Biology subjects

Mageeney, C. M.

Publications and source records attributed to Mageeney, C. M..

15 recordsLinked to original sources

bioRxiv2026

Genome-wide overexpression screen of Pseudomonas putida phage Emajogi

Bacteriophage genomes contain large fractions of uncharacterized genes, motivating high-throughput functional screens to illuminate their roles in host interaction and cytotoxicity. Here, we performed a genome wide overexpression assay for a Pseudomonas putida KT2440 infecting phage, Emajogi, and identified eight cytotoxic genes that reduce host growth under both basal and induced expression. We show that these proteins are conserved across phages that infect a diverse set of hosts. The most cytotoxic gene identified was a hypothetical protein (gp18) with no predicted functional domains. Transcriptomic profiling revealed that overexpression of gp18 caused a pronounced metabolic remodeling of the cell, likely shifting cell resources to phage production. Overall, this genome wide screen provides information about phage genes that were previously unannotated that can support biotechnology and novel antimicrobial development.

microbiology↗

bioRxiv2026

Chemical stressors differentially induce prophage replication in Acinetobacter baumannii strains

Prophages are prevalent genomic elements that profoundly shape microbial communities by controlling host bacterial populations through lysis or enabling genetic recombination via transduction. Standard induction protocols rely heavily on DNA-damaging agents like mitomycin C, failing to capture the complex chemical landscapes bacteria encounter in natural and clinical settings where diverse environmental insults trigger excision. Furthermore, the regulatory variations governing polylysogenic hosts containing multiple co-habiting prophages under identical stressors remain largely uncharacterized. In this study, we surveyed a panel of five clinical Acinetobacter baumannii strains containing zero to four resident prophages against a library of 57 diverse chemical stressors. We demonstrate that chemical treatments, including distinct classes of antibiotics, drive differential prophage induction patterns across different host backgrounds. This could lead to a greater understanding of the importance of prophages in antibiotic treatment regimes.

microbiology↗

bioRxiv2026

Mobile genetic elements are active and responsive to community context in model microbial consortium

Insertion and excision of genomic islands (GIs), chromosomally-integrated mobile genetic elements (MGEs), are major sources of microbial genome plasticity and can impact gene expression and phenotype of the host organism. GI mobilization also influences microbial communities beyond the host organism as GI excision generates MGEs that can be transferred between community members through horizontal gene transfer and induction of prophages can kill host populations, which impacts community structure. Established computational methods now enable precise GI mapping in genomes, as well as highly sensitive detection of GI excision from deep-genome sequencing data. We applied these approaches to metagenomic datasets from a defined soil microbial consortium grown on glass beads under hydration stress and compared GI activity with that observed in monoculture. Under these environmentally structured community growth conditions, GI excision was more abundant and involved a broader range of host species and GI types than under isolate growth conditions. Combined analysis with metatranscriptomic and metaproteomic data identified patterns of GI gene expression associated with induction. Three GIs showed particularly high excision together with strong transcription, numerous detected proteins, and evidence of association with potential transfer particles, including phages or vesicles. These results indicate that isolate studies can miss a substantial environmentally responsive layer of GI activity. More broadly, this work establishes a framework for mining existing community multi-omic datasets to quantify dynamic genome restructuring, identify active but poorly understood GIs, and generate mechanistic hypotheses about the processes that shape microbial genome plasticity and gene flow.

microbiology↗

bioRxiv2026

Phage proofing Pseudomonas putida uncovers novel broad-spectrum phage resistance protein Psh

Broad-spectrum phage resistance offers an important layer of protection against bacterial fermenter crashes during biomanufacturing processes, yet the underlying mechanisms are often poorly defined or come at a fitness cost. Using experimental evolution, we generated two Pseudomonas putida strains that were resistant to at least six phage genera. Genome sequencing revealed a single frameshift deletion in each strain that restored functionality of a Type I secretion system (T1SS) ATPase. We also found strong transcriptional upregulation of a nearby protein, PP_1794, which we coin Phage shielding helix rich protein (Psh). Overexpression experiments show that Psh protein is secreted by this restored T1SS to confer complete phage resistance. When compared to wild-type P. putida KT2440, no growth or expression defects were evident, but pyoverdine production was reduced. The Psh gene and T1SS are widely distributed across Gram-negative bacteria. These findings uncover a previously uncharacterized phage defense system in P. putida based on secretion-mediated receptor masking.

microbiology↗

bioRxiv2026

HtPIP: High-Throughput Phage Isolation Platform increases diversity and reduces isolation time using multiple bacteria

Bacteriophages are ubiquitous in nature, but relatively few have been isolated and characterized compared to the number of bacterial strains. Phage biotechnology applications benefit from a diverse library of isolated phages to kill or transfer genetic material to a bacterium of interest. However, scaling phage discovery for diverse bacterial hosts can be time consuming and costly. We developed an approach to capture novel phages for multiple bacteria strains in parallel from an environmental sample using commercially available 0.2-micron filter plates. Using this High-throughput Phage Isolation Platform (HtPIP), we isolated twelve novel phages spanning nine diverse bacterial host genera. Eleven of the isolated phages define new phage species with nine also defining new genera. We show the HtPIP can discover both DNA and RNA phages; including a Tectiviridae infecting Pseudomonas putida mt-2 and a Leviviricetes infecting a Microbacterium isolate, which represents the first cultured RNA phage infecting a host outside of proteobacteria. Using a metagenomic approach, we demonstrate that the HtPIP captures a higher proportion of novel phages compared to traditional low-throughput methods.

microbiology↗

bioRxiv2026

Discovery, induction, and screening of prophages in clinical Acinetobacter baumannii isolates

BackgroundAcinetobacter baumannii is a common bacterial pathogen in nosocomial infections. It has become one of the greatest threats to human health for its growing resistance to last resort antibiotics, which has led to a revival of phage therapy as a potential treatment. However, conventional methods for isolating A. baumannii-infecting phages are labor-intensive and often unsuccessful. MethodsOur approach involves a computational pipeline to identify temperate phages (prophages) integrated into A. baumannii genomes, followed by mitomycin C (MMC) induction of those strains to screen for active prophages. ResultsHere we show a prophage analysis for nearly 900 A. baumannii genomes. We observed MMC-triggered excision of nine prophages from eight A. baumannii strains by PCR and sequencing. Further we show four prophage form virions detectable by transmission electron microscopy, and two which can plaque on other A. baumannii isolates. ConclusionThis work demonstrates the utility and diversity of prophages for further development as therapeutics for antibiotic resistant A. baumannii.

microbiology↗

bioRxiv2025

Integrase-On-Demand: Bioprospecting integrases for targeted genomic insertion of genetic cargo

Integrases recognize defined sites within DNA and facilitate homologous recombination-independent transfer of genetic fragments. These innate capabilities render integrases as powerful biotechnology tools that mediate efficient site-specific integration of large genetic cargos. Given that genomes often lack the integration sites recognized by frequently utilized integrases, integrase technology has largely been restricted to genetic engineering of model organisms into which attB sites can be synthetically introduced. To enable single-step site-specific integrase-mediated genome editing in a broad spectrum of prokaryotes, we have devised the Integrase-On-Demand (IOD) method. IOD systematically identifies integrases, from bacteria and archaea, that can integrate into available attB sites in any target prokaryote. Computational results show that diverse bacteria generally have multiple potentially useable native attB/integrase pairs. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific genomic integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973 and measured efficiency of integration using suicide constructs. By eliminating the requirement to introduce non-native attB sites into the target genome, IOD may, when suitable transformation methods exist, allow facile genome integration of large constructs in non-model and possibly non-culturable bacteria. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/676606v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1442a69org.highwire.dtl.DTLVardef@3c91b1org.highwire.dtl.DTLVardef@8131f4org.highwire.dtl.DTLVardef@1f6a206_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Niche exclusion of a lung pathogen in mice with designed probiotic communities

For years, the airway microbiota have been theorized to be gatekeepers of respiratory health, as pathogens entering the airway make contact with resident microbes prior to or coincident with their interaction with host cells. Thus, modification of the native airway community may serve as a means of altering the local environment in favor of health. In this work, we hypothesize that synthetic bacterial communities introduced into the airway can serve as prophylactic countermeasures against infection by Burkholderia thailandensis in mice. We demonstrate that understanding of antagonistic interactions between a pathogen and airway microbiota in vitro can guide identification of probiotics with protective capabilities in vivo. Specifically, we show that niche overlap between the probiotic and pathogen is indicative of probiotic performance in vivo. This work serves as a foundation for the rational design of probiotic communities for protection against and treatment of respiratory infections.

microbiology↗

bioRxiv2023

Identification of a new antiphage system in Mycobacterium phage Butters

During lysogeny temperate phages establish a truce with the bacterial host. In this state, the phage genome (prophage) is maintained within the host environment. Consequently, many prophages have evolved systems to protect the host from heterotypic viral attack. This phenomenon of prophages mediating defense of their host against competitor phages is widespread among temperate mycobacteriophages. We previously showed that the Mycobacterium phage Butters prophage encodes a two-component system (gp30/31) that inhibits infection from a subset of mycobacteriophages that include PurpleHaze, but not Island3. Here we show that Butters gp57r is both necessary and sufficient to inhibit infection by Island3 and other phages. Gp57r acts post-DNA injection and its antagonism results in the impairment of Island3 DNA amplification. Gp57r inhibition of Island3 is absolute with no defense escape mutants. However, mutations mapping to minor tail proteins allow PurpleHaze to overcome gp57r defense. Gp57r has a HEPN domain which is present in many proteins involved in inter-genomic conflicts, suggesting that gp57r may inhibit heterotypic phage infections via its HEPN domain. We also show that Butters gp57r has orthologues in clinical isolates of Mycobacterium abscessus sp. including the phage therapy candidate strain GD91 which was found to be resistant to the panel of phages tested. It is conceivable that this GD91 orthologue of gp57r may mediate resistance to the subset of phages tested. Challenges of this nature underscore the importance of elucidating mechanisms of antiphage systems and mutations that allow for escape from inhibition. IMPORTANCEThe evolutionary arms race between phages and their bacteria host is ancient. During lysogeny, temperate phages establish a ceasefire with the host where they do not kill the host but derive shelter from it. Within the phenomenon of prophage-mediated defense, some temperate phages contribute genes that make their host more fit and resistant to infections by other phages. This arrangement has significance for both phage and bacterial evolutionary dynamics. Further, the prevalence of such antiphage systems poses a challenge to phage therapy. Thus, studies aimed at elucidating antiphage systems will further our understanding of phage-bacteria evolution as well as help with efforts to engineer therapeutic phages that circumvent antiphage systems.

microbiology↗

bioRxiv2022

An integrase clade that repeatedly targets prophage late genes, yielding helper-embedded satellites

Satellites are mobile genetic elements that rely on helper phages for their mobilization. The many known satellite-helper interactions are trans-regulatory, with gene products from one partner modulating the nucleic acid or protein activities of the other. We discovered a satellite type with a more intimate cis-regulatory configuration: integrated within, and co-oriented with, a late gene of its lambdoid helper prophage. This helper-embedded satellite (HES) configuration would delay expression of the interrupted helper late gene until the satellite excises; it also offers potential passive components to both HES replication and late transcription, driven by the helper. Induction of a helper-satellite composite was monitored; precise excision of the entire composite was observed, followed by its replication, and the excision of the satellite from it. We mapped 491 HESs to one of 14 sites in cognates of phage lambda late genes A, B, C, E, V, T, H, L and J. The associated integrases form a single phylogenetic clade with subclades respecting the 14 site groups, while the attP attachment site regions contained a new doubled DNA sequence motif. This clade thus exhibits a repeated tropism for prophage late genes as it develops new integration sites. HESs bear close genomic similarities to gram-negative phage-induced chromosomal islands (PICIs, of which we found many more integrated into fis and hpt genes). We describe four ordered zones in a general HES/PICI genome organization: an integration zone encoding integrase and AlpA, a Bro zone encoding members of the Bro-N network of domain-swapping DNA-interactive proteins and immunity repressor RNAs, a replication zone, and a late zone in which clusters as large as 18 consecutive helper late genes have been captured. Like the late zone, the Bro zone is dynamic, perhaps due to activity of the Bro proteins themselves.

bioinformatics↗

bioRxiv2021

Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies

The respiratory virus responsible for Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-2), has impacted nearly every aspect of life worldwide, claiming the lives of over 2.5 million people globally, at the time of this publication. Neutralizing nanobodies (VHH) represent a promising therapeutic intervention strategy to address the current SARS-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH antibodies were identified and evaluated for their capacity to tightly bind to the SARS-2 receptor-binding domain (RBD), to prevent binding of SARS-2 spike (S) to the cellular receptor Angiotensin-converting enzyme 2 (ACE2), and to neutralize viral infection. Preliminary preclinical evaluation of multiple nanobody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-2. The identified and characterized nanobodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic. Author SummaryTo fully address the on-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-2), it will be important to have both vaccines and therapeutic strategies to prevent and mitigate the effects of SARS-2. In this study, we describe the identification and characterization of potently neutralizing humanized single domain heavy chain (VHH) antibodies that have binding affinity for both the original Wuhan strain and widely circulating B.1.1.7/UK strain. VHH antibodies have the same therapeutic potential as conventional antibodies in half the size and with greater stability and solubility. Using a synthetic humanized high-diversity VHH phage library we identified several candidates with strong affinity for the SARS-2 spike that block the interaction of SARS-2 spike with the cellular receptor ACE2, and effectively neutralize infection with SARS-2 in vitro. By sequencing viral escape mutants generated in the presence of each VHH we mapped the binding sites of the VHH antibodies and assessed their affinity against newly emerging SARS-2 variants. Finally, we demonstrate that two of these VHH antibodies show prophylactic and therapeutic efficacy in vivo against challenge with SARS-2. This study establishes that screening highly diverse VHH phage libraries against viral threats can yield highly effective therapeutic agents in real time.

immunology↗

bioRxiv2020

Mycobacterium phage Butters-encoded proteins contribute to host defense against viral attack

A diverse set of prophage-mediated mechanisms protecting bacterial hosts from infection has been recently uncovered within Cluster N mycobacteriophages. In that context, we unveil a novel defense mechanism in Cluster N prophage Butters. By using bioinformatics analyses, phage plating efficiency experiments, microscopy, and immunoprecipitation assays, we show that Butters genes located in the central region of the genome play a key role in the defense against heterotypic viral attack. Our study suggests that a two component system articulated by interactions between protein products of genes 30 and 31 confers defense against heterotypic phage infection by PurpleHaze or Alma, but is insufficient to confer defense against attack by the heterotypic phage Island3. Therefore, based on heterotypic phage plating efficiencies on the Butters lysogen, additional prophage genes required for defense are implicated. IMPORTANCEMany sequenced bacterial genomes including pathogenic bacteria contain prophages. Some prophages encode defense systems that protect their bacterial host against heterotypic viral attack. Understanding the mechanisms undergirding these defense systems will be critical to development of phage therapy that circumvents these defenses. Additionally, such knowledge will help engineer phage-resistant bacteria of industrial importance.

microbiology↗

bioRxiv2020

Computational front end to diverse therapeutic phage cocktails

New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements (IGEs) in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five of these were engineered for non-lysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The {Delta}int phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection. SIGNIFICANCE STATEMENTThe antibiotic-resistance crisis in medicine and agriculture has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

microbiology↗

bioRxiv2020

New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements

Integrative genetic elements (IGEs) are mobile multigene DNA units that integrate into and excise from host bacterial chromosomes. Each IGE usually targets a specific site within a conserved host gene, integrating in a manner that preserves target gene function. However, a small number of bacterial genes are known to be inactivated upon IGE integration and reactivated upon excision, regulating phenotypes of virulence, mutation rate, and terminal differentiation in multicellular bacteria. The list of regulated gene integrity (RGI) cases has been slow-growing because IGEs have been challenging to precisely and comprehensively locate in genomes. We present software (TIGER) that maps IGEs with unprecedented precision and without attB site bias. TIGER uses a comparative genomic, ping-pong BLAST approach, based on the principle that the IGE integration module (i.e., its int-attP region) is cohesive. The resultant IGEs, along with integrase phylogenetic analysis and gene inactivation tests, revealed 19 new cases of genes whose integrity is regulated by IGEs (including dut, eccCa1, gntT, hrpB, merA, ompN, prkA, tqsA, traG, yifB, yfaT and ynfE), as well as recovering previously known cases (in sigK, spsM, comK, mlrA, and hlb genes). It also recovered known clades of site-promiscuous integrases and identified possible new ones.

bioinformatics↗

bioRxiv2020

Genome Sequences of Burkholderia thailandensis strains E421, E426, and DW503

We present the draft genome sequences of three Burkholderia thailandensis strains: E421, E426, and DW503. E421 consists of 90 contigs of 6,639,935bp and 67.73% GC content. E426 consists of 106 contigs of 6,587,853bp and 67.73% GC content. DW503 consists of 102 contigs of 6,458,767bp and 67.64% GC content.

genomics↗