Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens.

Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species.

Multifactorial Competition and Resistance in a Two-Species Bacterial System.

Microorganisms exist almost exclusively in interactive multispecies communities, but genetic determinants of the fitness of interacting bacteria, and accessible adaptive pathways, remain uncharacterized. Here, using a two-species system, we studied the antagonism of Pseudomonas aeruginosa against Escherichia coli. Our unbiased genome-scale approach enabled us to identify multiple factors that explained the entire antagonism observed. We discovered both forms of ecological competition-sequestration of iron led to exploitative competition, while phenazine exposure engendered interference competition. We used laboratory evolution to discover adaptive evolutionary trajectories in our system. In the presence of P. aeruginosa toxins, E. coli populations showed parallel molecular evolution and adaptive convergence at the gene-level. The multiple resistance pathways discovered provide novel insights into mechanisms of toxin entry and activity. Our study reveals the molecular complexity of a simple two-species interaction, an important first-step in the application of systems biology to detailed molecular dissection of interactions within native microbiomes.

Bacterial adaptation through loss of function.

The metabolic capabilities and regulatory networks of bacteria have been optimized by evolution in response to selective pressures present in each species' native ecological niche. In a new environment, however, the same bacteria may grow poorly due to regulatory constraints or biochemical deficiencies. Adaptation to such conditions can proceed through the acquisition of new cellular functionality due to gain of function mutations or via modulation of cellular networks. Using selection experiments on transposon-mutagenized libraries of bacteria, we illustrate that even under conditions of extreme nutrient limitation, substantial adaptation can be achieved solely through loss of function mutations, which rewire the metabolism of the cell without gain of enzymatic or sensory function. A systematic analysis of similar experiments under more than 100 conditions reveals that adaptive loss of function mutations exist for many environmental challenges. Drawing on a wealth of examples from published articles, we detail the range of mechanisms through which loss-of-function mutations can generate such beneficial regulatory changes, without the need for rare, specific mutations to fine-tune enzymatic activities or network connections. The high rate at which loss-of-function mutations occur suggests that null mutations play an underappreciated role in the early stages of adaption of bacterial populations to new environments.

Cheater-resistance is not futile.

Cooperative social systems are susceptible to cheating by individuals that reap the benefits of cooperation without incurring the costs. There are various theoretical mechanisms for the repression of cheating and many have been tested experimentally. One possibility that has not been tested rigorously is the evolution of mutations that confer resistance to cheating. Here we show that the presence of a cheater in a population of randomly mutated social amoebae can select for cheater-resistance. Furthermore, we show that this cheater-resistance can be a noble strategy because the resister strain does not necessarily exploit other strains. Thus, the evolution of resisters may be instrumental in preserving cooperative behaviour in the face of cheating.

Mutations in the Staphylococcus aureus Global Regulator CodY Confer Tolerance to an Interspecies Redox-Active Antimicrobial.

Bacteria often exist in multispecies communities where interactions among different species can modify individual fitness and behavior. Although many competitive interactions have been characterized, molecular adaptations that can counter this antagonism and preserve or increase fitness remain underexplored. Here, we characterize the adaptation of Staphylococcus aureus to pyocyanin, a redox-active interspecies antimicrobial produced by Pseudomonas aeruginosa , a co-infecting pathogen frequently isolated from wound and chronic lung infections with S. aureus . Using experimental evolution, we identified mutations in a conserved global transcriptional regulator, CodY, that confer tolerance to pyocyanin and thereby enhance survival of S. aureus . The transcriptional response of a pyocyanin tolerant CodY mutant to pyocyanin indicated a two-pronged defensive response compared to the wild type. Firstly, the CodY mutant strongly suppressed metabolism, by downregulating pathways associated with core metabolism, especially translation-associated genes, upon exposure to pyocyanin. Metabolic suppression via ATP depletion was sufficient to provide comparable protection against pyocyanin to the wild-type strain. Secondly, while both the wild-type and CodY mutant strains upregulated oxidative stress response pathways, the CodY mutant overexpressed multiple stress response genes compared to the wild type. We determined that catalase overexpression was critical to pyocyanin tolerance as its absence eliminated tolerance in the CodY mutant and overexpression of catalase was sufficient to impart tolerance to the wild-type strain. Together, these results suggest that both transcriptional responses likely contribute to pyocyanin tolerance in the CodY mutant. Our data thus provide new mechanistic insight into adaptation toward interbacterial antagonism via altered regulation that facilitates multifaceted protective cellular responses.

Interspecies surfactants serve as public goods enabling surface motility in Pseudomonas aeruginosa.

In most natural environments, bacteria live in polymicrobial communities where secreted molecules from neighboring species alter bacterial behaviors including motility, but such interactions are understudied. Pseudomonas aeruginosa is a motile opportunistic pathogen that exists in diverse multispecies environments such as the soil and is frequently found in human wound and respiratory tract co-infections with other bacteria including Staphylococcus aureus. Here we show that P. aeruginosa can co-opt secreted surfactants from other species for flagellar-based surface motility. We found that exogenous surfactants from S. aureus, other bacteria, and interkingdom species enabled P. aeruginosa to switch from swarming to an alternative surface spreading motility on semi-solid surfaces and allowed for the emergence of surface motility on hard agar where P. aeruginosa was otherwise unable to move. This motility was distinct from the response of other motile bacteria in the presence of exogenous surfactants. Mutant analysis indicated that this P. aeruginosa motility was similar to a previously described mucin-based motility, 'surfing', albeit with divergent regulation. Thus, our study demonstrates that secreted surfactants from the host as well as neighboring bacterial and interkingdom species act as public goods facilitating P. aeruginosa flagella-mediated surfing-like surface motility, thereby allowing it to access different environmental niches.

Cheating by exploitation of developmental prestalk patterning in Dictyostelium discoideum.

The cooperative developmental system of the social amoeba Dictyostelium discoideum is susceptible to exploitation by cheaters-strains that make more than their fair share of spores in chimerae. Laboratory screens in Dictyostelium have shown that the genetic potential for facultative cheating is high, and field surveys have shown that cheaters are abundant in nature, but the cheating mechanisms are largely unknown. Here we describe cheater C (chtC), a strong facultative cheater mutant that cheats by affecting prestalk differentiation. The chtC gene is developmentally regulated and its mRNA becomes stalk-enriched at the end of development. chtC mutants are defective in maintaining the prestalk cell fate as some of their prestalk cells transdifferentiate into prespore cells, but that defect does not affect gross developmental morphology or sporulation efficiency. In chimerae between wild-type and chtC mutant cells, the wild-type cells preferentially give rise to prestalk cells, and the chtC mutants increase their representation in the spore mass. Mixing chtC mutants with other cell-type proportioning mutants revealed that the cheating is directly related to the prestalk-differentiation propensity of the victim. These findings illustrate that a cheater can victimize cooperative strains by exploiting an established developmental pathway.

Efflux pump gene amplifications bypass necessity of multiple target mutations for resistance against dual-targeting antibiotic.

Antibiotics that have multiple cellular targets theoretically reduce the frequency of resistance evolution, but adaptive trajectories and resistance mechanisms against such antibiotics are understudied. Here we investigate these in methicillin resistant Staphylococcus aureus (MRSA) using experimental evolution upon exposure to delafloxacin (DLX), a novel fluoroquinolone that targets both DNA gyrase and topoisomerase IV. We show that selection for coding sequence mutations and genomic amplifications of the gene encoding a poorly characterized efflux pump, SdrM, leads to high DLX resistance, circumventing the requirement for mutations in both target enzymes. In the evolved populations, sdrM overexpression due to genomic amplifications containing sdrM and two adjacent genes encoding efflux pumps results in high DLX resistance, while the adjacent hitchhiking efflux pumps contribute to streptomycin cross-resistance. Further, lack of sdrM necessitates mutations in both target enzymes to evolve DLX resistance, and sdrM thus increases the frequency of resistance evolution. Finally, sdrM mutations and amplifications are similarly selected in two diverse clinical isolates, indicating the generality of this DLX resistance mechanism. Our study highlights that instead of reduced rates of resistance, evolution of resistance to multi-targeting antibiotics can involve alternate high-frequency evolutionary paths, that may cause unexpected alterations of the fitness landscape, including antibiotic cross-resistance.

Experimental systems biology approaches reveal interaction mechanisms in model multispecies communities.

Interactions between microorganisms in multispecies communities are thought to have substantial consequences for the community. Identifying the molecules and genetic pathways that contribute to such interplay is thus crucial to understand as well as modulate community dynamics. Here I focus on recent studies that utilize experimental systems biology techniques to study these phenomena in simplified model microbial communities. These unbiased biochemical and genomic approaches have identified novel interactions and described the underlying genetic and molecular mechanisms. I discuss the insights provided by these studies, describe innovative strategies used to investigate less tractable organisms and environments, and highlight the utility of integrating these and more targeted methods to comprehensively characterize interactions between species in microbial communities.

Extreme Antibiotic Persistence via Heterogeneity-Generating Mutations Targeting Translation.

Antibiotic persistence, the noninherited tolerance of a subpopulation of bacteria to high levels of antibiotics, is a bet-hedging phenomenon with broad clinical implications. Indeed, the isolation of bacteria with substantially increased persistence rates from chronic infections suggests that evolution of hyperpersistence is a significant factor in clinical therapy resistance. However, the pathways that lead to hyperpersistence and the underlying cellular states have yet to be systematically studied. Here, we show that laboratory evolution can lead to increase in persistence rates by orders of magnitude for multiple independently evolved populations of Escherichia coli and that the driving mutations are highly enriched in translation-related genes. Furthermore, two distinct adaptive mutations converge on concordant transcriptional changes, including increased population heterogeneity in the expression of several genes. Cells with extreme expression of these genes showed dramatic differences in persistence rates, enabling isolation of subpopulations in which a substantial fraction of cells are persisters. Expression analysis reveals coherent regulation of specific pathways that may be critical to establishing the hyperpersistence state. Hyperpersister mutants can thus enable the systematic molecular characterization of this unique physiological state, a critical prerequisite for developing antipersistence strategies.IMPORTANCE Bacterial persistence is a fascinating phenomenon in which a small subpopulation of bacteria becomes phenotypically tolerant to lethal antibiotic exposure. There is growing evidence that populations of bacteria in chronic clinical infections develop a hyperpersistent phenotype, enabling a substantially larger subpopulation to survive repeated antibiotic treatment. The mechanisms of persistence and modes of increasing persistence rates remain largely unknown. Here, we utilized experimental evolution to select for Escherichia coli mutants that have more than a thousandfold increase in persistence rates. We discovered that a variety of individual mutations to translation-related processes are causally involved. Furthermore, we found that these mutations lead to population heterogeneity in the expression of specific genes. We show that this can be used to isolate populations in which the majority of bacteria are persisters, thereby enabling systems-level characterization of this fascinating and clinically significant microbial phenomenon.

First among equals: competition between genetically identical cells.

Competition between genetically identical organisms is considered insignificant in evolutionary theory because it is presumed to have little selective consequence. We argue that competition between genetically identical cells could improve the fitness of a multicellular organism by directing fitter cells to the germ line or by eliminating unfit cells, and that cell-competition mechanisms have been conserved in multicellular organisms. We propose that competition between genetically identical or highly similar units could have similar selective advantages at higher organizational levels, such as societies.