Biology subjects

Weigele, P. R.

Publications and source records attributed to Weigele, P. R..

6 recordsLinked to original sources

bioRxiv2025

END nucleases: Antiphage defense systems targeting multiple hypermodified phage genomes

Prokaryotes carry clusters of phage defense systems in "defense islands" that have been extensively exploited bioinformatically and experimentally for discovery of immune functions. However, little effort has been dedicated to determining which specific system(s) within defense islands limit lytic phage reproduction in clinical bacterial strains. Here, we employed the CRISPR-based Cascade-Cas3 system to delete defense islands in a Pseudomonas aeruginosa clinical isolate to identify mechanisms of lytic phage antagonism. Deletion of one island in a cystic fibrosis-derived clinical isolate sensitized the strain to phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defense system is a Type IIS restriction endonuclease-like protein (ENDPaCF1), common in Pseudomonads, however it lacks an associated methyltransferase typical Type IIS R-M systems. ENDPaCF1 protects bacteria against phages with hypermodified DNA and is surprisingly agnostic to the specific structure of the modification, which is unlike typical type IV restriction endonucleases. In ENDPaCF1, the endonuclease domain is fused to a catalytically inactive Endonuclease III (iEndoIII), a domain that recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. We therefore propose that nucleases containing an iEndoIII domain (END nucleases) can sense diverse DNA hypermodifications. Our findings reveal modularity of the sensing and cleavage domains, as expected of a modification-dependent endonucleases. We further show that some hypermodified phages, including Pbunavirus family members and Wrowclawvirus family (Pa5oct-like) of jumbo phages, encode END nuclease inhibitors that directly bind to the nuclease, likely via the iEndoIII domain. These inhibitors are necessary for Pbunavirus to plaque on clinical isolates and sufficient to enable other hypermodified phages to plaque in the presence of this defense system.

microbiology↗

bioRxiv2024

The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution.

Mining phages for new enzymatic activities continues to be important for the development of new tools for biotechnology. In this study, we used MetaGPA--a method linking genotype to phenotype in metagenomic data--to identify deoxycytidine deaminases, a protein family highly associated with cytosine modifications in metaviromes. Unexpectedly, a subset of these deaminases exhibited a preference for 5-methylcytosine (5mC) over cytosine (C) in both mononucleotide and single-stranded DNA substrates. In a methylome sequencing workflow, preferential deamination of 5mC by these enzymes enabled direct conversion of methylated cytosine while completely eliminating any background deamination of unmodified cytosine. This direct conversion allows for precise identification of methylated sites at single-base resolution with unmatched sensitivity enabling broad applications for the simultaneous sequencing of genome and methylome.

genomics↗

bioRxiv2024

KlebPhaCol: A community-driven resource for Klebsiella research identified a novel gut phage order associated with the human gut

The growing threat of multidrug-resistant Klebsiella pneumoniae, coupled with its role in gut colonisation, has intensified the search for new treatments, including bacteriophage therapy. Despite increasing documentation of Klebsiella-targeting phages, clinical applications remain limited, with key phage-bacteria interactions still poorly understood. A major obstacle is fragmented access to well-characterised phage-bacteria pairings, restricting the collective advancement of therapeutic and mechanistic insights. To address this gap, we created the Klebsiella Phage Collection (KlebPhaCol), an open-source resource comprising 53 phages and 74 Klebsiella isolates, all fully characterised. These phages span five families - including a novel order, Felixvirales, associated with the human gut - and target 27 sequence types (including ST258, ST11, ST14) and 28 capsular-locus types (including KL1 and KL2), across six Klebsiella species. Freely accessible at www.klebphacol.org, KlebPhaCol invites the scientific community to both use and contribute to this resource, fostering collaborative research and a deeper understanding of Klebsiella-phage interactions beyond therapeutic use.

microbiology↗

bioRxiv2023

Modification of DNA by a viral enzyme and charged tRNA

Bacteriophage enzymes synthesize varied and complex DNA hypermodifications. The enzyme encoded by the phage Mu gene mom is necessary for post-replicative carbamoylmethyl addition to the exocyclic amine of deoxyadenosine in DNA during the lytic phase of the viral life-cycle. The molecular details of this modification reaction, including the molecular origins of the modification itself, have long eluded understanding. Here, we demonstrate that Mom co-opts the translational machinery of the host by harvesting activated glycine from charged tRNAGly to hypermodify adenine. Based on this insight, we report the first in vitro reconstitution of the Mu hypermodification from purified components. Using isotope labeling, we demonstrate that the carbamoyl nitrogen of the Mom modification is derived from the N6 of adenine, indicating an on-base rearrangement of the N6 aminoacylation product, possibly via a cyclic intermediate. Informed by the X-ray crystal structure of Mom, we have probed the location of the active site, identified a novel insertion, and established substrate specificities of the Mom enzyme.

biochemistry↗

bioRxiv2020

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 71 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (ImmI: ant, arc) or genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ~35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

genetics↗

bioRxiv2020

Biochemically diverse CRISPR-Cas9 orthologs

CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79 Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements. PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.

biochemistry↗