Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms. - Related Documents




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829101.0000Pseudomonas 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
960710.9997Transcriptome-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
831120.9997Perturbation 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
937730.9997Experimental 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
829540.9997Calcium Prevents Biofilm Dispersion in Bacillus subtilis. Biofilm dispersion is the final stage of biofilm development, during which biofilm cells actively escape from biofilms in response to deteriorating conditions within the biofilm. Biofilm dispersion allows cells to spread to new locations and form new biofilms in better locations. However, dispersal mechanisms have been elucidated only in a limited number of bacteria. Here, we investigated biofilm dispersion in Bacillus subtilis. Biofilm dispersion was clearly observed when B. subtilis was grown under static conditions in modified LB medium containing glycerol and manganese. Biofilm dispersion was synergistically caused by two mechanisms: decreased expression of the epsA operon encoding exopolysaccharide synthetases and the induction of sporulation. Indeed, constitutive expression of the epsA operon in the sporulation-defective ΔsigK mutant prevented biofilm dispersion. The addition of calcium to the medium prevented biofilm dispersion without significantly affecting the expression of the epsA operon and sporulation genes. In synthetic medium, eliminating calcium did not prevent the expression of biofilm matrix genes and, thereby, biofilm formation, but it attenuated biofilm architecture. These results indicate that calcium structurally stabilizes biofilms and causes resistance to biofilm dispersion mechanisms. Sporulation-dependent biofilm dispersion required the spoVF operon, encoding dipicolinic acid (DPA) synthase. During sporulation, an enormous amount of DPA is synthesized and stored in spores as a chelate with calcium. We speculate that, during sporulation, calcium bound to biofilm matrix components may be transported to spores as a calcium-DPA complex, which weakens biofilm structure and leads to biofilm dispersion. IMPORTANCE Bacteria growing as biofilms are notoriously difficult to eradicate and sometimes pose serious threats to public health. Bacteria escape from biofilms by degrading them when biofilm conditions deteriorate. This process, called biofilm dispersion, has been studied as a promising strategy for safely controlling biofilms. However, the regulation and mechanism of biofilm dispersion has been elucidated only in a limited number of bacteria. Here, we identified two biofilm dispersion mechanisms in the Gram-positive, spore-forming bacterium Bacillus subtilis. The addition of calcium to the medium stabilized biofilms and caused resistance to dispersal mechanisms. Our findings provide new insights into biofilm dispersion and biofilm control.202133927049
960550.9997Gene 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
961260.9997Using 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
937670.9997Historical 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
960480.9996Extreme 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
828690.9996RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence. RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications.202134440299
9621100.9996Bacterial 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
9381110.9996Cross-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
9620120.9996Determinants of Phage Host Range in Staphylococcus Species. Bacteria in the genus Staphylococcus are important targets for phage therapy due to their prevalence as pathogens and increasing antibiotic resistance. Here we review Staphylococcus outer surface features and specific phage resistance mechanisms that define the host range, the set of strains that an individual phage can potentially infect. Phage infection goes through five distinct phases: attachment, uptake, biosynthesis, assembly, and lysis. Adsorption inhibition, encompassing outer surface teichoic acid receptor alteration, elimination, or occlusion, limits successful phage attachment and entry. Restriction-modification systems (in particular, type I and IV systems), which target phage DNA inside the cell, serve as the major barriers to biosynthesis as well as transduction and horizontal gene transfer between clonal complexes and species. Resistance to late stages of infection occurs through mechanisms such as assembly interference, in which staphylococcal pathogenicity islands siphon away superinfecting phage proteins to package their own DNA. While genes responsible for teichoic acid biosynthesis, capsule, and restriction-modification are found in most Staphylococcus strains, a variety of other host range determinants (e.g., clustered regularly interspaced short palindromic repeats, abortive infection, and superinfection immunity) are sporadic. The fitness costs of phage resistance through teichoic acid structure alteration could make staphylococcal phage therapies promising, but host range prediction is complex because of the large number of genes involved, and the roles of many of these are unknown. In addition, little is known about the genetic determinants that contribute to host range expansion in the phages themselves. Future research must identify host range determinants, characterize resistance development during infection and treatment, and examine population-wide genetic background effects on resistance selection.201930902858
9623130.9996Prokaryotic toxin-antitoxin systems--the role in bacterial physiology and application in molecular biology. Bacteria have developed multiple complex mechanisms ensuring an adequate response to environmental changes. In this context, bacterial cell division and growth are subject to strict control to ensure metabolic balance and cell survival. A plethora of studies cast light on toxin-antitoxin (TA) systems as metabolism regulators acting in response to environmental stress conditions. Many of those studies suggest direct relations between the TA systems and the pathogenic potential or antibiotic resistance of relevant bacteria. Other studies point out that TA systems play a significant role in ensuring stability of mobile genetic material. The evolutionary origin and relations between various TA systems are still a subject of a debate. The impact of toxin-antitoxin systems on bacteria physiology prompted their application in molecular biology as tools allowing cloning of some hard-to-maintain genes, plasmid maintenance and production of recombinant proteins.201121394325
8248140.9996Coping with Environmental Eukaryotes; Identification of Pseudomonas syringae Genes during the Interaction with Alternative Hosts or Predators. Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success.201829690522
8292150.9996Exopolysaccharide anchoring creates an extreme resistance to sedimentation. By evolving strains of E. coli that hyper-resist sedimentation, we discovered an uncharacterized mechanism that bacteria can use to remain in suspension indefinitely without expending energy. This unusual phenotype was traced to the anchoring of long colanic acid polymers (CAP) that project from the cell surface. Although each characterized mutant activated this same mechanism, the genes responsible and the strengths of the phenotypes varied. Mutations in rcsC, lpp, igaA, or the yjbEFGH operon were sufficient to stimulate sedimentation resistance, while mutations altering the cps promoter, cdgI, or yjbF provided phenotypic enhancements. The sedimentation resistances changed in response to temperature, growth phase, and carbon source and each mutant exhibited significantly reduced biofilm formation. We discovered that the degree of colony mucoidy exhibited by these mutants was not related to the degree of Rcs pathways activation or to the amount of CAP that was produced; rather, it was related to the fraction of CAP that was shed as a true exopolysaccharide. Therefore, these and other mutations that activate this phenotype are likely to be absent from genetic screens that relied on centrifugation to harvest bacteria. We also found that this anchored CAP form is not linked to LPS cores and may not be attached to the outer membrane.IMPORTANCEBacteria can partition in aqueous environments between surface-dwelling, planktonic, sedimentary, and biofilm forms. Residence in each location provides an advantage depending on nutritional and environmental stresses and a community of a single species is often observed to be distributed throughout two or more of these niches. Another adaptive strategy is to produce an extracellular capsule, which provides an environmental shield for the microbe and can allow escape from predators and immune systems. We discovered that bacteria can either shed or stably anchor capsules to dramatically alter their propensity to sediment. The degree to which the bacteria anchor their capsule is controlled by a stress sensing system, suggesting that anchoring may be used as an adaptive response to severe environmental challenges.202133753470
8922160.9996Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches. Burkholderia pseudomallei, a soil and water saprophyte, is responsible for the tropical human disease melioidosis. A hundred years since its discovery, there is still much to learn about B. pseudomallei proteins that are essential for the bacterium's survival in and interaction with the infected host, as well as their roles within the bacterium's natural soil habitat. To address this gap, bacteria grown under conditions mimicking the soil environment were subjected to transcriptome sequencing (RNA-seq) analysis. A dual RNA-seq approach was used on total RNA from spleens isolated from a B. pseudomallei mouse infection model at 5 days postinfection. Under these conditions, a total of 1,434 bacterial genes were induced, with 959 induced in the soil environment and 475 induced in bacteria residing within the host. Genes encoding metabolism and transporter proteins were induced when the bacteria were present in soil, while virulence factors, metabolism, and bacterial defense mechanisms were upregulated during active infection of mice. On the other hand, capsular polysaccharide and quorum-sensing pathways were inhibited during infection. In addition to virulence factors, reactive oxygen species, heat shock proteins, siderophores, and secondary metabolites were also induced to assist bacterial adaptation and survival in the host. Overall, this study provides crucial insights into the transcriptome-level adaptations which facilitate infection by soil-dwelling B. pseudomallei. Targeting novel therapeutics toward B. pseudomallei proteins required for adaptation provides an alternative treatment strategy given its intrinsic antimicrobial resistance and the absence of a vaccine. IMPORTANCE Burkholderia pseudomallei, a soil-dwelling bacterium, is the causative agent of melioidosis, a fatal infectious disease of humans and animals. The bacterium has a large genome consisting of two chromosomes carrying genes that encode proteins with important roles for survival in diverse environments as well as in the infected host. While a general mechanism of pathogenesis has been proposed, it is not clear which proteins have major roles when the bacteria are in the soil and whether the same proteins are key to successful infection and spread. To address this question, we grew the bacteria in soil medium and then in infected mice. At 5 days postinfection, bacteria were recovered from infected mouse organs and their gene expression was compared against that of bacteria grown in soil medium. The analysis revealed a list of genes expressed under soil growth conditions and a different set of genes encoding proteins which may be important for survival, replication, and dissemination in an infected host. These proteins are a potential resource for understanding the full adaptation mechanism of this pathogen. In the absence of a vaccine for melioidosis and with treatment being reliant on combinatorial antibiotic therapy, these proteins may be ideal targets for designing antimicrobials to treat melioidosis.202336856434
9340170.9996Shigella viruses Sf22 and KRT47 require outer membrane protein C for infection. Viruses rely on hosts for their replication: thus, a critical step in the infection process is identifying a suitable host cell. Bacterial viruses, known as bacteriophages or phages, often use receptor binding proteins to discriminate between susceptible and non-susceptible hosts. By being able to evade predation, bacteria with modified or deleted receptor-encoding genes often undergo positive selection during growth in the presence of phage. Depending on the specific receptor(s) a phage uses, this may subsequently affect the bacteria's ability to form biofilms, its resistance to antibiotics, pathogenicity, or its phenotype in various environments. In this study, we characterize the interactions between two T4-like phages, Sf22 and KRT47, and their host receptor S. flexneri outer membrane protein C (OmpC). Results indicate that these phages use a variety of surface features on the protein, and that complete resistance most frequently occurs when hosts delete the ompC gene in full, encode premature stop codons to prevent OmpC synthesis, or eliminate specific regions encoding exterior loops.202235358430
9282180.9996Could DNA uptake be a side effect of bacterial adhesion and twitching motility? DNA acquisition promotes the spread of resistance to antibiotics and virulence among bacteria. It is also linked to several natural phenomena including recombination, genome dynamics, adaptation and speciation. Horizontal DNA transfer between bacteria occurs via conjugation, transduction or competence for natural transformation by DNA uptake. Among these, competence is the only mechanism of transformation initiated and entirely controlled by the chromosome of the recipient bacteria. While the molecular mechanisms allowing the uptake of extracellular DNA are increasingly characterized, the function of competence for natural transformation by DNA uptake, the selective advantage maintaining it and the reasons why bacteria take up DNA in the first place are still debated. In this synthesis, I review some of the literature and discuss the four hypotheses on how and why do bacteria take up DNA. I argue that DNA uptake by bacteria is an accidental by-product of bacterial adhesion and twitching motility. Adhesion and motility are generally increased in stressful conditions, which may explain why bacteria increase DNA uptake in these conditions. In addition to its fundamental scientific relevance, the new hypothesis suggested here has significant clinical implications and finds further support from the fact that antibiotics sometimes fail to eliminate the targeted bacterium while inevitably causing stress to others. The widespread misuse of antibiotics may thus not only be selecting for resistant strains, but may also be causing bacteria to take up more DNA with the consequent increase in the chances of acquiring drug resistance and virulence-a scenario in full concordance with the previously reported induction of competence genes by antibiotics in Streptococcus pneumoniae and Legionella pneumophila.201323381940
9619190.9996Phage "delay" towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages. In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their "vigor" when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such "less-robust" mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically "running away" to more phage-free locations. Delay mechanisms-to the extent that they are less specific in terms of what phages are targeted-collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.201731294157