Evolutionary consequences of bacterial resistance to a flagellotropic phage. - Related Documents




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936101.0000Evolutionary consequences of bacterial resistance to a flagellotropic phage. Bacteria often rapidly evolve resistance to bacteriophages (phages) by mutating or suppressing the phage-receptors, the factors that phages first target to initiate infection. Flagellotropic phages infect bacteria by initially binding to the flagellum. Since motility is an important fitness factor that allows bacteria to efficiently explore their environment, losing flagellar function to evade infection by flagellotropic phages represents a crucial trade-off. In this study, we investigated the evolutionary responses of Escherichia coli when exposed to the flagellotropic phage χ. Using an experimental evolution approach, E. coli cells were repeatedly subjected to environments rich in phage χ but selective for motility. Unlike traditional well-mixed cultures, we employed swim-plate assays to simulate spatial confinement and promote motility. Whole genome sequencing of evolved populations revealed early emergence of non-motile, χ-resistant mutants with mutations disrupting motility-related genes. Motile mutants emerged in later passages, possessing mutations in the flagellin gene fliC. Swim-plate assays showed a diverse range of motility among these mutants, with some displaying slower, and others faster, expansion speeds compared to the ancestral strain. Single-cell tracking experiments indicated an increased tumble bias in χ-resistant mutants, suggesting an adaptive response involving altered flagellar rotation. Our findings demonstrate that motility can undergo trade-offs and trade-ups with phage resistance, shedding light on the complex evolutionary dynamics between motile bacteria and flagellotropic phages.202540654869
937710.9995Experimental Evolution of the TolC-Receptor Phage U136B Functionally Identifies a Tail Fiber Protein Involved in Adsorption through Strong Parallel Adaptation. Bacteriophages have received recent attention for their therapeutic potential to treat antibiotic-resistant bacterial infections. One particular idea in phage therapy is to use phages that not only directly kill their bacterial hosts but also rely on particular bacterial receptors, such as proteins involved in virulence or antibiotic resistance. In such cases, the evolution of phage resistance would correspond to the loss of those receptors, an approach termed evolutionary steering. We previously found that during experimental evolution, phage U136B can exert selection pressure on Escherichia coli to lose or modify its receptor, the antibiotic efflux protein TolC, often resulting in reduced antibiotic resistance. However, for TolC-reliant phages like U136B to be used therapeutically, we also need to study their own evolutionary potential. Understanding phage evolution is critical for the development of improved phage therapies as well as the tracking of phage populations during infection. Here, we characterized phage U136B evolution in 10 replicate experimental populations. We quantified phage dynamics that resulted in five surviving phage populations at the end of the 10-day experiment. We found that phages from all five surviving populations had evolved higher rates of adsorption on either ancestral or coevolved E. coli hosts. Using whole-genome and whole-population sequencing, we established that these higher rates of adsorption were associated with parallel molecular evolution in phage tail protein genes. These findings will be useful in future studies to predict how key phage genotypes and phenotypes influence phage efficacy and survival despite the evolution of host resistance. IMPORTANCE Antibiotic resistance is a persistent problem in health care and a factor that may help maintain bacterial diversity in natural environments. Bacteriophages ("phages") are viruses that specifically infect bacteria. We previously discovered and characterized a phage called U136B, which infects bacteria through TolC. TolC is an antibiotic resistance protein that helps bacteria pump antibiotics out of the cell. Over short timescales, phage U136B can be used to evolutionarily "steer" bacterial populations to lose or modify the TolC protein, sometimes reducing antibiotic resistance. In this study, we investigate whether U136B itself evolves to better infect bacterial cells. We discovered that the phage can readily evolve specific mutations that increase its infection rate. This work will be useful for understanding how phages can be used to treat bacterial infections.202337191555
893420.9995A tradeoff between bacteriophage resistance and bacterial motility is mediated by the Rcs phosphorelay in Escherichia coli. Across the tree of life, pleiotropy is thought to constrain adaptation through evolutionary tradeoffs. However, few examples of pleiotropy exist that are well explained at the genetic level, especially for pleiotropy that is mediated by multiple genes. Here, we describe a set of pleiotropic mutations that mediate two key fitness components in bacteria: parasite resistance and motility. We subjected Escherichia coli to strong selection by phage U136B to obtain 27 independent mucoid mutants. Mucoidy is a phenotype that results from excess exopolysaccharide and can act as a barrier against viral infection but can also interfere with other cellular functions. We quantified the mutants' phage resistance using efficiency of plaquing assays and swimming motility using swim agar plates, and we sequenced the complete genomes of all mutants to identify mucoid-causing mutations. Increased phage resistance co-occurred with decreased motility. This relationship was mediated by highly parallel (27/27) mutations to the Rcs phosphorelay pathway, which senses membrane stress to regulate exopolysaccharide production. Together, these results provide an empirical example of a pleiotropic relationship between two traits with intermediate genetic complexity.202439194382
829130.9995Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms. Phage tail-like bacteriocins (tailocins) are bacterially produced protein toxins that mediate competitive interactions between cocolonizing bacteria. Both theoretical and experimental research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive cells, sensitive cells outcompete resistant cells, and resistant cells outcompete producers. These so-called rock-paper-scissors dynamics explain how all three populations occupy the same environment, without one driving the others extinct. Using Pseudomonas syringae as a model, we demonstrate that otherwise sensitive cells survive bacteriocin exposure through a physiological mechanism. This mechanism allows cells to survive bacteriocin killing without acquiring resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival during a subsequent exposure. Tailocin persister cells were more prevalent in stationary- rather than log-phase cultures. Of the fraction of cells that gained detectable resistance, there was a range from complete (insensitive) to incomplete (partially sensitive) resistance. By using genomic sequencing and genetic engineering, we showed that a mutation in a hypothetical gene containing 8 to 10 transmembrane domains causes tailocin high persistence and that genes of various glycosyltransferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result indicates that bacteria likely utilize persistence to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance.IMPORTANCE Bacteriocins are bacterially produced protein toxins that are proposed as antibiotic alternatives. However, a deeper understanding of the responses of target bacteria to bacteriocin exposure is lacking. Here, we show that target cells of Pseudomonas syringae survive lethal bacteriocin exposure through both physiological persistence and genetic resistance mechanisms. Cells that are not growing rapidly rely primarily on persistence, whereas those growing rapidly are more likely to survive via resistance. We identified various mutations in lipopolysaccharide biogenesis-related regions involved in tailocin persistence and resistance. By assessing host fitness of various classes of mutants, we showed that persistence and subtle resistance are mechanisms P. syringae uses to survive competition and preserve host fitness. These results have important implications for developing bacteriocins as alternative therapeutic agents.202032312747
937540.9995Multistep diversification in spatiotemporal bacterial-phage coevolution. The evolutionary arms race between phages and bacteria, where bacteria evolve resistance to phages and phages retaliate with resistance-countering mutations, is a major driving force of molecular innovation and genetic diversification. Yet attempting to reproduce such ongoing retaliation dynamics in the lab has been challenging; laboratory coevolution experiments of phage and bacteria are typically performed in well-mixed environments and often lead to rapid stagnation with little genetic variability. Here, co-culturing motile E. coli with the lytic bacteriophage T7 on swimming plates, we observe complex spatiotemporal dynamics with multiple genetically diversifying adaptive cycles. Systematically quantifying over 10,000 resistance-infectivity phenotypes between evolved bacteria and phage isolates, we observe diversification into multiple coexisting ecotypes showing a complex interaction network with both host-range expansion and host-switch tradeoffs. Whole-genome sequencing of these evolved phage and bacterial isolates revealed a rich set of adaptive mutations in multiple genetic pathways including in genes not previously linked with phage-bacteria interactions. Synthetically reconstructing these new mutations, we discover phage-general and phage-specific resistance phenotypes as well as a strong synergy with the more classically known phage-resistance mutations. These results highlight the importance of spatial structure and migration for driving phage-bacteria coevolution, providing a concrete system for revealing new molecular mechanisms across diverse phage-bacterial systems.202236577749
960750.9995Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution. Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress response processes known as adaptive resistance. Adaptive resistance fosters transient tolerance increases and the emergence of mutations conferring heritable drug resistance. In order to extend the applicable lifetime of new antibiotics, we must seek to hinder the occurrence of bacterial adaptive resistance; however, the regulation of adaptation is difficult to identify due to immense heterogeneity emerging during evolution. This study specifically seeks to generate heterogeneity by adapting bacteria to different stresses and then examines gene expression trends across the disparate populations in order to pinpoint key genes and pathways associated with adaptive resistance. The targets identified here may eventually inform strategies for impeding adaptive resistance and prolonging the effectiveness of antibiotic treatment.201728217741
937660.9994Historical Contingency Drives Compensatory Evolution and Rare Reversal of Phage Resistance. Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria-phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution.202235994371
962170.9994Bacterial biodiversity drives the evolution of CRISPR-based phage resistance. About half of all bacteria carry genes for CRISPR-Cas adaptive immune systems(1), which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome(2). Whereas CRISPR loci evolve rapidly in natural environments(3,4), bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions(5,6). Here we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments(7,8). Specifically, by using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with the mutation of phage receptors, and therefore tips the balance in favour of the evolution of CRISPR-based resistance. We also demonstrate that this has important knock-on effects for the virulence of P. aeruginosa, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.201931645729
960580.9994Gene Expression Variability Underlies Adaptive Resistance in Phenotypically Heterogeneous Bacterial Populations. The root cause of the antibiotic resistance crisis is the ability of bacteria to evolve resistance to a multitude of antibiotics and other environmental toxins. The regulation of adaptation is difficult to pinpoint due to extensive phenotypic heterogeneity arising during evolution. Here, we investigate the mechanisms underlying general bacterial adaptation by evolving wild-type Escherichia coli populations to dissimilar chemical toxins. We demonstrate the presence of extensive inter- and intrapopulation phenotypic heterogeneity across adapted populations in multiple traits, including minimum inhibitory concentration, growth rate, and lag time. To search for a common response across the heterogeneous adapted populations, we measured gene expression in three stress-response networks: the mar regulon, the general stress response, and the SOS response. While few genes were differentially expressed, clustering revealed that interpopulation gene expression variability in adapted populations was distinct from that of unadapted populations. Notably, we observed both increases and decreases in gene expression variability upon adaptation. Sequencing select genes revealed that the observed gene expression trends are not necessarily attributable to genetic changes. To further explore the connection between gene expression variability and adaptation, we propagated single-gene knockout and CRISPR (clustered regularly interspaced short palindromic repeats) interference strains and quantified impact on adaptation to antibiotics. We identified significant correlations that suggest genes with low expression variability have greater impact on adaptation. This study provides evidence that gene expression variability can be used as an indicator of bacterial adaptive resistance, even in the face of the pervasive phenotypic heterogeneity underlying adaptation.201527623410
938090.9994Coevolution between marine Aeromonas and phages reveals temporal trade-off patterns of phage resistance and host population fitness. Coevolution of bacteria and phages is an important host and parasite dynamic in marine ecosystems, contributing to the understanding of bacterial community diversity. On the time scale, questions remain concerning what is the difference between phage resistance patterns in marine bacteria and how advantageous mutations gradually accumulate during coevolution. In this study, marine Aeromonas was co-cultured with its phage for 180 days and their genetic and phenotypic dynamics were measured every 30 days. We identified 11 phage resistance genes and classified them into three categories: lipopolysaccharide (LPS), outer membrane protein (OMP), and two-component system (TCS). LPS shortening and OMP mutations are two distinct modes of complete phage resistance, while TCS mutants mediate incomplete resistance by repressing the transcription of phage genes. The co-mutation of LPS and OMP was a major mode for bacterial resistance at a low cost. The mutations led to significant reductions in the growth and virulence of bacterial populations during the first 60 days of coevolution, with subsequent leveling off. Our findings reveal the marine bacterial community dynamics and evolutionary trade-offs of phage resistance during coevolution, thus granting further understanding of the interaction of marine microbes.202337814126
9364100.9994Predictable properties of fitness landscapes induced by adaptational tradeoffs. Fitness effects of mutations depend on environmental parameters. For example, mutations that increase fitness of bacteria at high antibiotic concentration often decrease fitness in the absence of antibiotic, exemplifying a tradeoff between adaptation to environmental extremes. We develop a mathematical model for fitness landscapes generated by such tradeoffs, based on experiments that determine the antibiotic dose-response curves of Escherichia coli strains, and previous observations on antibiotic resistance mutations. Our model generates a succession of landscapes with predictable properties as antibiotic concentration is varied. The landscape is nearly smooth at low and high concentrations, but the tradeoff induces a high ruggedness at intermediate antibiotic concentrations. Despite this high ruggedness, however, all the fitness maxima in the landscapes are evolutionarily accessible from the wild type. This implies that selection for antibiotic resistance in multiple mutational steps is relatively facile despite the complexity of the underlying landscape.202032423531
8896110.9994Nonoptimal Gene Expression Creates Latent Potential for Antibiotic Resistance. Bacteria regulate genes to survive antibiotic stress, but regulation can be far from perfect. When regulation is not optimal, mutations that change gene expression can contribute to antibiotic resistance. It is not systematically understood to what extent natural gene regulation is or is not optimal for distinct antibiotics, and how changes in expression of specific genes quantitatively affect antibiotic resistance. Here we discover a simple quantitative relation between fitness, gene expression, and antibiotic potency, which rationalizes our observation that a multitude of genes and even innate antibiotic defense mechanisms have expression that is critically nonoptimal under antibiotic treatment. First, we developed a pooled-strain drug-diffusion assay and screened Escherichia coli overexpression and knockout libraries, finding that resistance to a range of 31 antibiotics could result from changing expression of a large and functionally diverse set of genes, in a primarily but not exclusively drug-specific manner. Second, by synthetically controlling the expression of single-drug and multidrug resistance genes, we observed that their fitness-expression functions changed dramatically under antibiotic treatment in accordance with a log-sensitivity relation. Thus, because many genes are nonoptimally expressed under antibiotic treatment, many regulatory mutations can contribute to resistance by altering expression and by activating latent defenses.201830169679
9612120.9994Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages. Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.201121509046
9381130.9994Cross-resistance is modular in bacteria-phage interactions. Phages shape the structure of natural bacterial communities and can be effective therapeutic agents. Bacterial resistance to phage infection, however, limits the usefulness of phage therapies and could destabilise community structures, especially if individual resistance mutations provide cross-resistance against multiple phages. We currently understand very little about the evolution of cross-resistance in bacteria-phage interactions. Here we show that the network structure of cross-resistance among spontaneous resistance mutants of Pseudomonas aeruginosa evolved against each of 27 phages is highly modular. The cross-resistance network contained both symmetric (reciprocal) and asymmetric (nonreciprocal) cross-resistance, forming two cross-resistance modules defined by high within- but low between-module cross-resistance. Mutations conferring cross-resistance within modules targeted either lipopolysaccharide or type IV pilus biosynthesis, suggesting that the modularity of cross-resistance was structured by distinct phage receptors. In contrast, between-module cross-resistance was provided by mutations affecting the alternative sigma factor, RpoN, which controls many lifestyle-associated functions, including motility, biofilm formation, and quorum sensing. Broader cross-resistance range was not associated with higher fitness costs or weaker resistance against the focal phage used to select resistance. However, mutations in rpoN, providing between-module cross-resistance, were associated with higher fitness costs than mutations associated with within-module cross-resistance, i.e., in genes encoding either lipopolysaccharide or type IV pilus biosynthesis. The observed structure of cross-resistance predicted both the frequency of resistance mutations and the ability of phage combinations to suppress bacterial growth. These findings suggest that the evolution of cross-resistance is common, is likely to play an important role in the dynamic structure of bacteria-phage communities, and could inform the design principles for phage therapy treatments.201830281587
9378140.9994Inferring strain-level mutational drivers of phage-bacteria interaction phenotypes arising during coevolutionary dynamics. The enormous diversity of bacteriophages and their bacterial hosts presents a significant challenge to predict which phages infect a focal set of bacteria. Infection is largely determined by complementary - and largely uncharacterized - genetics of adsorption, injection, cell take-over and lysis. Here we present a machine learning approach to predict phage-bacteria interactions trained on genome sequences of and phenotypic interactions amongst 51 Escherichia coli strains and 45 phage λ strains that coevolved in laboratory conditions for 37 days. Leveraging multiple inference strategies and without a priori knowledge of driver mutations, this framework predicts both who infects whom and the quantitative levels of infections across a suite of 2,295 potential interactions. We found that the most effective approach inferred interaction phenotypes from independent contributions from phage and bacteria mutations, accurately predicting 86% of interactions while reducing the relative error in the estimated strength of the infection phenotype by 40% . Feature selection revealed key phage λ and E. coli mutations that have a significant influence on the outcome of phage-bacteria interactions, corroborating sites previously known to affect phage λ infections, as well as identifying mutations in genes of unknown function not previously shown to influence bacterial resistance. The method's success in recapitulating strain-level infection outcomes arising during coevolutionary dynamics may also help inform generalized approaches for imputing genetic drivers of interaction phenotypes in complex communities of phage and bacteria.202438260415
9604150.9993Extreme 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.202031964772
8991160.9993Salicylate Increases Fitness Cost Associated with MarA-Mediated Antibiotic Resistance. Antibiotic resistance is generally associated with a fitness deficit resulting from the burden of producing and maintaining resistance machinery. This additional cost suggests that resistant bacteria will be outcompeted by susceptible bacteria in conditions without antibiotics. However, in practice, this process is slow in part because of regulation that minimizes expression of these genes in the absence of antibiotics. This suggests that if it were possible to turn on their expression, the cost would increase, thereby accelerating removal of resistant strains. Experimental and theoretical studies have shown that environmental chemicals can change the fitness cost associated with resistance and therefore have a significant impact on population dynamics. The multiple antibiotic resistance activator (MarA) is a clinically important regulator in Escherichia coli that activates downstream genes to increase resistance against multiple classes of antibiotics. Salicylate is an inducer of MarA that can be found in the environment and derepresses marA's expression. In this study, we sought to unravel the interplay between salicylate and the fitness cost of MarA-mediated antibiotic resistance. Using salicylate as an inducer of MarA, we found that a wide spectrum of concentrations can increase burden in resistant strains compared to susceptible strains. Induction resulted in rapid exclusion of resistant bacteria from mixed populations of antibiotic-resistant and susceptible cells. A mathematical model captures the process and predicts its effect in various environmental conditions. Our work provides a quantitative understanding of salicylate exposure on the fitness of different MarA variants and suggests that salicylate can lead to selection against MarA-mediated resistant strains. More generally, our findings show that natural inducers may serve to bias population membership and could impact antibiotic resistance and other important phenotypes.201931349991
8311170.9993Perturbation of Quorum Sensing after the Acquisition of Bacteriophage Resistance Could Contribute to Novel Traits in Vibrio alginolyticus. Bacteria employ a wide range of molecular mechanisms to confer resistance to bacteriophages, and these mechanisms are continuously being discovered and characterized. However, there are instances where certain bacterial species, despite lacking these known mechanisms, can still develop bacteriophage resistance through intricate metabolic adaptation strategies, potentially involving mutations in transcriptional regulators or phage receptors. Vibrio species have been particularly useful for studying the orchestrated metabolic responses of Gram-negative marine bacteria in various challenges. In a previous study, we demonstrated that Vibrio alginolyticus downregulates the expression of specific receptors and transporters in its membrane, which may enable the bacterium to evade infection by lytic bacteriophages. In our current study, our objective was to explore how the development of bacteriophage resistance in Vibrio species disrupts the quorum-sensing cascade, subsequently affecting bacterial physiology and metabolic capacity. Using a real-time quantitative PCR (rt-QPCR) platform, we examined the expression pattern of quorum-sensing genes, auto-inducer biosynthesis genes, and cell density regulatory proteins in phage-resistant strains. Our results revealed that bacteriophage-resistant bacteria downregulate the expression of quorum-sensing regulatory proteins, such as LuxM, LuxN, and LuxP. This downregulation attenuates the normal perception of quorum-sensing peptides and subsequently diminishes the expression of cell density regulatory proteins, including LuxU, aphA, and LuxR. These findings align with the diverse phenotypic traits observed in the phage-resistant strains, such as altered biofilm formation, reduced planktonic growth, and reduced virulence. Moreover, the transcriptional depletion of aphA, the master regulator associated with low cell density, was linked to the downregulation of genes related to virulence. This phenomenon appears to be phage-specific, suggesting a finely tuned metabolic adaptation driven by phage-host interaction. These findings contribute to our understanding of the role of Vibrio species in microbial marine ecology and highlight the complex interplay between phage resistance, quorum sensing, and bacterial physiology.202337764117
9601180.9993Phage steering in the presence of a competing bacterial pathogen. The rise of antibiotic-resistant bacteria has necessitated the development of alternative therapeutic strategies, such as bacteriophage therapy, where viruses infect bacteria, reducing bacterial burden. However, rapid bacterial resistance to phage treatment remains a critical challenge, potentially leading to failure. Phage steering, which leverages the evolutionary dynamics between phage and bacteria, offers a novel solution by driving bacteria to evolve away from virulence factors or resistance mechanisms. In this study, we examined whether phage steering using bacteriophage Luz19 could function in the presence of a competing pathogen, Staphylococcus aureus (SA) (USA300), while targeting Pseudomonas aeruginosa (PAO1). Through in vitro co-evolution experiments with and without the competitor, we observed that Luz19 consistently steered P. aeruginosa away from the Type IV pilus (T4P), a key virulence factor, without interference from SA. Genomic analyses revealed mutations in T4P-associated genes, including pilR and pilZ, which conferred phage resistance. Our findings suggest that phage steering remains effective even in polymicrobial environments, providing a promising avenue for enhancing bacteriophage therapy efficacy in complex infections.IMPORTANCEPhage steering-using phages that bind essential virulence or resistance-associated structures-offers a promising solution by selecting for resistance mutations that attenuate pathogenic traits. However, it remains unclear whether this strategy remains effective in polymicrobial contexts, where interspecies interactions may alter selective pressures. Here, we demonstrate that Pseudomonas aeruginosa evolves phage resistance via loss-of-function mutations in Type IV pilus (T4P) when challenged with the T4P-binding phage Luz19 and that this evolutionary trajectory is preserved even in the presence of a competing pathogen, Staphylococcus aureus. Phage resistance was phenotypically confirmed via twitching motility assays and genotypically via whole-genome sequencing. These findings support the robustness of phage steering under interspecies competition, underscoring its translational potential for managing complex infections-such as those seen in cystic fibrosis-where microbial diversity is the norm.202540492711
8923190.9993The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli. Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms. IMPORTANCE: With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress.201627879333