Biology subjects

Weigele, P.

Publications and source records attributed to Weigele, P..

5 recordsLinked to original sources

bioRxiv2026

Unlocking the secrets of paired terminal genomic repeats utilised by the Burkholderia jumbo phage Matilda

Bacteriophages (phages) are viruses that infect bacteria. Jumbo phages have genomes exceeding 220 kb, which can support greater functional independence and complexity. This work identified vB_BceM_Matilda ({phi}Matilda), a novel Burkholderia-targeting jumbo phage. Before injecting its genome into the host bacterium, {phi}Matilda binds to the bacterial flagellum, the rotation of which is required to drive a productive phage infection. Its linear 267 kb dsDNA genome has a previously undescribed tripartite architecture, with bidirectional terminal elements of 1.3 kb and 25 kb, each flanked by distinct inverted repeats of 1.5 kb and 18 kb, respectively. These architectural elements bookend the genome. Transcriptomic and proteomic analyses tracked the phage infection process and confirmed that genes within these terminal elements are transcribed and translated during infection. The presence of bidirectional terminal sequences in related jumbo phages suggests that the tripartite genome architecture may be conserved and functionally important. This work represents the first description of this genome architecture in any organism, expanding our understanding of genome organisation and raising new questions about the replication, packaging, and evolution of jumbo phage genomes.

microbiology↗

bioRxiv2026

A methylome-derived m6-dAMP trigger assembles a PUA-Cal-HAD immune filament that depletes dNTPs to abort phage infection

Bacteria must distinguish phage attack from normal homeostatic processes, yet the danger signals that trigger many defence systems remain unknown. Here, we show that a PUA-Calcineurin-CE-HAD module from Escherichia coli ECOR28 confers broad anti-phage protection by binding Dam-methylated deoxyadenosine monophosphate (m6-dAMP) generated during phage-induced chromosome degradation. Ligand binding converts a preassembled PUA-Calcineurin-CE hexamer loaded with six HAD phosphatases into a polymerising filament. The filament acts as a high-flux dNTP sink through a two-enzyme cascade: HAD first dephosphorylates dATP to dADP, and Calcineurin-CE then converts dADP to dAMP. dNTP collapse halts phage replication and enforces abortive infection. Multiple mobile-element DNA mimic proteins block filament assembly, revealing a direct phage counter-defence. More broadly, our findings extend a conserved, cross-kingdom paradigm of immune filament assembly to nucleotide-depletion antiviral defence and suggest modified-nucleotide sensing by related PUA-Calcineurin-CE modules as a widespread, underappreciated bacterial strategy.

microbiology↗

bioRxiv2025

Biosynthesis of glycosylated 5-hydroxycytosine in the DNA of diverse viruses

Characterizing viral strategies for circumventing cellular defenses is critical to understanding the biology and therapeutic application of bacteriophages as antimicrobials. Many bacteriophages synthesize complex modifications (i.e., hypermodifications) to the nucleobases of their virion DNA in order to circumvent the endonuclease-based defenses of their hosts. To date, most hypermodified pyrimidines in viruses are synthesized via group transfer to a pre-existing hydroxyl moiety in DNA during the later stages of lytic development leading up to packaging of the viral chromosome into the capsid. Typically, these occur at hydroxymethylcytosine or hydroxymethyluracil. We find mono- and poly-arabinosylated cytidines completely replacing canonical cytidine in the native DNA of the Escherichia phage RB69 and Acinetobacter baumannii phage DLP3. In both cases, an arabinosyl moiety is connected directly to the pyrimidine through an ether linkage with the bridging oxygen originating from 5-hydroxycytosine (5hoC) in DNA. The 5hoC is synthesized as a mononucleotide by a virally encoded thymidylate synthase homolog. We find diverse virally encoded thymidylate synthase homologs, including the Rhizobium phage RL38J1, are capable of producing 5hoC suggesting this base modification is a starting point for glycodiverse cytosine derivatives. Characterizing these cytosine derivatives adds to our understanding of viral anti-defense mechanisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/659550v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@fcc165org.highwire.dtl.DTLVardef@1fced2aorg.highwire.dtl.DTLVardef@369ceaorg.highwire.dtl.DTLVardef@177df60_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Domainator, a flexible software suite for domain-based annotation and neighborhood analysis, identifies proteins involved in antiviral systems

The availability of large databases of biological sequences presents an opportunity for in-depth exploration of gene diversity and function. Bacterial defense systems are a rich source of diverse, but difficult to annotate genes with biotechnological applications. In this work, we present Domainator, a flexible and modular software suite for domain-based gene neighborhood and protein search, extraction, and clustering. We demonstrate the utility of Domainator through three examples related to bacterial defense systems. First, we cluster CRISPR-associated Rossman fold (CARF) containing proteins with difficult to annotate effector domains, classifying most of them as likely transcriptional regulators and a subset as likely RNAses. Second, we extract and cluster P4-like phage satellite defense hotspots and identify an abundant system related to Lamassu phage defense systems. Third, we integrate a protein language model into Domainator and use it to identify restriction enzymes with low homology to known reference sequences, validating the activity of one example in-vitro. Domainator is made available as an open-source package with detailed documentation and usage examples.

bioinformatics↗

bioRxiv2023

Four additional natural 7-deazaguanine derivatives in phages and how to make them

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2-deoxy-7-deazaguanine (dDG), and 2-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN), and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

genomics↗