María Luisa Galbis‐Martínez

How CRISPR-Cas and cyclic oligonucleotide–based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend Myxococcus xanthus against a phage. Cryo–electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)–caspase HetF–associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ ₂ and σ ₄ domains, undergoing σ ₄ -induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti–σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.

Summary: Light‐induced carotenogenesis in Myxococcus xanthus is controlled by the B ₁₂ ‐based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B ₁₂ ‐independent CarH paralogue CarA and various other proteins, some eukaryotic‐like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA ‐ carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium , which lack CarA but retain all other factors. We experimentally show B ₁₂ ‐mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro ) is a dimer that combines direct CarH‐like B ₁₂ ‐based photoregulation with CarA‐like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B ₁₂ ‐dependent photoreceptors. It further establishes the B ₁₂ ‐dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B ₁₂ ‐independent circuit.