision steps and offering laboratory and bioinformatic workflow recommendations, while also delineating those aspects that require further testing. Our goal is to ultimately facilitate the streamlining of microbiome-based analyses within the existing forensic framework to provide alternate lines of evidence, thereby improving the quality of investigations. [ABSTRACT FROM AUTHOR]

Cellular heterogeneity in clonal bacterial populations is widespread. Division of labor and bet hedging are common adaptive explanations for the function of such heterogeneity. We suggest group-level phenotypes via shareable molecules and variation in cellular vigor as two alternative evolutionary explanations for bacterial cellular heterogeneity. [ABSTRACT FROM AUTHOR]

There has been great progress in understanding how bacterial groups coordinate social actions, such as biofilm formation and public-goods secretion. Less clear is whether the seemingly coordinated group-level responses actually mirror what individual cells do. Here, we use a microscopy approach to simultaneously quantify the investment of individual cells of the bacterium Pseudomonas aeruginosa into two public goods, the siderophores pyochelin and pyoverdine. Using gene expression as a proxy for investment, we initially observe no coordination but high heterogeneity and bimodality in siderophore investment across cells. With increasing cell density, gene expression becomes more homogenized across cells, accompanied by a moderate shift from pyochelin to pyoverdine expression. We find positive associations in the expression of pyochelin and pyoverdine genes across cells, with cell-to-cell variation correlating with cellular metabolic states. Our work suggests that siderophore-mediated signalling aligns behaviour of individuals over time and spurs a coordinated three-phase siderophore investment cycle. Tracking of growth and siderophore gene expression at the single-cell level in growing populations of the bacterium Pseudomonas aeruginosa provides insight into bacterial social behaviours among cells in clonal groups. [ABSTRACT FROM AUTHOR]

A common way for bacteria to cooperate is via the secretion of beneficial public goods (proteases, siderophores, biosurfactants) that can be shared amongst individuals in a group. Bacteria often simultaneously deploy multiple public goods with complementary functions. This raises the question whether natural selection could favour division of labour where subpopulations or species specialize in the production of a single public good, whilst sharing the complementary goods at the group level. Here we use an experimental system, where we mix engineered specialists of the bacterium Pseudomonas aeruginosa that can each only produce one of the two siderophores, pyochelin or pyoverdine and explore the conditions under which specialization can lead to division of labour. When growing pyochelin and pyoverdine specialists at different mixing ratios under different levels of iron limitation, we found that specialists could only successfully complement each other in environments with moderate iron limitation and grow as good as the generalist wildtype but not better. Under more stringent iron limitation, the dynamics in specialist communities was characterized by mutual cheating and with higher proportions of pyochelin producers greatly compromising group productivity. Nonetheless, specialist communities remained stable through negative frequency‐dependent selection. Our work shows that specialization in a bacterial community can be spurred by cheating and does not necessarily result in beneficial division of labour. We propose that natural selection might favour fine‐tuned regulatory mechanisms in generalists over division of labour because the former enables generalists to remain flexible and adequately adjust public good investments in fluctuating environments. [ABSTRACT FROM AUTHOR]

Bacterial infections often involve more than one pathogen. While it is well established that polymicrobial infections can impact disease outcomes, we know little about how pathogens interact and affect each other's behaviour and fitness. Here, we used a microscopy approach to explore interactions between Pseudomonas aeruginosa and six human opportunistic pathogens that often co‐occur in polymicrobial infections: Acinetobacter baumannii, Burkholderia cenocepacia, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. When following growing microcolonies on agarose pads over time, we observed a broad spectrum of species‐specific ecological interactions, ranging from mutualism to antagonism. For example, P. aeruginosa engaged in a mutually beneficial interaction with E. faecium but suffered from antagonism by E. coli. While we found little evidence for active directional growth towards or away from cohabitants, we observed that some pathogens increased growth in double layers in response to competition and that physical forces due to fast colony expansion had a major impact on fitness. Overall, our work provides an atlas of pathogen interactions, highlighting the diversity of potential species dynamics that may occur in polymicrobial infections. We discuss possible mechanisms driving pathogen interactions and offer predictions of how the different ecological interactions could affect virulence. [ABSTRACT FROM AUTHOR]

Cooperation can be favoured through the green‐beard mechanism, where a set of linked genes encodes both a cooperative trait and a phenotypic marker (green beard), which allows carriers of the trait to selectively direct cooperative acts to other carriers. In theory, the green‐beard mechanism should favour cooperation even when interacting partners are totally unrelated at the genome level. Here, we explore such an extreme green‐beard scenario between two unrelated bacterial species—Pseudomonas aeruginosa and Burkholderia cenocepacia, which share a cooperative locus encoding the public good pyochelin (an iron‐scavenging siderophore) and its cognate receptor (green beard) required for iron–pyochelin uptake. We show that pyochelin, when provided in cell‐free supernatants, can be mutually exchanged between species and provide fitness benefits under iron limitation. However, in co‐culture we observed that these cooperative benefits vanished and communities were dominated by P. aeruginosa, regardless of strain background and species starting frequencies. Our results further suggest that P. aeruginosa engages in interference competition to suppress B. cenocepacia, indicating that inter‐species conflict arising from dissimilarities at the genome level overrule the aligned cooperative interests at the pyochelin locus. Thus, green‐beard cooperation is subdued by competition, indicating that interspecific siderophore cooperation is difficult to evolve and to be maintained. [ABSTRACT FROM AUTHOR]

Bacterial communities in the environment and in infections are typically diverse, yet we know little about the factors that determine interspecies interactions. Here, we apply concepts from ecological theory to understand how biotic and abiotic factors affect interaction patterns between the two opportunistic human pathogens Pseudomonas aeruginosa and Staphylococcus aureus, which often cooccur in polymicrobial infections. Specifically, we conducted a series of short- and long-term competition experiments between P. aeruginosa PAO1 (as our reference strain) and three different S. aureus strains (Cowan I, 6850, and JE2) at three starting frequencies and under three environmental (culturing) conditions. We found that the competitive ability of P. aeruginosa strongly depended on the strain background of S. aureus, whereby P. aeruginosa dominated against Cowan I and 6850 but not against JE2. In the latter case, both species could end up as winners depending on conditions. Specifically, we observed strong frequency-dependent fitness patterns, including positive frequency dependence, where P. aeruginosa could dominate JE2 only when common (not when rare). Finally, changes in environmental (culturing) conditions fundamentally altered the competitive balance between the two species in a way that P. aeruginosa dominance increased when moving from shaken to static environments. Altogether, our results highlight that ecological details can have profound effects on the competitive dynamics between coinfecting pathogens and determine whether two species can coexist or invade each others’ populations from a state of rare frequency. Moreover, our findings might parallel certain dynamics observed in chronic polymicrobial infections. [ABSTRACT FROM AUTHOR]

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore overproducing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signalling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies. [ABSTRACT FROM AUTHOR]

Policing occurs in insect, animal and human societies, where it evolved as a mechanism maintaining cooperation. Recently, it has been suggested that policing might even be relevant in enforcing cooperation in much simpler organisms such as bacteria. Here, we used individual‐based modelling to develop an evolutionary concept for policing in bacteria and identify the conditions under which it can be adaptive. We modelled interactions between cooperators, producing a beneficial public good, cheaters, exploiting the public good without contributing to it, and public good‐producing policers that secrete a toxin to selectively target cheaters. We found that toxin‐mediated policing is favoured when (a) toxins are potent and durable, (b) toxins are cheap to produce, (c) cell and public good diffusion is intermediate, and (d) toxins diffuse farther than the public good. Although our simulations identify the parameter space where toxin‐mediated policing can evolve, we further found that policing decays when the genetic linkage between public good and toxin production breaks. This is because policing is itself a public good, offering protection to toxin‐resistant mutants that still produce public goods, yet no longer invest in toxins. Our work thus highlights that not only specific environmental conditions are required for toxin‐mediated policing to evolve, but also strong genetic linkage between the expression of public goods, toxins and toxin resistance is essential for this mechanism to remain evolutionarily stable in the long run. [ABSTRACT FROM AUTHOR]

Background: A common form of cooperation in bacteria is based on the secretion of beneficial metabolites, shareable as public good among cells within a group. Because cooperation can be exploited by "cheating" mutants, which contribute less or nothing to the public good, there has been great interest in understanding the conditions required for cooperation to remain evolutionarily stable. In contrast, much less is known about whether cheats, once fixed in the population, are able to revert back to cooperation when conditions change. Here, we tackle this question by subjecting experimentally evolved cheats of Pseudomonas aeruginosa, partly deficient for the production of the iron-scavenging public good pyoverdine, to conditions previously shown to favor cooperation. Results: Following approximately 200 generations of experimental evolution, we screened 720 evolved clones for changes in their pyoverdine production levels. We found no evidence for the re-evolution of full cooperation, even in environments with increased spatial structure, and reduced costs of public good production - two conditions that have previously been shown to maintain cooperation. In contrast, we observed selection for complete abolishment of pyoverdine production. The patterns of complete trait degradation were likely driven by "cheating on cheats" in unstructured, iron-limited environments where pyoverdine is important for growth, and selection against a maladaptive trait in iron-rich environments where pyoverdine is superfluous. Conclusions: Our study shows that the path to re-evolve public-goods cooperation can be constrained. While a limitation of the number of mutational targets potentially leading to reversion might be one reason for the observed pattern, an alternative explanation is that the selective conditions required for revertants to spread from rarity are much more stringent than those needed to maintain cooperation. [ABSTRACT FROM AUTHOR]

Bacteria secrete a variety of compounds important for nutrient scavenging, competition mediation and infection establishment. While there is a general consensus that secreted compounds can be shared and therefore have social consequences for the bacterial collective, we know little about the physical limits of such bacterial social interactions. Here, we address this issue by studying the sharing of iron-scavenging siderophores between surface-attached microcolonies of the bacterium Pseudomonas aeruginosa. Using single-cell fluorescence microscopy, we show that siderophores, secreted by producers, quickly reach non-producers within a range of 100 µm, and significantly boost their fitness. Producers in turn respond to variation in sharing efficiency by adjusting their pyoverdine investment levels. These social effects wane with larger cell-to-cell distances and on hard surfaces. Thus, our findings reveal the boundaries of compound sharing, and show that sharing is particularly relevant between nearby yet physically separated bacteria on soft surfaces, matching realistic natural conditions such as those encountered in soft tissue infections. [ABSTRACT FROM AUTHOR]

Bacteria typically rely on secreted metabolites, potentially shareable at the community level, to scavenge resources from the environment. The evolution of diffusible, shareable metabolites is, however, difficult to explain because molecules can get lost, or be exploited by cheating mutants. A key question is whether natural selection can act on molecule structure to control loss and shareability. We tested this possibility by collating information on diffusivity properties of 189 secreted iron-scavenging siderophores and the natural habitats occupied by the siderophore-producing species. In line with evolutionary theory, we found that highly diffusible siderophores have preferentially evolved in species living in structured habitats, such as soil and hosts, because structuring can keep producers and their shareable goods together. Poorly diffusible siderophores, meanwhile, have preferentially evolved in species living in unstructured habitats, such as seawater, indicating that these metabolites are less shareable and more likely provide direct benefits to the producers. [ABSTRACT FROM AUTHOR]

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 host Galleria mellonella with 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 first target the most virulent species to reduce host morbidity, a prediction we validated experimentally. [ABSTRACT FROM AUTHOR]

Over the past decade, there has been enormous interest in understanding the great diversity of microbial cooperative behaviors, including communication, group-based swarming, fruiting-body formation, and the secretion of group-beneficial enzymes and food-scavenging molecules. Zhang and Rainey, henceforth Z&R, recently contended that sociomicrobiologists have been overzealous in their casting of microbes as inherently social organisms, and too hasty in interpreting microbial behaviors in a social evolutionary framework. This challenge accompanied a set of experiments in which they revisited one of the best-studied social behaviors in bacteria-the production of diffusible, sharable iron-scavenging siderophore molecules. Z&R posit that their findings challenge the view that siderophore production is a cooperative trait. Here, we demonstrate that their arguments are flawed, and stem from both technical mistakes and misunderstandings of social evolution theory. [ABSTRACT FROM AUTHOR]

Adaptations by hosts in response to parasitism are generally believed to reduce the susceptibility of the adapted individual. However, recent work on Escherichia coli showed that bacteria can fight deadly phage attacks by committing altruistic suicide upon infection, in order to prevent parasite transmission to nearby relatives. Here, we compare the efficiency of suicidal host defense with individual-based resistance. We show that in unstructured environments suicidal host defense is futile since suicide cannot preferentially protect relatives, whereas individual-based resistance is highly efficient in defying phages. In contrast, we found that in structured environments suicidal host defense and individual-based resistance were both efficient in withstanding phages, with the latter type performing slightly better. We propose that the putative lower efficiency of suicidal host defense might be compensated by the fact that suicidal systems usually do not bear pleiotropic costs of resistance, as it is usually the case for individual-based resistance mechanisms. [ABSTRACT FROM AUTHOR]

In social groups where relatedness among interacting individuals is low, cooperation can often only be maintained through mechanisms that repress competition among group members. Repression-of-competition mechanisms, such as policing and punishment, seem to be of particular importance in human societies, where cooperative interactions often occur among unrelated individuals. In line with this view, economic games have shown that the ability to punish defectors enforces cooperation among humans. Here, I examine a real-world example of a repression-of-competition system, the police institutions common to modern human societies. Specifically, I test evolutionary policing theory by comparing data on policing effort, per capita crime rate, and similarity (used as a proxy for genetic relatedness) among citizens across the 26 cantons of Switzerland. This comparison revealed full support for all three predictions of evolutionary policing theory. First, when controlling for policing efforts, crime rate correlated negatively with the similarity among citizens. This is in line with the prediction that high similarity results in higher levels of cooperative self-restraint (i.e. lower crime rates) because it aligns the interests of individuals. Second, policing effort correlated negatively with the similarity among citizens, supporting the prediction that more policing is required to enforce cooperation in low-similarity societies, where individuals' interests diverge most. Third, increased policing efforts were associated with reductions in crime rates, indicating that policing indeed enforces cooperation. These analyses strongly indicate that humans respond to cues of their social environment and adjust cheating and policing behaviour as predicted by evolutionary policing theory. [ABSTRACT FROM AUTHOR]

While adaptive adjustment of sex ratio in the function of colony kin structure and food availability commonly occurs in social Hymenoptera, long-term studies have revealed substantial unexplained between-year variation in sex ratio at the population level. In order to identify factors that contribute to increased between-year variation in population sex ratio, we conducted a comparative analysis across 47 Hymenoptera species differing in their breeding system. We found that between-year variation in population sex ratio steadily increased as one moved from solitary species, to primitively eusocial species, to single-queen eusocial species, to multiple-queen eusocial species. Specifically, between-year variation in population sex ratio was low (6.6% of total possible variation) in solitary species, which is consistent with the view that in solitary species, sex ratio can vary only in response to fluctuations in ecological factors such as food availability. In contrast, we found significantly higher (19.5%) between- year variation in population sex ratio in multiple-queen eusocial species, which supports the view that in these species, sex ratio can also fluctuate in response to temporal changes in social factors such as queen number and queen-worker control over sex ratio, as well as factors influencing caste determination. The simultaneous adjustment of sex ratio in response to temporal fluctuations in ecological and social factors seems to preclude the existence of a single sex ratio optimum. The absence of such an optimum may reflect an additional cost associated with the evolution of complex breeding systems in Hymenoptera societies. [ABSTRACT FROM AUTHOR]

Numerous theoretical studies have investigated how limited dispersal may provide an explanation for the evolution of cooperation, by leading to interactions between relatives. However, despite considerable theoretical attention, there has been a lack of empirical tests. In this article, we test how patterns of dispersal influence the evolution of cooperation, using iron-scavenging in the bacterium Pseudomonas aeruginosa as our cooperative trait. We found that relatively limited dispersal does not favor cooperation. The reason for this is that although limited dispersal increases the relatedness between interacting individuals, it also leads to increased local competition for resources between relatives. This result supports Taylor's prediction that in the simplest possible scenario, the effects of increased relatedness and local competition exactly cancel out. In contrast, we show that one way for cooperation to be favored is if individuals disperse in groups (budding dispersal), because this maintains high relatedness while reducing local competition between relatives (relatively global competition). [ABSTRACT FROM AUTHOR]

Due to their haplo-diploid sex determination system and the resulting conflict over optimal sex allocation between queens and workers, social Hymenoptera have become important model species to study variation in sex allocation. While many studies indeed reported sex allocation to be affected by social factors such as colony kin structure or queen number, others, however, found that sex allocation was impacted by ecological factors such as food availability. In this paper, we present one of the rare studies that simultaneously investigated the effects of social and ecological factors on social insect nest reproductive parameters (sex and reproductive allocation, nest productivity) across several years. We found that the sex ratio was extremely male biased in a polygynous (multiple queens per nest) population of the ant Formica exsecta. Nest-level sex allocation followed the pattern predicted by the queen-replenishment hypothesis, which holds that gynes (new queens) should only be produced and recruited in nests with low queen number (i.e. reduced local resource competition) to ensure nest survival. Accordingly, queen number (social factor) was the main determinant on whether a nest produced gynes or males. However, ecological factors had a large impact on nest productivity and therefore on a nest's resource pool, which determines the degree of local resource competition among co-breeding queens and at what threshold in queen number nests should switch from male to gyne production. Additionally, our genetic data revealed that gynes are recruited back to their parental nests after mating. However, our genetic data are also consistent with some adult queens dispersing on foot from nests where they were produced to nests that never produced queens. As worker production is reduced in gyne-producing nests, queen migration might be offset by workers moving in the other direction, leading to a nest network characterized by reproductive division of labour. Altogether our study shows that both, social and ecological factors can influence long-term nest reproductive strategies in insect societies. [ABSTRACT FROM AUTHOR]

The theory of inclusive fitness provides a powerful explanation for reproductive altruism in social insects, whereby workers gain inclusive fitness benefit by rearing the brood of related queens. Some ant species, however, have unicolonial population structures where multiple nests, each containing numerous queens, are interconnected and individuals move freely between nests. In such cases, nestmate relatedness values may often be indistinguishable from zero, which is problematic for inclusive fitness-based explanations of reproductive altruism. We conducted a detailed population genetic study in the polygynous ant Formica exsecta, which has been suggested to form unicolonial populations in its native habitat. Analyses based on adult workers indeed confirmed a genetic structuring consistent with a unicolonial population structure. However, at the population level the genetic structuring inferred from worker pupae was not consistent with a unicolonial population structure, but rather suggested a multicolonial population structure of extended family-based nests. These contrasting patterns suggest limited queen dispersal and free adult worker dispersal. That workers indeed disperse as adults was confirmed by mark–recapture measures showing consistent worker movement between nests. Together, these findings describe a new form of social organization, which possibly also characterizes other ant species forming unicolonial populations in their native habitats. Moreover, the genetic analyses also revealed that while worker nestmate relatedness was indistinguishable from zero at a small geographical scale, it was significantly positive at the population level. This highlights the need to consider the relevant geographical scale when investigating the role of inclusive fitness as a selective force maintaining reproductive altruism. [ABSTRACT FROM AUTHOR]

A key feature differentiating animal societies is the partitioning of reproduction among breeders. We studied how reproduction is partitioned among nestmate queens of the ant Formica exsecta in laboratory colonies. In polygynous (multiple-queen) colonies, queens can increase their reproductive success by laying more eggs or by increasing the proportion of eggs that develop into reproductive individuals instead of workers. We found that reproductive skew among queens for egg production was low, but that 84% of all queens contributed exclusively to one type of brood, either males or workers (no new queens are produced in the laboratory). Furthermore, our data revealed that the degree of reproductive specialization among queens significantly increased during brood development. Contrary to predictions of most reproductive skew models, the extent of reproductive skew was not associated with relatedness among co-breeding queens. We also found no association between the pattern of queen specialization and relatedness between queens and their mates. Such an association would occur if queens mated to related males specialize in male production to avoid the cost of inbreeding. Altogether, our findings show a yet undescribed pattern of reproductive specialization among nestmate queens in ants and emphasizes the need to investigate reproductive contributions of queens for each type of offspring separately. [Copyright &y& Elsevier]

The relationship between social rank and reproductive success is one of the key questions for understanding differences in primate social group structures. We determined the paternity of 18 infants in a social group of Barbary macaques (Macaca sylvanus) born over a period of 6 yr in the provisioned, free-ranging colony in Gibraltar. We successfully used 13 pairs of primers of variable microsatellite loci to amplify DNA from blood and hair samples and applied the computer programs CERVUS 2.0 and KINSHIP 1.3 to assign paternity to 13 candidate males. We collected data for 19 females that had given birth to 66 infants over a period of 7 yr. We used paternity analyses and female birth records to test the hypothesis that social rank is correlated with reproductive success. Results showed that numbers of paternities and maternities were equally distributed among all reproducing individuals in the social group regardless of rank. Subadult males reproduced as often as adult males. High-ranking females did not start to reproduce earlier than low-ranking females. Interestingly, there was a tendency toward a positive correlation between the ranks of mothers and the ranks of the corresponding fathers. It might be concluded either that a correlation between social rank and reproductive success is generally absent in Barbary macaques or that artificially favorable environmental conditions in Gibraltar preclude any correlation between social rank and reproductive success. [ABSTRACT FROM AUTHOR]

Microbial secondary metabolites are a rich source for pharmaceutical discoveries and play crucial ecological functions. While tools exist to identify secondary metabolite clusters in genomes, precise sequencetofunction mapping remains challenging because neither function nor substrate specificity of biosynthesis enzymes can accurately be predicted. Here, we developed a knowledgeguided bioinformatic pipeline to solve these issues. We analyzed 1928 genomes of Pseudomonas bacteria and focused on iron-scavenging pyoverdines as model metabolites. Our pipeline predicted 188 chemically different pyoverdines with nearly 100% structural accuracy and the presence of 94 distinct receptor groups required for the uptake of ironloaded pyoverdines. Our pipeline unveils an enormous yet overlooked diversity of siderophores (151 new structures) and receptors (91 new groups). Our approach, combining feature sequence with phylogenetic approaches, is extendable to other metabolites and microbial genera, and thus emerges as powerful tool to reconstruct bacterial secondary metabolism pathways based on sequence data. [ABSTRACT FROM AUTHOR]

Microbial secondary metabolites represent a rich source for drug discovery, plant protective agents, and biotechnologically relevant compounds. Among them are siderophores, iron-chelating molecules, that show a great influence on bacterial community assembly and the potential to control pathogen invasions. One of such a siderophore is pyoverdine that is produced by fluorescent Pseudomonas members and consists of different peptide chains specific to each bacterial species. The identification and structural elucidation of such suites of siderophores remain widely underexplored as general high-throughput analytical protocols are missing. Therefore, a dedicated method was established allowing a rapid localization and structural elucidation of pyoverdines. Liquid bacterial culture samples were purified by an easy small-scale solid-phase extraction (SPE). Ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) separated highly polar pyoverdines and their derivatives. All ion fragmentation (AIF) generated mass spectra containing the characteristic fragments of the biological precursor of pyoverdine, ferribactin. This led to the revelation of the mass of secreted pyoverdines. Targeted MS/MS experiments at multiple collision energies accomplished the full structure elucidation of the pyoverdine peptide chain. A mass calculator and a fragmentation predictor facilitated greatly the interpretation of MS/MS spectra by providing accurate masses for a straightforward comparison of measured and theoretical values. The method was successfully validated using four well-known pyoverdines with various peptide chains. Finally, the applicability was proven by the analysis of 13 unknown pyoverdines secreted by sampled bacterial cultures. Among these, 4 novel pyoverdine peptide chains were discovered and are herein reported for the first time. [ABSTRACT FROM AUTHOR]

Most habitats host diverse bacterial communities, offering opportunities for inter-species interactions. While competition might often dominate such interactions, little is known about whether bacteria can sense competitors and mount adequate responses. The competition sensing hypothesis proposes that bacteria can use cues such as nutrient stress and cell damage to prepare for battle. Here, we tested this hypothesis by measuring transcriptome changes in Pseudomonas aeruginosa exposed to the supernatant of its competitor Burkholderia cenocepacia. We found that P. aeruginosa exhibited significant growth-medium-dependent transcriptome changes in response to competition. In an iron-rich medium, P. aeruginosa upregulated genes encoding the type-VI secretion system and the siderophore pyoverdine, whereas genes encoding phenazine toxins and hydrogen cyanide were upregulated under iron-limited conditions. Moreover, general stress response and quorum sensing regulators were upregulated upon supernatant exposure. Altogether, our results reveal nuanced competitive responses of P. aeruginosa when confronted with B. cenocepacia supernatant, integrating both environmental and social cues. Transcriptomic data show that the bacterium Pseudomonas aeruginosa reacts to secreted compounds from its competitor Burkholderia cenocepacia by upregulating a set of competitive traits that vary as a function of iron availability in the medium. [ABSTRACT FROM AUTHOR]

The zebrafish Danio rerio has become a popular model host to explore disease pathology caused by infectious agents. A main advantage is its transparency at an early age, which enables live imaging of infection dynamics. While multispecies infections are common in patients, the zebrafish model is rarely used to study them, although the model would be ideal for investigating pathogen-pathogen and pathogen-host interactions. This may be due to the absence of an established multispecies infection protocol for a defined organ and the lack of suitable image analysis pipelines for automated image processing. To address these issues, we developed a protocol for establishing and tracking single and multispecies bacterial infections in the inner ear structure (otic vesicle) of the zebrafish by imaging. Subsequently, we generated an image analysis pipeline that involved deep learning for the automated segmentation of the otic vesicle, and scripts for quantifying pathogen frequencies through fluorescence intensity measures. We used Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, three of the difficult-to-treat ESKAPE pathogens, to show that our infection protocol and image analysis pipeline work both for single pathogens and pairwise pathogen combinations. Thus, our protocols provide a comprehensive toolbox for studying single and multispecies infections in real-time in zebrafish. [ABSTRACT FROM AUTHOR]

Microbial invasions can compromise ecosystem services and spur dysbiosis and disease in hosts. Nevertheless, the mechanisms determining invasion outcomes often remain unclear. Here, we examine the role of iron‐scavenging siderophores in driving invasions of Pseudomonas aeruginosa into resident communities of environmental pseudomonads. Siderophores can be 'public goods' by delivering iron to individuals possessing matching receptors; but they can also be 'public bads' by withholding iron from competitors lacking these receptors. Accordingly, siderophores should either promote or impede invasion, depending on their effects on invader and resident growth. Using supernatant feeding and invasion assays, we show that invasion success indeed increased when the invader could use its siderophores to inhibit (public bad) rather than stimulate (public good) resident growth. Conversely, invasion success decreased the more the invader was inhibited by the residents' siderophores. Our findings identify siderophores as a major driver of invasion dynamics in bacterial communities under iron‐limited conditions. [ABSTRACT FROM AUTHOR]

Bacteria often cooperate by secreting molecules that can be shared as public goods between cells. Because the production of public goods is subject to cheating by mutants that exploit the good without contributing to it, there has been great interest in elucidating the evolutionary forces that maintain cooperation. However, little is known about how bacterial cooperation evolves under conditions where cheating is unlikely to be of importance. Here we use experimental evolution to follow changes in the production of a model public good, the iron‐scavenging siderophore pyoverdine, of the bacterium Pseudomonas aeruginosa. After 1200 generations of evolution in nine different environments, we observed that cheaters only reached high frequency in liquid medium with low iron availability. Conversely, when adding iron to reduce the cost of producing pyoverdine, we observed selection for pyoverdine hyperproducers. Similarly, hyperproducers also spread in populations evolved in highly viscous media, where relatedness between interacting individuals is increased. Whole‐genome sequencing of evolved clones revealed that hyperproduction is associated with mutations involving genes encoding quorum‐sensing communication systems, while cheater clones had mutations in the iron‐starvation sigma factor or in pyoverdine biosynthesis genes. Our findings demonstrate that bacterial social traits can evolve rapidly in divergent directions, with particularly strong selection for increased levels of cooperation occurring in environments where individual dispersal is reduced, as predicted by social evolution theory. Moreover, we establish a regulatory link between pyoverdine production and quorum‐sensing, showing that increased cooperation with respect to one trait (pyoverdine) can be associated with the loss (quorum‐sensing) of another social trait. [ABSTRACT FROM AUTHOR]

In heterogenous, spatially structured habitats, individuals within populations can become adapted to the prevailing conditions in their local environment. Such local adaptation has been reported for animals and plants, and for pathogens adapting to hosts. There is increasing interest in applying the concept of local adaptation to microbial populations, especially in the context of microbe–microbe interactions. Here, we tested whether cooperation and cheating on cooperation can spur patterns of local adaptation in soil and pond communities of Pseudomonas bacteria, collected across a geographical scale of 0.5 to 50 m. We focussed on the production of pyoverdines, a group of secreted iron‐scavenging siderophores that often differ among pseudomonads in their chemical structure and the receptor required for their uptake. A combination of supernatant‐feeding and competition assays between isolates from four distance categories revealed tremendous variation in the extent to which pyoverdine non‐ and low‐producers can benefit from pyoverdines secreted by producers. However, this variation was not explained by geographical distance, but primarily depended on the phylogenetic relatedness between interacting isolates. A notable exception occurred in local pond communities, where the effect of phylogenetic relatedness was eroded in supernatant assays, probably due to the horizontal transfer of receptor genes. While the latter result could be a signature of local adaptation, our results overall indicate that common ancestry and not geographical distance is the main predictor of siderophore‐mediated social interactions among pseudomonads. [ABSTRACT FROM AUTHOR]

The author comments on the implications of cheat invasion on the bacterial traits of lung infection of patients in Zurich, Switzerland. The topics discussed include the conventional understanding of the natural selection principle of English geologist and naturalist Charles Darwin, the reasons why organisms lose bacterial traits, and the Pseudomonas aeruginosa's long-term evolution in cystic fibrosis lungs.

Abstract: Phenotypic plasticity in response to competition is a well‐described phenomenon in higher organisms. Here, we show that also bacteria have the ability to sense the presence of competitors and mount fine‐tuned responses to match prevailing levels of competition. In our experiments, we studied interspecific competition for iron between the bacterium Pseudomonas aeruginosa (PA) and its competitor Burkholderia cenocepacia (BC). We focused on the ability of PA to phenotypically adjust the production of pyoverdine, an iron‐scavenging siderophore. We found that PA upregulates pyoverdine production early on during competition under condition of low iron availability. This plastic upregulation was fine‐tuned in response to the level of competition imposed by BC, and seems to confer a relative fitness benefit to PA in the form of an earlier initiation of growth. At later time points, however, PA showed reduced growth in mixed compared to monoculture, suggesting that competitive responses are costly. Altogether, our results demonstrate that phenotypic plasticity in siderophore production plays an important role in interspecific competition for iron. Upregulating siderophore production may be a powerful strategy to lock iron away from competing species, and to reserve this nutrient for strain members possessing the compatible receptor for uptake. [ABSTRACT FROM AUTHOR]

Summary: Phenotypic noise and social evolution in microbes have recently attracted huge interdisciplinary interest. A new study highlights the interaction between these phenomena and its implications for self-destructive cooperation. [Copyright &y& Elsevier]

Bacteria frequently cooperate by sharing secreted metabolites such as enzymes and siderophores. The expression of such 'public good' traits can be interdependent, and studies on laboratory systems have shown that trait linkage affects eco-evolutionary dynamics within bacterial communities. Here, we examine whether linkage among social traits occurs in natural habitats by examining investment levels and correlations between five public goods (biosurfactants, biofilm components, proteases, pyoverdines and toxic compounds) in 315 Pseudomonas isolates from soil and freshwater communities. Our phenotypic assays revealed that (i) social trait expression profiles varied dramatically; (ii) correlations between traits were frequent, exclusively positive and sometimes habitat-specific; and (iii) heterogeneous (specialised) trait repertoires were rarer than homogeneous (unspecialised) repertoires. Our results show that most isolates lie on a continuum between a 'social' type producing multiple public goods, and an 'asocial' type showing low investment into social traits. This segregation could reflect local adaptation to different microhabitats, or emerge from interactions between different social strategies. In the latter case, our findings suggest that the scope for competition among unspecialised isolates exceeds the scope for mutualistic exchange of different public goods between specialised isolates. Overall, our results indicate that complex interdependencies among social traits shape microbial lifestyles in nature. [ABSTRACT FROM AUTHOR]

Antibiotics are losing efficacy due to the rapid evolution and spread of resistance. Treatments targeting bacterial virulence factors have been considered as alternatives because they target virulence instead of pathogen viability, and should therefore exert weaker selection for resistance than conventional antibiotics. However, antivirulence treatments rarely clear infections, which compromises their clinical applications. Here, we explore the potential of combining antivirulence drugs with antibiotics against the opportunistic human pathogen Pseudomonas aeruginosa. We combined two antivirulence compounds (gallium, a siderophore quencher, and furanone C-30, a quorum sensing [QS] inhibitor) together with four clinically relevant antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) in 9×9 drug concentration matrices. We found that drug-interaction patterns were concentration dependent, with promising levels of synergies occurring at intermediate drug concentrations for certain drug pairs. We then tested whether antivirulence compounds are potent adjuvants, especially when treating antibiotic resistant (AtbR) clones. We found that the addition of antivirulence compounds to antibiotics could restore growth inhibition for most AtbR clones, and even abrogate or reverse selection for resistance in five drug combination cases. Molecular analyses suggest that selection against resistant clones occurs when resistance mechanisms involve restoration of protein synthesis, but not when efflux pumps are up-regulated. Altogether, our work provides a first systematic analysis of antivirulence-antibiotic combinatorial treatments and suggests that such combinations have the potential to be both effective in treating infections and in limiting the spread of antibiotic resistance. This microbiology study reveals that compounds that disarm bacterial pathogens by targeting their virulence factors can be potent adjuvants to antibiotics, as they remain active against—and can reduce the selective advantage of—antibiotic resistant strains. [ABSTRACT FROM AUTHOR]

Explaining how cooperation can persist in the presence of cheaters, exploiting the cooperative acts, is a challenge for evolutionary biology. Microbial systems have proved extremely useful to test evolutionary theory and identify mechanisms maintaining cooperation. One of the most widely studied system is the secretion and sharing of iron‐scavenging siderophores by Pseudomonas bacteria, with many insights gained from this system now being considered as hallmarks of bacterial cooperation. Here, we introduce siderophore secretion by the bacterium Burkholderia cenocepacia H111 as a novel parallel study system, and show that this system behaves differently. For ornibactin, the main siderophore of this species, we discovered a novel mechanism of how cheating can be prevented. Particularly, we found that secreted ornibactin cannot be exploited by ornibactin‐defective mutants because ornibactin receptor and synthesis genes are co‐expressed from the same operon, such that disruptive mutations in synthesis genes compromise receptor availability required for siderophore uptake and cheating. For pyochelin, the secondary siderophore of this species, we found that cheating was possible, but the relative success of cheaters was positive frequency dependent, thus diametrically opposite to the Pseudomonas and other microbial systems. Altogether, our results highlight that expanding our repertoire of microbial study systems leads to new discoveries and suggest that there is an enormous diversity of social interactions out there in nature, and we might have only looked at the tip of the iceberg so far. [ABSTRACT FROM AUTHOR]

How unicellular organisms optimize the production of compounds is a fundamental biological question. While it is typically thought that production is optimized at the individual‐cell level, secreted compounds could also allow for optimization at the group level, leading to a division of labor where a subset of cells produces and shares the compound with everyone. Using mathematical modeling, we show that the evolution of such division of labor depends on the cost function of compound production. Specifically, for any trait with saturating benefits, linear costs promote the evolution of uniform production levels across cells. Conversely, production costs that diminish with higher output levels favor the evolution of specialization–especially when compound shareability is high. When experimentally testing these predictions with pyoverdine, a secreted iron‐scavenging compound produced by Pseudomonas aeruginosa, we found linear costs and, consistent with our model, detected uniform pyoverdine production levels across cells. We conclude that for shared compounds with saturating benefits, the evolution of division of labor is facilitated by a diminishing cost function. More generally, we note that shifts in the level of selection from individuals to groups do not solely require cooperation, but critically depend on mechanistic factors, including the distribution of compound synthesis costs. [ABSTRACT FROM AUTHOR]

Summary: Many bacteria rely on the secretion of siderophores to scavenge iron from the environment. Laboratory studies revealed that abiotic and biotic factors together determine how much siderophores bacteria make, and whether siderophores can be exploited by non‐producing cheaters or be deployed by producers to inhibit competitors. Here, we explore whether these insights apply to natural communities, by comparing the production of the siderophore pyoverdine among 930 Pseudomonas strains from 48 soil and pond communities. We found that pH, iron content, carbon concentration and community diversity determine pyoverdine production levels, and the extent to which strains are either stimulated or inhibited by heterologous (non‐self) pyoverdines. While pyoverdine non‐producers occurred in both habitats, their prevalence was higher in soils. Environmental and genetic analyses suggest that non‐producers can evolve as cheaters, exploiting heterologous pyoverdine, but also due to pyoverdine disuse in environments with increased iron availability. Overall, we found that environmental factors explained between‐strain variation in pyoverdine production much better in soils than in ponds, presumably because high strain mixing in ponds impedes local adaption. Our study sheds light on the complexity of natural bacterial communities, and provides first insights into the multivariate nature of siderophore‐based iron acquisition and competition among environmental pseudomonads. [ABSTRACT FROM AUTHOR]

Given the rise of bacterial resistance against antibiotics, we urgently need alternative strategies to fight infections. Some propose we should disarm rather than kill bacteria, through targeted disruption of their virulence factors. It is assumed that this approach (i) induces weak selection for resistance because it should only minimally impact bacterial fitness, and (ii) is specific, only interfering with the virulence factor in question. Given that pathogenicity emerges from complex interactions between pathogens, hosts and their environment, such assumptions may be unrealistic. To address this issue in a test case, we conducted experiments with the opportunistic human pathogen Pseudomonas aeruginosa, where we manipulated the availability of a virulence factor, the iron-scavenging pyoverdine, within the insect host Galleria mellonella. We observed that pyoverdine availability was not stringently predictive of virulence and affected bacterial fitness in nonlinear ways. We show that this complexity could partly arise because pyoverdine availability affects host responses and alters the expression of regulatorily linked virulence factors. Our results reveal that virulence factor manipulation feeds back on pathogen and host behaviour, which in turn affects virulence. Our findings highlight that realizing effective and evolutionarily robust antivirulence therapies will ultimately require deeper engagement with the intrinsic complexity of host-pathogen systems. [ABSTRACT FROM AUTHOR]

A hallmark of bacterial sociality is that groups can coordinate cooperative actions through a cell-to-cell communication process called quorum sensing (QS). QS regulates key bacterial phenotypes such as virulence in infections and digestion of extracellular compounds in the environment. Although QS responses are typically studied as group-level phenotypes, it is unclear whether individuals coordinate their actions at the single-cell level or whether group phenotypes simply reflect the sum of their noisy members. Here, we studied the behavior of Pseudomonas aeruginosa individuals by tracking their temporal commitments to the two intertwined Las and Rhl-QS systems, from low to high population density. Using chromosomally integrated fluorescent gene reporters, we found that QS gene expression (signal, receptor, and cooperative exoproduct) was noisy with heterogeneity peaking during the build-up phase of QS. Moreover, we observed the formation of discrete subgroups of cells that transiently segregate into two gene expression states: low Las-receptor expressers that instantly activate exoproduct production and high Las-receptor expressers with delayed exoproduct production. Later, gene expression activities converged with all cells fully committing to QS. We developed general mathematical models to show that gene expression segregation can mechanistically be spurred by molecular resource limitations during the initiation phase of regulatory cascades such as QS. Moreover, our models indicate that gene expression segregation across cells can operate as a built-in brake enabling a temporary bet-hedging strategy in unpredictable environments. Altogether, our work reveals that studying the behavior of bacterial individuals is key to understanding emergent collective actions at the group level. [Display omitted] • P. aeruginosa bacteria show high heterogeneity in their communication activities • Cells in clonal groups temporarily split into instant and delayed communicators • Limitation of regulatory resources can induce the split in communication activities • Communication segregation can serve as a temporary bet-hedging strategy Using double gene expression reporter assays, Jayakumar et al. show that cells of the bacterium Pseudomonas aeruginosa express high heterogeneity in quorum-sensing communication activities. Cells in clonal groups temporarily split into instant and delayed communicators, possibly reflecting a bet-hedging strategy in fluctuating environments. [ABSTRACT FROM AUTHOR]

Bacteria often possess multiple siderophore-based iron uptake systems for scavenging this vital resource from their environment. However, some siderophores seem redundant, because they have limited iron-binding efficiency and are seldom expressed under iron limitation. Here, we investigate the conundrum of why selection does not eliminate this apparent redundancy. We focus on Pseudomonas aeruginosa, a bacterium that can produce two siderophores—the highly efficient but metabolically expensive pyoverdine, and the inefficient but metabolically cheap pyochelin. We found that the bacteria possess molecular mechanisms to phenotypically switch from mainly producing pyoverdine under severe iron limitation to mainly producing pyochelin when iron is only moderately limited. We further show that strains exclusively producing pyochelin grew significantly better than strains exclusively producing pyoverdine under moderate iron limitation, whereas the inverse was seen under severe iron limitation. This suggests that pyochelin is not redundant, but that switching between siderophore strategies might be beneficial to trade off efficiencies versus costs of siderophores. Indeed, simulations parameterized from our data confirmed that strains retaining the capacity to switch between siderophores significantly outcompeted strains defective for one or the other siderophore under fluctuating iron availabilities. Finally, we discuss how siderophore switching can be viewed as a form of collective decision-making, whereby a coordinated shift in behaviour at the group level emerges as a result of positive and negative feedback loops operating among individuals at the local scale. [ABSTRACT FROM AUTHOR]

An abstract of the study "Altruism Can Evolve When Relatedness Is Low: Evidence From Bacteria Committing Suicide Upon Phage Infection," by Dominik Refardt, et al. is presented.

Summary Organisms as simple as bacteria can engage in complex collective actions, such as group motility and fruiting body formation. Some of these actions involve a division of labor, where phenotypically specialized clonal subpopulations or genetically distinct lineages cooperate with each other by performing complementary tasks. Here, we combine experimental and computational approaches to investigate potential benefits arising from division of labor during biofilm matrix production. We show that both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation in the soil bacterium Bacillus subtilis . In this species, biofilm matrix consists of two major components, exopolysaccharides (EPSs) and TasA. We observed that clonal groups of B. subtilis phenotypically segregate into three subpopulations composed of matrix non-producers, EPS producers, and generalists, which produce both EPSs and TasA. This incomplete phenotypic specialization was outperformed by a genetic division of labor, where two mutants, engineered as specialists, complemented each other by exchanging EPSs and TasA. The relative fitness of the two mutants displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency reaching a stable equilibrium at 30% TasA producers, corresponding exactly to the population composition where group productivity is maximized. Using individual-based modeling, we show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix compounds generate for the collective and that genetic division of labor can be favored when it breaks metabolic constraints associated with the simultaneous production of two matrix components. [ABSTRACT FROM AUTHOR]