Proximity-ligation-based sequencing from 111 samples and 5 environments reveals that a substantial proportion of phages infect multiple species.

Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use diff…

Humans and animals are ubiquitously colonized by Enterobacteriaceae , a bacterial family that contains both commensals and clinically significant pathogens. Here, we report Enterobacteriaceae megaplasmids of up to 1.58 Mbp in length in infant and adult guts, and other microbiomes. Of the 19 complete plasmid genomes, one was reconstructed from an Escherichia coli isolate; others were linked to species of Citrobacter and Enterobacter via analysis of genome modification patterns. The detection of related plasmids in different Enterobacteriaceae , conjugation machinery, and more diverse modified motifs in certain plasmids compared to hosts suggests that these elements are self-transmissible, with a broad host range. The plasmids encode multi-drug efflux systems and potential secreted effectors. Up to 208 tRNAs are encoded, and include sequence variants that may counter tRNA-centric defense mechanisms. Overall, the vast megaplasmid coding capacity may broaden host range, increase competitiveness, control invasion by other elements, and counter programmed cell death. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, 5R01AI092531 Swiss National Science Foundation, IZJFZ3-177614 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/X011011/1, BBS/E/QU/230002A, BB/T008717/1 Chan Zuckerberg Initiative (United States), https://ror.org/02qenvm24 Finnish Multidisciplinary Centre of Excellence in Antimicrobial Resistance Research, 346127

Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness. ### Competing Interest Statement M.W.G.W. is a co-founder of Can9 Bioengineering. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits. A patent application has been filed related to this work (PCT Patent Application No. PCT/US24/40027). The remaining authors declare no competing interests. National Science Foundation, https://ror.org/021nxhr62, DGE-2036197, CAREER 2239685 National Institutes of Health, NIH DP2AI177904, R01CA259634, U24GM129539 Spain Ministry of Science Innovation and Universities Grant, PID2023-147463NB-I00 Simons Foundation, SF349247 Kinship Foundation Pew Biomedical Scholarship Irma T. Hirschl Career Scientist Award Howard Hughes Medical Institute Columbia University Irving Medical Center Dean’s Office Vagelos Precision Medicine Fund

Bacteria, despite their diversity, use conserved cytoskeletal systems for their intracellular organization. In unicellular bacteria, the ParMRC DNA partitioning apparatus is well known for forming act...

Streptococcus pyogenes Cas9 is perhaps the most widely used CRISPR-Cas enzyme. Here, cryo-EM structures provide insight into the conformational landscape of Cas9 under multi-turnover conditions, sugge...

Abstract. In double-membraned bacteria, non-equilibrium processes that occur at the outer membrane are typically coupled to the chemiosmotically energized

Job details

Phages and bacteria are locked in a molecular arms race, with phage anti-defence proteins (ADPs) enabling them to evade bacterial immune systems. To streamline access to information on ADPs, we develo...

Simple guidelines and a checklist are provided for performing high-quality equilibrium binding measurements.

Merold and Bekere et al. reveal that OAS2 exists as a zinc-mediated dimer in an autoinhibited state, preventing autoreactivity to short RNAs. Dimer-to-monomer transition, dsRNA binding to a non-canonical interface, and targeting to the Golgi by myristoylation are necessary for OAS2 activation and restriction of viruses that form double membrane vesicles.