Biology subjects

Cooper, V. S.

Publications and source records attributed to Cooper, V. S..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

The nutritional environment is sufficient to select coexisting biofilm and quorum-sensing mutants of Pseudomonas aeruginosa

The evolution of bacterial populations during infections can be influenced by various factors including available nutrients, the immune system, and competing microbes, rendering it difficult to identify the specific forces that select on evolved traits. The genomes of Pseudomonas aeruginosa isolated from the airway of patients with cystic fibrosis (CF), for example, have revealed commonly mutated genes, but which phenotypes led to their prevalence is often uncertain. Here, we focus on effects of nutritional components of the CF airway on genetic adaptations by P. aeruginosa grown in either well-mixed (planktonic) or biofilm-associated conditions. After only 80 generations of experimental evolution in a simple medium with glucose, lactate, and amino acids, all planktonic populations diversified into lineages with mutated genes common to CF infections: morA, encoding a regulator of biofilm formation, or lasR, encoding a quorum sensing regulator that modulates the expression of virulence factors. Although mutated quorum sensing is often thought to be selected in vivo due to altered virulence phenotypes or social cheating, isolates with lasR mutations demonstrated increased fitness when grown alone and outcompeted the ancestral PA14 strain. Nonsynonymous SNPs in morA increased fitness in a nutrient concentration-dependent manner during planktonic growth and surprisingly also increased biofilm production. Populations propagated in biofilm conditions also acquired mutations in loci associated with chronic infections, including lasR and cyclic-di-GMP regulators roeA and wspF. These findings demonstrate that nutrient conditions and biofilm selection are alone sufficient to select mutants with problematic clinical phenotypes including increased biofilm and altered quorum sensing. ImportancePseudomonas aeruginosa produces dangerous chronic infections that are known for their rapid diversification and recalcitrance to treatment. We performed evolution experiments to identify adaptations selected by two specific aspects of the CF respiratory environment: nutrient levels and surface attachment. Propagation of P. aeruginosa in nutrients present within the CF airway was alone sufficient to drive diversification into subpopulations with identical mutations in regulators of biofilm and quorum sensing to those arising during infection. Thus, the adaptation of opportunistic pathogens to nutrients found in the host may select mutants with phenotypes that complicate treatment and clearance of infection.

microbiology↗

bioRxiv2021

Excess dietary sugar impairs colonic epithelial regeneration in response to damage.

The colonic epithelium requires continuous renewal by intestinal stem cells (ISCs) to restore the barrier after damage and proliferation of epithelial cells is modulated by dietary metabolites. We demonstrate that mice fed a high sugar diet failed to repair colonic barrier damage, resulting in increased intestinal pathology. Culturing ISCs in excess sugar limited murine and human colonoid development, indicating that dietary sugar can directly affect colonic epithelial proliferation. Similarly, in vivo lineage tracing experiments and transcriptomic analysis indicated that dietary sugar impeded the proliferative potential of ISCs. ISCs and their immediate daughter cells predominantly rely on mitochondrial respiration for energy; however, metabolic analysis of colonic crypts revealed that a high sugar diet primed the epithelium for glycolysis without a commensurate increase in aerobic respiration. Colonoids cultured in high-glucose conditions accumulated glycolytic metabolites but not TCA cycle intermediates, indicating that the two metabolic pathways may not be coupled in proliferating intestinal epithelium. Accordingly, biochemically inducing pyruvate flux through the TCA cycle by inhibiting pyruvate dehydrogenase kinase rescued sugar-impaired colonoid development. Our results indicate that excess dietary sugar can directly inhibit epithelial proliferation in response to damage and may inform diets that better support the treatment of acute intestinal injury.

immunology↗

bioRxiv2021

SprayNPray: user-friendly taxonomic profiling of genome and metagenome contigs

Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semiautomated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.

bioinformatics↗

bioRxiv2021

Evolutionary divergence of the Wsp signal transduction system in β- and γ-proteobacteria

Bacteria rapidly adapt to their environment by integrating external stimuli through diverse signal transduction systems. Pseudomonas aeruginosa, for example, senses surface-contact through the Wsp signal transduction system to trigger the production of cyclic di-GMP. Diverse mutations in wsp genes that manifest enhanced biofilm formation are frequently reported in clinical isolates of P. aeruginosa, and in biofilm studies of Pseudomonas spp. and Burkholderia cenocepacia. In contrast to the convergent phenotypes associated with comparable wsp mutations, we demonstrate that the Wsp system in B. cenocepacia does not impact intracellular cyclic di-GMP levels unlike that in Pseudomonas spp. Our current mechanistic understanding of the Wsp system is entirely based on the study of four Pseudomonas spp. and its phylogenetic distribution remains unknown. Here, we present the first broad phylogenetic analysis to date to show that the Wsp system originated in the {beta}-proteobacteria then horizontally transferred to Pseudomonas spp., the sole member of the {gamma}-proteobacteria. Alignment of 794 independent Wsp systems with reported mutations from the literature identified key amino acid residues that fall within and outside annotated functional domains. Specific residues that are highly conserved but uniquely modified in B. cenocepacia likely define mechanistic differences among Wsp systems. We also find the greatest sequence variation in the extracellular sensory domain of WspA, indicating potential adaptations to diverse external stimuli beyond surface-contact sensing. This study emphasizes the need to better understand the breadth of functional diversity of the Wsp system as a major regulator of bacterial adaptation beyond B. cenocepacia and select Pseudomonas spp. ImportanceThe Wsp signal transduction system serves as an important model system for studying how bacteria adapt to living in densely structured communities known as biofilms. Biofilms frequently cause chronic infections and environmental fouling, and they are very difficult to eradicate. In Pseudomonas aeruginosa, the Wsp system senses contact with a surface, which in turn activates specific genes that promote biofilm formation. We demonstrate that the Wsp system in Burkholderia cenocepacia regulates biofilm formation uniquely from that in Pseudomonas species. Furthermore, a broad phylogenetic analysis reveals the presence of the Wsp system in diverse bacterial species, and sequence analyses of 794 independent systems suggest that the core signaling components function similarly but with key differences that may alter what or how they sense. This study shows that Wsp systems are highly conserved and more broadly distributed than previously thought, and their unique differences likely reflect adaptations to distinct environments.

microbiology↗

bioRxiv2021

Immunosuppression broadens evolutionary pathways to treatment failure during Acinetobacter baumannii pneumonia

Acinetobacter baumannii is increasingly refractory to antibiotic treatment in healthcare settings. As is true of most human pathogens, the genetic path to antimicrobial resistance (AMR) and the role that the immune system plays in modulating AMR during disease are poorly understood. Here we reproduced several routes to fluoroquinolone resistance, performing evolution experiments using sequential lung infections in mice that are replete or depleted of neutrophils, providing two key insights into the evolution of drug resistance. First, neutropenic hosts acted as reservoirs for the accumulation of drug resistance during drug treatment. Selection for variants with altered drug sensitivity profiles arose readily in the absence of neutrophils, while immunocompetent animals restricted the appearance of these variants. Secondly, antibiotic treatment failure in the immunocompromised host was shown to occur without clinically defined resistance, an unexpected result that provides a model for how antibiotic failure occurs clinically in the absence of AMR. The genetic mechanism underlying both these results is initiated by mutations activating the drug egress pump regulator AdeL, which drives persistence in the presence of antibiotic. Therefore, antibiotic persistence mutations present a two-pronged risk during disease, causing drug treatment failure in the immunocompromised host while simultaneously increasing the emergence of high-level AMR.

microbiology↗

bioRxiv2021

Rampant prophage movement among transient competitors drives rapid adaptation during infection

Interactions between bacteria, their close competitors, and viral parasites are common in infections but understanding of these eco-evolutionary dynamics is limited. Most examples of adaptations caused by phage lysogeny are through the acquisition of new genes. However, integrated prophages can also insert into functional genes and impart a fitness benefit by disrupting their expression, a process called active lysogeny. Here, we show that active lysogeny can fuel rapid, parallel adaptations in establishing a chronic infection. These recombination events repeatedly disrupted genes encoding global regulators, leading to increased cyclic-di-GMP levels and elevated biofilm production. The implications of prophage-mediated adaptation are broad, as even transient members of microbial communities can alter the course of evolution and generate persistent phenotypes associated with poor clinical outcomes. One Sentence SummaryBacteriophage act as genetic regulators that are key to establishing chronic infections and are rapidly shared among co-infecting strains.

microbiology↗

bioRxiv2021

Polygenic adaptation, clonal interference, and the evolution of mutators in experimental Pseudomonas aeruginosa populations

In bacterial populations, switches in lifestyle from motile, planktonic growth to surface-grown biofilm is associated with persistence in both infections and non-clinical biofilms. Studies have identified the first steps of adaptation to biofilm growth but have yet to replicate the extensive genetic diversity observed in chronic infections or in the natural environment. We conducted a 90-day long evolution experiment with Pseudomonas aeruginosa PA14 in growth media that promotes biofilm formation in either planktonic culture or in a biofilm bead model. Surprisingly, all populations evolved extensive genetic diversity with hundreds of mutations maintained at intermediate frequencies, while fixation events were rare. Instead of the expected few beneficial mutations rising in frequency through successive sweeps, we observe a remarkable 40 genes with parallel mutations spanning both environments and often on coexisting genotypes within a population. Additionally, the evolution of mutator genotypes (mutS or mutL mutator alleles) that rise to high frequencies in as little as 25 days contribute to the extensive genetic variation and strong clonal interference. Parallelism in several transporters (including pitA, pntB, nosD, and pchF) indicate probable adaptation to the arginine media that becomes highly alkaline during growth. Further, genes involved in signal transduction (including gacS, aer2, bdlA, and PA14_71750) reflect likely adaptations to biofilm-inducing conditions. This experiment shows how extensive genetic and phenotypic diversity can arise and persist in microbial populations despite strong selection that would normally purge diversity. ImportanceHow biodiversity arises and is maintained in clonally reproducing organisms like microbes remains unclear. Many models presume that beneficial genotypes will outgrow others and purge variation via selective sweeps. Environmental structure like biofilms may oppose this process and preserve variation. We tested this hypothesis by evolving P. aeruginosa populations in biofilm-promoting media for three months and found both adaptation and diversification that were mostly uninterrupted by fixation events that eliminate diversity. Genetic variation tended to be greater in lines grown using a bead model of biofilm growth but many lineages also persisted in planktonic lines. Convergent evolution affecting dozens of genes indicates that selection acted on a wide variety of traits to improve fitness, causing many adapting lineages to co-occur and persist. This result demonstrates that some environments may expose a large fraction of the genome to selection and select for many adaptations at once, causing enduring diversity.

evolutionary biology↗

bioRxiv2020

Experimental evolution to identify undescribed mechanisms of resistance to a novel cationic peptide antibiotic

A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and mutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. The results show how the experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice.

microbiology↗

bioRxiv2020

Evolution and adaptation of Pseudomonas aeruginosa in the paranasal sinuses of people with cystic fibrosis

People with the genetic disorder cystic fibrosis (CF) harbor lifelong respiratory infections, with morbidity and mortality frequently linked to chronic lung infections dominated by the opportunistically pathogenic bacterium Pseudomonas aeruginosa. During chronic CF lung infections, a single clone of P. aeruginosa can persist for decades and dominate end-stage CF lung disease due to its propensity to adaptively evolve to the respiratory environment, a process termed "pathoadaptation". Chronic rhinosinusitis (CRS), chronic inflammation and infection of the sinonasal space, is highly prevalent in CF and the sinuses may serve as the first site in the respiratory tract to become colonized by bacteria that then proceed to seed lung infections. We identified three evolutionary genetic routes by which P. aeruginosa evolves in the sinuses of people with CF, including through the evolution of mutator lineages and proliferative insertion sequences and culminating in early genomic signatures of host-restriction. Our findings raise the question of whether a significant portion of the pathoadaptive phenotypes previously thought to have evolved in response to selective pressures in the CF lungs may have first arisen in the sinuses and underscore the link between sinonasal and lung disease in CF. Graphical abstract and highlightsO_LIPseudomonas aeruginosa undergoes adaptive evolution in the sinuses of people with CF C_LIO_LIOver time, pathoadapted strains display early signatures of genome degradation consistent with recent host restriction C_LIO_LIMutations previously thought to occur in CF lungs may have first evolved in sinuses C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/359844v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@13b36aborg.highwire.dtl.DTLVardef@8230e1org.highwire.dtl.DTLVardef@1570c3eorg.highwire.dtl.DTLVardef@1e4e530_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

microbiology↗

bioRxiv2020

The roles of history, chance, and natural selection in the evolution of antibiotic resistance

History, chance, and selection are the fundamental factors that drive and constrain evolution. We designed evolution experiments to disentangle and quantify effects of these forces on the evolution of antibiotic resistance. History was established by prior antibiotic selection of the pathogen Acinetobacter baumannii in both structured and unstructured environments, selection occurred in increasing concentrations of new antibiotics, and chance differences arose as random mutations among replicate populations. The effects of history were reduced by increasingly strong selection in new drugs, but not erased, at times producing important contingencies. Selection in structured environments constrained resistance to new drugs and led to frequent loss of resistance to the initial drug. This research demonstrates that despite strong selective pressures of antibiotics leading to genetic parallelism, history can etch potential vulnerabilities to orthogonal drugs.

evolutionary biology↗

bioRxiv2020

Quorum sensing provides a molecular mechanism for evolution to tune and maintain investment in cooperation

As selection frequently favors non-cooperating defectors in mixed populations with cooperators, mechanisms that promote cooperation stability clearly exist. One potential mechanism is bacterial cell-to-cell communication, quorum sensing (QS), which can allow cooperators to prevent invasion by defectors. However, the impact of QS on widespread maintenance of cooperation in well-mixed conditions has not been experimentally demonstrated over extended evolutionary timescales. Here, we use wild-type ( WT) Vibrio harveyi that regulates cooperation with QS and an unconditionally cooperating ( UC) mutant to examine the evolutionary origins and subsequent dynamics of novel defectors during a long-term evolution experiment. We found that UC lineages were completely outcompeted by defectors, whereas functioning QS enabled the maintenance of cooperative variants in most WT populations. Sequencing of evolved populations revealed multiple luxR mutations that swept the UC lineages. However, the evolution of mutant lineages with reduced levels of bioluminescence ( dims) occurred in many WT lineages. These dim variants also decreased other cooperative phenotypes regulated by QS, such as protease production, indicating they result from changes to QS regulation. This diminished investment phenotype optimizes a trade-off between cooperative input and growth output, allowing cooperation to be maintained under QS control even in the presence of evolved defectors.

evolutionary biology↗

bioRxiv2020

Quantitative mapping of mRNA 3' ends in Pseudomonas aeruginosa reveals putative riboregulators and a pervasive role for transcription termination in response to azithromycin

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3 ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3 end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3 ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3 end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3 end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

microbiology↗

bioRxiv2020

Genotypic diversity between surgical and nasal Staphylococcus aureus isolates

Staphylococcus aureus is a common organism in periprosthetic joint infection (PJI). Little is known about S. aureus genetic diversity in PJI as compared to nasal carriage. We hypothesized PJI S. aureus strains would be associated with increased virulence as compared to those from nasal carriage. Whole genome sequencing and multilocus sequence typing (MLST) was performed to genotype these two populations at high resolution. MLST revealed a variety of genotypes in both populations but many belonged to the most common clonal complexes. In nasal cultures, 69% of strains were of clonal complexes CC5, CC8, and CC30. In PJI cultures, only 51% could be classified in these common clonal complexes. Remaining strains were atypical, and these atypical strains in PJI were associated with poor host status and compromised immune conditions. Mutations in genes involved in fibronectin binding (ebh, fnbA, clfA, clfB) systematically distinguished later PJI isolates from the first PJI isolate from each patient. S. aureus isolated from nasal carriage and PJI specimens differ significantly, with the latter being more diverse. Strains associated with lower pathogenicity tended to be found in immunocompromised patients, suggesting the host immune system plays an important role in preventing PJI. Repeated mutations in S. aureus genes associated with extracellular matrix binding were identified suggesting an adaptive, parallel evolution in S. aureus during the development of PJI.

microbiology↗

bioRxiv2020

SARS-CoV-2 genome evolution exposes early human adaptations

The set of mutations observed at the outset of the SARS-CoV-2 pandemic may illuminate how the virus will adapt to humans as it continues to spread. Viruses are expected to quickly acquire beneficial mutations upon jumping to a new host species. Advantageous nucleotide substitutions can be identified by their parallel occurrence in multiple independent lineages and are likely to result in changes to protein sequences. Here we show that SARS-CoV-2 is acquiring mutations more slowly than expected for neutral evolution, suggesting purifying selection is the dominant mode of evolution during the initial phase of the pandemic. However, several parallel mutations arose in multiple independent lineages and may provide a fitness advantage over the ancestral genome. We propose plausible reasons for several of the most frequent mutations. The absence of mutations in other genome regions suggests essential components of SARS-CoV-2 that could be the target of drug development. Overall this study provides genomic insights into how SARS-CoV-2 has adapted and will continue to adapt to humans. SUMMARYIn this study we sought signals of evolution to identify how the SARS-CoV-2 genome has adapted at the outset of the COVID-19 pandemic. We find that the genome is largely undergoing purifying selection that maintains its ancestral sequence. However, we identified multiple positions on the genome that appear to confer an adaptive advantage based on their repeated evolution in independent lineages. This information indicates how SARS-CoV-2 will evolve as it diversifies in an increasing number of hosts.

evolutionary biology↗

bioRxiv2020

Host adaptations in the fixLJ pathway of the Burkholderia cepacia complex increase virulence

The Burkholderia cepacia complex (BCC) is composed of multiple species, including B. multivorans and B. dolosa, that are significant pathogens for people with cystic fibrosis (CF) and are extensively resistant to many antibiotics. The fixL gene of the fixLJ 2-component system (TCS) in these BCC species shows evidence of positive selection for nonsynonymous mutations during chronic lung infection in CF. Previous work showed that the B. dolosa fixLJ system regulates 11% of the genome and modulates biofilm formation, motility, persistence within macrophages, and virulence in a murine pneumonia model. Here, we assess the impacts of clinically observed FixL evolved variants in fixLJ pathway-mediated phenotypes in B. dolosa and B. multivorans. BCC carrying the ancestral fixL sequence are less pathogenic than constructs carrying evolved variants in both a macrophage infection model and a murine pneumonia model. In vitro phospho-transfer experiments demonstrate that the evolved B. dolosa FixL variants are able to reduce fixLJ pathway activity by either having lower levels of kinase activity or increased phosphatase activity. Notably, the ancestral fixL genotype has increased ability to survive within the soil compared to isogenic constructs with evolved fixL genotypes, demonstrating that increased virulence comes at an expense. Modulation of the FixLJ system has profound effects on many BCC phenotypes including full pathogenicity, and this modulation is critical for BCC adaptation to the host.

microbiology↗

bioRxiv2020

One gene, multiple ecological strategies: a biofilm regulator is a capacitor for sustainable diversity

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR. RpfR uniquely integrates two major signaling systems -- quorum sensing and the motile-sessile switch mediated by cyclic-d-GMP -- by two domains that sense, respond to, and control synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions. Significance statementMany organisms including bacteria live in fluctuating environments requiring attachment and dispersal. These lifestyle decisions require multiple external signals to be processed by several genetic pathways, but how they are integrated is largely unknown. We conducted multiple evolution experiments totaling >20,000 generations with Burkholderia cenocepacia populations grown in a model of the biofilm life cycle and identified parallel mutations in one gene, rpfR, that is a conserved central regulator. Because RpfR has multiple sensor and catalytic domains, different mutations can produce different ecological strategies that can coexist and even increase net growth. This study demonstrates that a single gene may coordinate complex life histories in biofilm-dwelling bacteria and that selection in defined environments can reshape niche breadth by single mutations.

microbiology↗

bioRxiv2020

Experimental Evolution in vivo to Identify Selective Pressures During Pneumococcal Colonization

Experimental evolution is a powerful technique to understand how populations evolve from selective pressures imparted by the surrounding environment. With the advancement of whole-population genomic sequencing it is possible to identify and track multiple contending genotypes associated with adaptations to specific selective pressures. This approach has been used repeatedly with model species in vitro, but only rarely in vivo. Herein we report results of replicate experimentally evolved populations of Streptococcus pneumoniae propagated by repeated murine nasal colonization with the aim of identifying gene products under strong selection as well as the population-genetic dynamics of infection cycles. Frameshift mutations in one gene, dltB, responsible for incorporation of D-alanine into teichoic acids on the bacterial surface, evolved repeatedly and swept to high frequency. Targeted deletions of dltB produced a fitness advantage during initial nasal colonization coupled with a corresponding fitness disadvantage in the lungs during pulmonary infection. The underlying mechanism behind the fitness tradeoff between these two niches was found to be enhanced adherence to respiratory cells balanced by increased sensitivity to host-derived antimicrobial peptides, a finding recapitulated in the murine model. Additional mutations were also selected that are predicted to affect trace metal transport, central metabolism and regulation of biofilm production and competence. These data indicate that experimental evolution can be applied to murine models of pathogenesis to gain insight into organism-specific tissue tropisms. ImportanceEvolution is a powerful force that can be experimentally harnessed to gain insight into how populations evolve in response to selective pressures. Herein we tested the applicability of experimental evolutionary approaches to gain insight into how the major human pathogen Streptococcus pneumoniae responds to repeated colonization events using a murine model. These studies revealed the population dynamics of repeated colonization events and demonstrated that in vivo experimental evolution resulted in highly reproducible trajectories that reflect the environmental niche encountered during nasal colonization. Mutations impacting the surface charge of the bacteria were repeatedly selected during colonization and provided a fitness benefit in this niche that was counterbalanced by a corresponding fitness defect during lung infection. These data indicate that experimental evolution can be applied to models of pathogenesis to gain insight into organism-specific tissue tropisms.

microbiology↗