Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

Chair

Prof.

Viral-induced compartmentalization of a bacterial cell

Invited speaker

Invited lecture

Bacterial antiviral defense by a ubiquitin-like system

Prof.Dr

Integration of chemical defense in the context of the bacterial immune system

FG Viruses of microbes

Under the constant threat of predation by (bacterio-)phages, bacteria evolved a vast array of anti-phage defense mechanism. Besides protein- and RNA-based defense systems, recent studies have highlighted the role of Streptomyces-derived secondary metabolites, such as the DNA-intercalating anthracyclines1 as well as several aminoglycoside antibiotics as further facet of bacterial immunity2-3. Molecules of both classes have been shown to inhibit infection of various dsDNA phages by interfering with an early stage of the phage life cycle3.Here, we focus on the influence of antibiotic resistance genes on the molecular versatility of aminoglycosides. Remarkably, we showed that drug modification via different acetyltransferases as well as target site modification via 16S rRNA methylation enables an uncoupling of the antibacterial and antiviral properties of these compounds, providing the prerequisite for community-wide antiphage defense. Inhibition of a Streptomyces phage was also achieved using the supernatants from natural aminoglycoside producers, hinting at a broad physiological significance of the antiviral properties of aminoglycosides.4Additionally, we aim to assess specific phage characteristics that render them susceptible to chemical defense mechanisms. To this end, a diverse set of Streptomyces phages as well as the BASEL phage collection, comprising 69 fully characterized E. coli phages5, was screened against known antiphage small molecules. This screening revealed distinct, taxonomically-related clusters of phages, which are particularly sensitive to certain compounds. Moreover, we intend to explore the integration of chemical defense strategies within the broader context of the bacterial immunity by investigating potential synergistic interactions with other defense mechanisms.We expect the outcomes of this project to deepen our understanding of multicellular defense via small molecules and provide a more comprehensive view of bacterial antiphage immunity.1 Kronheim et al. (2018), Nature 564, 283-286, doi:10.1038/s41586-018-0767-x.2 Kever et al. (2022), mBio 13, e00783-00722, doi:10.1128/mbio.00783-22.3 Hardy et al. (2023), Trends Microbiol 31, 92-106, doi:10.1016/j.tim.2022.08.001.4 Kever et al. (2024), µLife 5, uqae015, doi: 10.1093/femsml/uqae015.5 Maffei et al. (2021), PLoS Biol 19, e3001424, doi:10.1371/journal.pbio.3001424

Short lecture

The role of a phage defense island in enterohemorrhagic Escherichia coli

Introduction: Enterohemorrhagic Escherichia coli (EHEC) O175:H7 strain EDL933 is a foodborne pathogen that can cause life-threatening infections, such as bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). We found that EDL933 is highly resistant to most phages that efficiently lyse E. coli K12 strains. Using DefenseFinder, three nuclease/helicase-based phage defense systems were predicted to be encoded on an O-island in EHEC strains, including Zorya-II, Druantia-III and Serket-I. Goals: We hypothesize that this phage defense island contributes to the strong phage resistance of EHEC and that the three systems act synergistically in the anti-phage defense. Thus, we aimed to determine the phage spectrum protected by each defense system and to study their synergistic interactions. We are further interested in the molecular mechanisms of the anti-phage defense. Materials & Methods: We used EOP and growth assays under phage infections to analyze the phage spectrum and synergy of the defense systems. Biochemical and structural analyses were applied to study the molecular mechanism of the Druantia-III and Serket-I antiphage defense. Results: EOP assays with druHE, zorABE and serABCD-deficient strains revealed that the three defense systems protect to different extents against a broad spectrum of restriction-sensitive phages, but not against hypermodified Tevenvirinae. Using double system mutants, we found that Zorya-II, Druantia-III and Serket-I provide additive resistance and synergy towards different phages. Biochemical and structural analysis established that DruE functions as 3"-5"-directional DNA helicase and nuclease containing unique molecular tools, which affect its molecular activities. Using in vivo complementation studies with DruE variants, we established that the DUF1998 domain with its conserved 4Cys-Zn2+ cluster is essential for the anti-phage defense, while the PLD1 and PLD2 domains are dispensable for functionality. Summary: Overall, we hypothesize that the three defense systems cooperate in phage DNA sensing, discriminate phage and host DNA by methylation, cleave non-methylated phage DNA at specific restriction sites and subsequently degrade phage DNA.

Prof. Dr.

Molecular sensing mechanisms of phage infection mediated through the Zorya anti-bacteriophage defense system

In the dynamic battle between bacteriophages (phages) and their bacterial hosts, sophisticated anti-phage defense systems are pivotal for bacterial survival. Among these, the recently discovered Zorya system is notable for its unique approach to phage deterrence. The Zorya type I system in Escherichia coli consists of the membrane proteins ZorAB, which are homologous to the MotAB stator units of the bacterial flagellum, and the cytoplasmic effector proteins ZorCD. In this project, we aim to elucidate the molecular mechanisms that enable the Zorya system to detect and counteract phage infections. Utilizing a combination of structure-based mutagenesis, functional assays and live-cell total-internal reflection fluorescence (TIRF) microscopy, we investigate how the Zorya system responds to phage infection. Further, we explore the activation mechanism of the Zorya system and monitor the recruitment of Zorya components to the site of infection, which is crucial for inhibiting phage replication. Our research anticipates revealing detailed insights into the molecular sensing mechanisms and interactions within the Zorya system, thus contributing significantly to our understanding of bacterial immune strategies and potentially guiding the development of novel antibacterial therapies.

Chimalliviruses bend membranes and translation

On first contact, bacterial viruses called phages inject their genetic payload into the host. Strikingly, some jumbo phages of the Chimalliviridae family form a self-made early phage infection (EPI) vesicle, creating an additional cellular boundary for their injected genome together with an encapsulated virion RNA polymerase. This EPI vesicle protects the genome and compartmentalises transcription. Transcripts are produced from the EPI vesicle and released into the cytosol for immediate translation. Previously, we identified and structurally characterised the phage factor ΦKZ014, which is immediately produced and occupies translating ribosomes. ΦKZ014 is reported to be recruited to the EPI vesicle together with ribosomes and is thought to play a role in the host stress response. It remains unclear how Chimalliviridae organise membrane structures and orchestrate localised translation events at the molecular level. To elucidate the intricate coupling between transcription and translation at the interface of the EPI vesicle membrane, we imaged snapshots shortly after infection using cryo-ET. In ongoing work, we are inferring the internal organisation of phage-infected cells at the interface of cellular boundaries. Our goal is to understand how phages can create cellular structures and compartments inside bacteria and how translating ribosomes are coupled to interfaces. Insights into the EPI vesicle-ribosome interplay may pave the way for novel delivery approaches and the design of artificial compartments for biotechnological and medical applications.ReferencesGerovac et al. 2024 bioRxivWannasrichan et al. 2024 bioRxivGerovac et al. 2024 Nature Microbiology

Lecture hall 3 | HZO-60

Mini symposium

FG Viruses of microbes

Termin

Chair

Programm