Biology subjects

Duerkop, B. A.

Publications and source records attributed to Duerkop, B. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Perturbations shift the composition of bacterial DNA carried by virus-like particles in the murine gut microbiome

Horizontal gene transfer (HGT) is a driving force in microbial evolution that allows community members to rapidly evolve to cope with environmental stressors and competition. Despite the importance of HGT for the generation of genetic diversity, little is known about the specific mechanisms or dynamics of transfer in complex communities. Transductomics is a sequencing based technique which identifies potential HGT by bacteriophages (transduction) through sequencing of the transductome - the DNA carried by bacteriophages and other virus-like particles in a sample. We analyzed the murine gut transductome before and after perturbations with antibiotics and Clostridioides difficile infection (CDI). We found that several bacterial families - the Oscillospiraceae, Butyricoccaceae, and Turicibactericeae - disproportionally contributed to the transductome. Some families, like the Butyricicoccaceae, were frequent transducers in both the baseline and perturbed murine gut microbiome while other taxa displayed condition-specific transduction indicating that there may be specific transducing subpopulations or regulatory mechanisms controlling transduction frequency. Additionally, we found a diversity of highly abundant and enriched mobile genetic elements (MGEs) in the transductome including plasmids, integrative conjugative elements, phage satellites and transposons. The detection of MGEs containing conjugative elements suggest that some MGEs may spread through both transduction and conjugation. Overall, our work reveals a complex network of gene exchange occurring through transduction in the gut microbiome.

microbiology↗

bioRxiv2026

TrIdent - An R package to automate transductomics analysis of virus-like particle mediated DNA mobilization

BackgroundTransduction is a form of horizontal gene transfer in which bacterial DNA is packaged and transferred by virus-like particles (VLPs). Transductomics is a sequencing-based method used to detect DNA carried by VLPs. During transductomics analysis, reads from a samples ultra-purified VLPs are mapped to metagenomic contigs assembled from the same samples whole-community. The read mapping produces coverage patterns that require a time-consuming manual inspection and classification process which makes the methods use unfeasible for datasets with many samples. ResultsWe developed a novel algorithm, TrIdent (Transduction Identification), that uses pattern-matching to automate the transductomics data analysis and that is available as an R package (https://jlmaier12.github.io/TrIdent/). There is no software equivalent to TrIdent so we compared TrIdents classifications of transductomics datasets to classifications made by human classifiers. TrIdents classifications were generally comparable to the manual classifications on a previously generated, manually classified transductomics dataset. When applied to newly generated transductomics data from the murine microbiota, TrIdent agreed with two independent human classifiers as much as the two independent human classifications agreed with each other. TrIdent classified transductomics datasets in a fraction of the time needed by human classifiers, and the classifications produced by TrIdent are fully reproducible. We used TrIdent to explore three murine gut transductomes and found that bacterial DNA associated with the Oscillospiraceae and Turicibacteraceae families was highly enriched in the DNA packaged by VLPs as compared to the whole community metagenomes. ConclusionsThe TrIdent software is a more accessible, more efficient, and more reproducible alternative to the manual inspection of read coverage patterns previously required for transductomics data analysis. To demonstrate the application of TrIdent, we analyzed transductomics datasets from murine fecal pellets and showed that specific low abundance bacterial families appear to be heavily involved in transduction.

genomics↗

bioRxiv2025

Serine recombinases are conserved genetic markers of antiphage defense systems

Renewed interest in phage therapy has highlighted a need to understand how bacteria subvert phage infection through antiphage defense systems. Traditionally, strategies to identify antiphage defense systems lack throughput or have limitations for bacterial species where antiphage defense systems are understudied. Herein, we developed a bioinformatic pipeline that uses a small serine recombinase to identify known and unknown antiphage defense systems. Using this approach to query reference genomes and metagenomes, we show that small serine recombinase genes are genetically linked to antiphage defense systems and serve as bait for finding these systems across diverse bacterial phyla. Using co-transcription predictions and statistical analysis of protein domain abundances, we experimentally validated our bioinformatic approach by discovering that KAP P-loop NTPases are fused to putative antiphage domains and reinforce prokaryotic Schlafen proteins as a new class of antiphage defense. Our work shows that small serine recombinases are a reliable genetic marker for the discovery of antiphage defenses across diverse bacterial phyla.

microbiology↗

bioRxiv2025

Enterococcus faecium sagA mutants have cell envelope defects influencing antibiotic resistance and bacteriophage susceptibility

Enterococcus faecium is a Gram-positive bacterium that is resident to the intestines of animals including humans. E. faecium is also an opportunistic pathogen that causes multidrug resistant (MDR) infections. Bacteriophages (phages) have been proposed as therapeutics for the treatment of MDR infections, however, an obstacle for phage therapy is the emergence of phage resistance. Despite this, the development of phage resistance can impact bacterial fitness, thus, understanding the molecular basis of fitness costs associated with phage resistance can likely be leveraged as an antimicrobial strategy. We discovered that phage resistant E. faecium harbor mutations in the cell wall hydrolase gene sagA. SagA cleaves crosslinked peptidoglycan (PG) involved in PG remodeling. We show that mutations in sagA compromise E. faecium PG hydrolysis rendering them sensitive to {beta}-lactam antibiotics. sagA mutants have cell envelope integrity defects, increased cellular permeability, and aberrant distribution of penicillin binding proteins. This corresponds to a growth defect where cells have abnormal division septa, membrane blebbing, and the formation of mini cells. The dysregulation of the cell envelope in sagA mutants alters the binding of phages to the E. faecium cell surface. Our data support a model where phage infection of E. faecium requires phages to localize to sites of peptidoglycan remodeling at the cell poles and division septa. Our findings show that by altering the function of a single PG hydrolase, E. faecium loses intrinsic {beta}-lactam resistance. This indicates that phage therapy could help revive certain antibiotics when used in combination.

microbiology↗

bioRxiv2025

A prophage-encoded sRNA limits lytic phage infection of adherent-invasive E. coli

Prophages are prevalent features of bacterial genomes that can reduce susceptibility to lytic phage infection, yet the mechanisms involved are often elusive. Here, we identify a small RNA (svsR) encoded by the lambdoid prophage NC-SV in adherent-invasive Escherichia coli (AIEC) strain NC101 that confers resistance to lytic coliphages. Comparative genomic analyses revealed that NC-SV-like prophages and svsR homologs are conserved across diverse Enterobacteriaceae. Transcriptional analyses reveal that svsR represses maltodextrin transport genes, including lamB, which encodes the outer membrane maltoporin LamB--a known receptor for multiple phages. Nutrient supplementation experiments show that maltodextrin enhances phage adsorption, while glucose suppresses it, consistent with established effects of these sugars on lamB expression. In vivo, we compared wild-type NC101 and a prophage-deletion strain (NC101{Delta}NC-SV) in mice to assess the impact of NC-SV on lytic phage susceptibility. Although intestinal E. coli densities remained stable across groups, animals colonized with NC101 exhibited markedly reduced phage burdens in both the intestinal lumen and mucosa compared to mice colonized with NC101{Delta}NC-SV. This reduced phage pressure was associated with increased dissemination of NC101 to extraintestinal tissues, including the spleen and liver. Together, these findings highlight a nutrient-responsive, prophage-encoded mechanism that protects AIEC from phage predation and may promote bacterial persistence and dissemination in the inflamed gut.

microbiology↗

bioRxiv2025

Chemical inhibition of a bacterial immune system

The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-bacteriophage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report that anti-phage systems can be selectively inhibited by small molecules, thereby sensitizing phage-resistant bacteria to phages. We discovered a class of chemical inhibitors that inhibit the type II Thoeris anti-phage immune system. These inhibitors block the biosynthesis of a histidine-ADPR intracellular alarm signal by ThsB and prevent ThsA from arresting phage replication. These inhibitors promiscuously inhibit type II Thoeris systems from diverse bacteria--including antibiotic-resistant pathogens. Chemical inhibition of the Thoeris defense improved the efficacy of a model phage therapy against a phage-resistant strain of P. aeruginosa in a mouse infection, suggesting a therapeutic potential. Furthermore, these inhibitors may be employed as chemical tools to dissect the importance of the Thoeris system for phage defense in natural microbial communities.

biochemistry↗

bioRxiv2024

Enterococcal quorum-controlled protease alters phage infection

Increased prevalence of multidrug resistant bacterial infections has sparked interest in alternative antimicrobials, including bacteriophages (phages). Limited understanding of the phage infection process hampers our ability to utilize phages to their full therapeutic potential. To understand phage infection dynamics we performed proteomics on Enterococcus faecalis infected with the phage VPE25. We discovered numerous uncharacterized phage proteins are produced during phage infection of Enterococcus faecalis. Additionally, we identified hundreds of changes in bacterial protein abundances during infection. One such protein, enterococcal gelatinase (GelE), an fsr quorum sensing regulated protease involved in biofilm formation and virulence, was reduced during VPE25 infection. Plaque assays showed that mutation of either the fsrA or gelE resulted in plaques with a "halo" morphology and significantly larger diameters, suggesting decreased protection from phage infection. GelE-associated protection during phage infection is dependent on the murein hydrolase regulator LrgA and antiholin-like protein LrgB, whose expression have been shown to be regulated by GelE. Our work may be leveraged in the development of phage therapies that can modulate the production of GelE thereby altering biofilm formation and decreasing E. faecalis virulence.

microbiology↗

bioRxiv2024

Pseudo-pac site sequences used by phage P22 in generalized transduction of Salmonella

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains-LT2 and 14028S- for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems. ImportanceBacteriophage P22 has been a genetic tool and a key model for the study of generalized transduction in Salmonella since the 1950s, yet certain components of the generalized transduction molecular mechanism remain unknown. Specifically, the locations and sequences of pseudo-pac sites, hypothesized to facilitate packaging of Salmonella DNA by P22, to date have not been determined. In this study, we identified the specific locations and sequences of the pseudo-pac sites frequently recognized by P22 in Salmonella genomes. The identification of highly efficient pseudo-pac sites in Salmonella provides fundamental insights into the sequence specificity necessary for P22 pac site recognition and opens the door to more targeted use of generalized transduction with P22.

microbiology↗

bioRxiv2023

Evolution of a small phage protein confers resistance to antiphage defense in Enterococcus faecalis

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

microbiology↗

bioRxiv2023

Insertion sequence driven evolution of the microbiota revealed by a novel metagenomics pipeline

Insertion sequence (IS) elements are mobile genetic elements in bacterial genomes that support adaptation. We developed a database of IS elements coupled to a computational pipeline that identifies IS element insertions in the microbiota. We discovered that diverse IS elements insert into the genomes of intestinal bacteria regardless of human host lifestyle. These insertions target bacterial accessory genes that aid in their adaptation to unique environmental conditions. Using IS expansion in Bacteroides, we show that IS activity leads to insertion "hot spots" in accessory genes. We show that IS insertions are stable and can be transferred between humans. Extreme environmental perturbations force IS elements to fall out of the microbiota and many fail to rebound following homeostasis. Our work shows that IS elements drive bacterial genome diversification within the microbiota and establishes a framework for understanding how strain level variation within the microbiota impacts human health.

genomics↗

bioRxiv2023

Aging-associated augmentation of gut microbiome virulence capability drives sepsis severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host, but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition, but an overabundance of genomic virulence factors that have functional consequence on host immune evasion. One Sentence SummaryThe severity of sepsis in the aged host is in part mediated by longevity-associated increases in gut microbial virulence.

microbiology↗

bioRxiv2022

Targeted IS-element sequencing uncovers transposition dynamics during selective pressure in enterococci

Insertion sequences (IS) are simple transposons implicated in the genome evolution of diverse pathogenic bacterial species. Enterococci have emerged as important human intestinal pathogens with newly adapted virulence potential and antibiotic resistance. These genetic features arose in tandem with large-scale genome evolution mediated by mobile elements. Pathoadaptation in enterococci is thought to be mediated in part by the IS element IS256 through gene inactivation and recombination events. However, the regulation of IS256 and the mechanisms controlling its activation are not well understood. Here, we adapt an IS256-specfic deep sequencing method to describe how chronic lytic phage infection drives widespread diversification of IS256 in E. faecalis and how antibiotic exposure is associated with IS256 diversification in both E. faecalis and E. faecium during a clinical human infection. We show through comparative genomics that IS256 is primarily found in hospital-adapted enterococcal isolates. Analyses of IS256 transposase gene levels reveal that IS256 mobility is regulated at the transcriptional level by multiple mechanisms in E. faecalis, indicating tight control of IS256 activation in the absence of selective pressure. Our findings reveal that stressors such as phages and antibiotic exposure drives rapid genome-scale transposition in the enterococci. IS256 diversification can therefore explain how selective pressures mediate evolution of the enterococcal genome, ultimately leading to the emergence of dominant nosocomial lineages that threaten human health. Author SummaryInsertion sequence (IS) elements are simple transposons that are ubiquitous in bacteria. In the enterococci, which includes medically relevant species such as Enterococcus faecalis and Enterococcus faecium, the IS element IS256 is widespread and has been implicated in pathogenesis and antibiotic resistance. Despite the importance of IS256 to the biology of the enterococci, we know very about how this element is regulated and diversifies enterococcal genomes. Here, we show that IS256 is preferentially found in hospital-adapted and virulent strains of the enterococci. In E. faecalis V583, a vancomycin resistant blood isolate, IS256 is regulated by multiple transcriptional mechanisms. To understand how IS256 is mobilized, we adapted an Illumina-based deep sequencing method called IS-Seq to find novel IS256 insertions when applying the selective pressure of bacteriophage (phage) predation. Using this method, we found that chronic phage infection drives IS256 diversification of the E. faecalis V583 genome. Additionally, we tracked IS256 insertional activity during a human E. faecium infection and found that increased IS256 diversity was associated with specific antibiotic usage. Together, our results demonstrate that the enterococci control IS256 activity to diversify their genomes which may lead to the emergence of hospital-adapted strains that threaten human health.

microbiology↗

bioRxiv2021

Genetically distant bacteriophages elicit unique genomic changes in Enterococcus faecalis

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages promote genetic alterations in medically relevant bacteria is important as phages continue to become established biologics for the treatment of multidrug-resistant (MDR) bacterial infections. Before phages are used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages, to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole genome sequencing revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in several genes previously unreported to be associated with phage infection in E. faecalis. Intriguingly, there was only one shared mutation in the E. faecalis genome in response to each of the two phages tested, demonstrating that infection by genetically distinct phages results in different host responses. This study shows that infection of the same host by disparate phages leads to evolutionary trajectories that result in distinct genetic changes. This implies that bacteria respond to phage pressure through host responses that are tailored to specific phages. This work serves as the basis for the study of E. faecalis genome evolution during phage infection and will inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis. IMPORTANCEStudies characterizing the genome evolution of bacterial pathogens following phage selective pressure are lacking. Phage therapy is experiencing a rebirth in Western medicine. Such studies are critical for understanding how bacteria subvert phage infection and how phages evolve to counter such mutations. This study utilizes comparative genomic analyses to demonstrate how a pathogenic strain of Enterococcus faecalis responds to infection by two genetically distant phages. We show that genetic alterations in the E. faecalis genome accumulate in a manner that is specific to the infecting phage with little to no overlap in shared fixed mutations. This suggests that bacterial genome evolution in response to phage infection is uniquely tied to phage genotype, and sets a precedence for investigations into how phages drive bacterial genome evolution relevant to phage therapeutic applications.

microbiology↗

bioRxiv2021

Individuals at risk for developing rheumatoid arthritis harbor differential intestinal bacteriophage communities with distinct metabolic potential

Rheumatoid arthritis (RA) is an autoimmune disease characterized in seropositive individuals by the presence of anti-cyclic citrullinated protein (CCP) antibodies. RA is linked to the intestinal microbiota, yet the association of microbes with CCP serology and their contribution to RA is unclear. We describe intestinal phage communities of individuals at risk for developing RA, with or without anti-CCP antibodies, whose first degree relatives have been diagnosed with RA. We show that at-risk individuals harbor intestinal phage compositions that diverge based on CCP serology, are dominated by Lachnospiraceae phages, and originate from disparate ecosystems. These phages encode unique repertoires of auxiliary metabolic genes (AMGs) which associate with anti-CCP status, suggesting that these phages directly influence the metabolic and immunomodulatory capability of the microbiota. This work sets the stage for the use of phages as preclinical biomarkers and provides insight into a possible microbial-based causation of RA disease development.

microbiology↗

bioRxiv2020

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could potentially serve as antimicrobial adjuvants in treating E. faecium infections.

microbiology↗

bioRxiv2020

Phage infection mediates inhibition of bystander bacteria

Bacteriophages (phages) are being considered as alternative therapeutics for the treatment of multidrug resistant bacterial infections. Considering phages have narrow host-ranges, it is generally accepted that therapeutic phages will have a marginal impact on non-target bacteria. We have discovered that lytic phage infection induces transcription of type VIIb secretion system (T7SS) genes in the pathobiont Enterococcus faecalis. Membrane damage during phage infection induces T7SS gene expression resulting in cell contact dependent antagonism of different Gram positive bystander bacteria. Deletion of essB, a T7SS structural component, abrogates phage-mediated killing of bystanders. A predicted immunity gene confers protection against T7SS mediated inhibition, and disruption of its upstream LXG toxin gene rescues growth of E. faecalis and Staphylococcus aureus bystanders. Phage induction of T7SS gene expression and bystander inhibition requires IreK, a serine/threonine kinase, and OG1RF_11099, a predicted GntR-family transcription factor. Additionally, sub-lethal doses of membrane targeting and DNA damaging antibiotics activated T7SS expression independent of phage infection, triggering T7SS antibacterial activity against bystander bacteria. Our findings highlight how phage infection and antibiotic exposure of a target bacterium can affect non-target bystander bacteria and implies that therapies beyond antibiotics, such as phage therapy, could impose collateral damage to polymicrobial communities. Author SummaryRenewed interest in phages as alternative therapeutics to combat multi-drug resistant bacterial infections, highlights the importance of understanding the consequences of phage-bacteria interactions in the context of microbial communities. Although it is well established that phages are highly specific for their host bacterium, there is no clear consensus on whether or not phage infection (and thus phage therapy) would impose collateral damage to non-target bacteria in polymicrobial communities. Here we provide direct evidence of how phage infection of a clinically relevant pathogen triggers an intrinsic type VII secretion system (T7SS) antibacterial response that consequently restricts the growth of neighboring bacterial cells that are not susceptible to phage infection. Phage induction of T7SS activity is a stress response and in addition to phages, T7SS antagonism can be induced using sub-inhibitory concentrations of antibiotics that facilitate membrane or DNA damage. Together these data show that a bacterial pathogen responds to diverse stressors to induce T7SS activity which manifests through the antagonism of neighboring non-kin bystander bacterial cells.

microbiology↗

bioRxiv2020

Genome-wide mutagenesis identifies factors involved in Enterococcus faecalis vaginal adherence and persistence

Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro, but did not contribute to colonization in vivo. Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5 and 8 days post-inoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract. IMPORTANCEDespite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is known about the bacterial factors that influence E. faecalis vaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.

microbiology↗

bioRxiv2020

Microbial DNA on the move: sequencing based detection and analysis of transduced DNA in pure cultures and microbial communities

Horizontal gene transfer (HGT) plays a central role in microbial evolution. Our understanding of the mechanisms, frequency and taxonomic range of HGT in polymicrobial environments is limited, as we currently rely on historical HGT events inferred from genome sequencing and studies involving cultured microorganisms. We lack approaches to observe ongoing HGT in microbial communities. To address this knowledge gap, we developed a DNA sequencing based "transductomics" approach that detects and characterizes microbial DNA transferred via transduction. We validated our approach using model systems representing a range of transduction modes and show that we can detect numerous classes of transducing DNA. Additionally, we show that we can use this methodology to obtain insights into DNA transduction among all major taxonomic groups of the intestinal microbiome. This work extends the genomic toolkit for the broader study of mobile DNA within microbial communities and could be used to understand how phenotypes spread within microbiomes. Significance StatementMicrobes can rapidly evolve new capabilities by acquiring genes from other organisms through a process called horizontal gene transfer (HGT). HGT occurs via different routes, one of which is by the transfer of DNA carried by microbe infecting viruses (phages) or virus-like agents. This process is called transduction and has primarily been studied in the lab using pure cultures or indirectly in environmental communities by analyzing signatures in microbial genomes revealing past transduction events. The transductomics approach that we present here, allows for the detection and characterization of genes that are potentially transferred between microbes in complex microbial communities at the time of measurement and thus provides insights into real-time ongoing horizontal gene transfer.

microbiology↗