Biology subjects

Geier, R.

Publications and source records attributed to Geier, R..

3 recordsLinked to original sources

bioRxiv2026

Insights into Genome Ejection by a Therapeutic phiKMV-like Bacteriophage

Ar-KM is a phiKMV-like therapeutic bacteriophage used in clinical candidate phage therapy cocktails to treat lung infections caused by Pseudomonas aeruginosa. Here, we present an integrative structural atlas of Ar-KM proteins using cryo-EM, proteomics, and bioinformatics. From a single purified Ar-KM preparation, we identified three distinct populations: mature DNA-filled virions, open-nozzle particles with ejection proteins extending from the tail, and closed-nozzle empty particles. Near-atomic-resolution reconstructions of all three states enabled us to build atomic models for eleven structural proteins. The mature virion revealed the pre-ejection conformation of three ejection proteins, gp41, gp42, and gp43, homologous to coliphage T7s gp14, gp15, and gp16, respectively. Unlike T7, peptidoglycan hydrolase activity associated with the ejectosome resides in the gp15-like periplasmic tunnel protein gp42, whereas in T7 the lysozyme-like domain is located at the N-terminus of gp16, underscoring the structural plasticity and evolutionary mosaicity of ejection proteins. We further identified a short -helical factor, gp34, present in eight copies at the mismatched interface between the portal barrel and gp41. Gp34 forms a cage within the nozzle, acting as a molecular wedge that stabilizes the open conformation and permits gp41 to assemble into a hexameric channel during ejection. Evolutionarily, gp34 appears to be an ortholog of the essential gene gp7.3 in phage T7 and is conserved across sequenced phiKMV-like phages. We propose that this protein functions as an ejection protein assembly factor, stabilizing the open nozzle during infection and allowing the coordinated exit of ejection proteins and their assembly into a DNA-ejectosome. HighlightsO_LIStructural polymorphism of phage Ar-KM defines open and closed nozzle states C_LIO_LIEjection proteins gp43, gp42, and gp41 assemble in a 4:8:8 stoichiometry C_LIO_LILysozyme domain shows modular positioning within the ejectosome C_LIO_LIPortal protein acts as the primary barrier to genome leakage C_LIO_LIWedge protein gp34 (T7 gp7.3 ortholog) bridges portal and gp41 C_LI

biophysics↗

bioRxiv2025

High-resolution cryo-EM analysis of the therapeutic Pseudomonas phage Pa223

Cryogenic electron microscopy (cryo-EM) analysis of bacteriophages is a valuable method for deciphering virus composition and conformational plasticity. In this study, we present a high-resolution structural atlas of the Pseudomonas virus Pa223, a phage from the Bruynoghevirus genus that has recently been used in clinical cocktails for treating cystic fibrosis and non-cystic fibrosis bronchiectasis, as well as for compassionate care. By combining bioinformatics, proteomics, cryo-EM single particle analysis, and localized reconstruction, we annotated and built atomic models for eight structural polypeptide chains that form the icosahedral capsid and noncontractile tail. We discovered that the Pa223 capsid is decorated by a spike protein that features a unique triple-{beta} helix fold with no structural homologs in the database. The Pa223 tail features six trimeric tail fibers extending upwards, similar to, but shorter than, those found in phage T7. Unlike T7, the Pa223 tail is extended by two head-to-tail adaptors and sealed by a trimeric tail needle, similar to P22-like phages. We identified a protein bound around the outer perimeter of the portal protein, positioned similarly to the ejection protein gp72, which was identified in the Pseudomonas phage DEV, a Litunavirus phage and member of the reclassified Schitoviridae family. This structural hint led us to identify the Pa223 ejection proteins gp53, gp54, and gp56, which bioinformatically resemble those of T7-like phages more closely than Schitoviridae. Thus, phage Pa223 contains diverse structural elements found in P22-like, T7-like, and Litunavirus phages, providing a framework for understanding the diversification and evolution of ejection proteins in Bruynogheviruses. HighlightsO_LIThe high-resolution structure of Bruynoghevirus Pa223 reveals hybrid structural features that are shared among P22-like, T7-like, and Litunavirus phages. C_LIO_LIThe Pa223 capsid is decorated with a trimeric spike asymmetrically bound at the icosahedral 3-fold axes. C_LIO_LIThe Pa223 tail features two quasi-equivalent conformations of the head-to-tail adaptor protein arranged into two coaxial rings. C_LIO_LIIdentification of the ejection protein gp54 through structural similarity to gp72 from the Litunavirus DEV. C_LIO_LIBioinformatic mapping of the Pa223 ejection proteins gp53 and gp56 validated through mass spectrometry analysis of infectious virions. C_LI

biochemistry↗

bioRxiv2018

Alternative hydrogen uptake pathways suppress methane production in ruminants

Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic, and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H2 metabolism. Two thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyze H2 production or consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g. Ruminococcus) accounted for half of all hydrogenase transcripts. Various H2 uptake pathways were also expressed, including methanogenesis (Methanobrevibacter), fumarate reduction and nitrate ammonification (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic fumarate reducer Wolinella succinogenes in co-culture compared to pure culture. We conclude that H2 metabolism is a more complex and widespread trait among rumen microorganisms than previously recognized. There is evidence that alternative hydrogenotrophs, including acetogens and selenomonads, can prosper in the rumen and effectively compete with methanogens for H2 in low methane yield ruminants. Strategies to increase flux through alternative H2 uptake pathways, including animal selection, dietary supplementation, and methanogenesis inhibitors, may lead to sustained methane mitigation.

microbiology↗