A bacterial CARD–NLR-like immune system controls the release of gene transfer agents

nature microbiology Article https://doi.org/10.1038/s41564-026-02316-4 A bacterial CARD–NLR-like immune system controls the release of gene transfer agents Received: 23 May 2025 Accepted: 5 March 2026 Published online: xx xx xxxx Check for updates Emma J. Banks Boris Stojilković 1 , Pavol Bárdy 2, Ngat T. Tran1, Phuong M. Nguyen , Kevin Gozzi4, Abbas Maqbool5 & Tung B. K. Le 1 , 2 3 Bacteria use immune systems to detect and defend against mobile genetic elements including phages. Gene transfer agents (GTAs) are domesticated prophages with phage-like characteristics including the ability to induce host cell lysis for gene transfer. Whether GTAs elicit or avoid bacterial immune systems is poorly understood. Here, a transposon mutagenesis with deep sequencing screen in Caulobacter crescentus identified a tripartite system, LypABC, essential for GTA-mediated cell lysis and gene transfer. LypABC resembles a caspase recruitment domain–nucleotide-binding leucine-rich repeat (CARD–NLR) anti-phage defence system. LypABC is dispensable for DNA packaging into GTA particles but required for host cell lysis, involving the peptidase domains of LypA and LypC, and the ATPase domain of LypB. As LypABC overproduction is toxic, strict regulation through the transcriptional repressor CdxB is required. CdxB binds the promoters of lypABC and of essential GTA activator genes, coupling GTA activation to host cell lysis. Our findings suggest that bacterial immune systems can be co-opted to support horizontal gene transfer by GTAs. Mobile genetic elements (MGEs), such as bacteriophages, plasmids and transposons, are double-edged swords; while they can confer certain adaptive advantages to their host through horizontal gene transfer, they often act selfishly, exploiting the host for their own propagation1. Bacteria are therefore engaged in an arms race against MGEs and have evolved an extraordinary diversity of immune systems to detect and defend against MGEs, including 150 distinct anti-MGE systems that have been identified in recent years2–8. Although immune systems are traditionally considered antagonistic to MGEs, it remains unclear whether some immune systems might be versatile and, in certain contexts, may facilitate rather than prevent MGE propagation. Amid the constant conflict between bacteria and phages, GTAs are exceptions. GTAs are selfless virus-like MGEs that have been domesticated from ancient viruses to provide beneficial functions to their hosts9–11. GTAs9,12–14 are encoded by gene clusters within a wide variety of bacterial and archaeal genomes, and are deeply integrated with their host physiology15,16. GTAs transition through a series of life stages: GTA gene cluster activation17, GTA particle assembly18, non-selective encapsulation of host DNA into GTA particles19,20, GTA particle release by host cell lysis21,22 and, finally, transfer of host DNA into recipient bacteria18,23,24. Most notably, unlike bacteriophages, GTA capsid heads are too small to package complete GTA gene clusters (for example, the Caulobacter crescentus GTA can package only ~8.3 kb of DNA yet its encoding GTA cluster is >15 kb (ref. 25)). Consequently, GTAs are unable to self-multiply and be infectious26. Despite GTA domestication, the phage-like origin and appearance of GTAs—together with life stages that include host cell lysis—are factors that may inadvertently trigger host immunity. It remains unclear how GTAs might avoid, subvert or even adopt host immune systems to complete their life stages. Here, by studying GTA-mediated host cell lysis in C. crescentus, we identified a potential bacterial immune system, LypABC, that has been adopted to control the release of GTA particles. LypABC resemble components of CARD–NLR anti-phage defence systems27. First described in Lysobacter enzymogenes, CARD–NLR immunity occurs through abortive infection that involves sacrificial death of phage-infected cells, preventing the release of mature phage particles Department of Molecular Microbiology, John Innes Centre, Norwich, UK. 2York Structural Biology Laboratory, University of York, York, UK. 3Department of Cell and Developmental Biology, John Innes Centre, Norwich, UK. 4The Rowland Institute at Harvard, Harvard University, Cambridge, MA, USA. 5 Department of Biochemistry and Metabolism, John Innes Centre, Norwich, UK. e-mail: ; 1 Nature Microbiology Article and thereby curbing infection27. The L. enzymogenes CARD–NLR system senses phage infection, somehow activating a CARD-containing protein component, which interacts with an NLR-like protein27. Cell death occurs through proteolysis-based activation of a gasdermin effector, which directly causes cell lysis by forming membrane pores and permeabilizing the cell membrane28,29. In animals, many NLR-based inflammatory responses also contain CARD components that signal to caspases30,31, which then proteolytically cleave and activate gasdermin effectors, leading to the release of pro-inflammatory cytokines and cell death32–34. Here we find that predicted anti-phage defence domains of LypABC are essential for cell lysis. We further show that LypABC specifically mediates cell lysis for GTA release, but is dispensable for DNA packaging into GTA particles. Overproduction of LypABC is highly toxic to both GTA-producing and non-producing cells, highlighting the need for this system to be tightly regulated. Lastly, we identify a transcriptional regulator, CdxB, that directly represses genes encoding GTA-activating factors and LypABC, thereby coupling GTA gene cluster activation and host cell lysis. In summary, we have identified a CARD–NLR-like system that may benefit MGEs and promote horizontal gene transfer. Results GTA-mediated host cell lysis results in ghost cell formation C. crescentus GTA synthesis is repressed under standard laboratory conditions but can be activated by deleting the master repressor gene, rogA (ref. 25; Fig. 1a). This relieves RogA-mediated repression of the gafYZ operon, which is essential for GTA activation25. The transcriptional activator GafY, together with integration host factor (IHF), co-activates the expression of GTA gene clusters and accessory genes elsewhere on the chromosome. Meanwhile, GafZ enables RNA polymerase to bypass internal transcription terminators within the core GTA gene cluster, ensuring complete expression of an entire biosynthetic gene cluster25,35,36 (Fig. 1a). To investigate the consequences of GTA activation and how this leads to host cell lysis, we observed wild-type (GTA-off) and ΔrogA (GTA-on) C. crescentus strains during stationary phase by phase-contrast microscopy (Fig. 1b). While the wild-type strain comprised almost entirely phase-dark cells with only 0.1 ± 0.1% phase-light cells (that is, ghost remnants of lysed cells), the ΔrogA mutant population was heterogeneous, consisting of a mixture of phase-dark cells and a substantially higher proportion of phase-light ghost cells (51.6 ± 2.5%) than the wil (...truncated)