Biology subjects

Millan, V.

Publications and source records attributed to Millan, V..

3 recordsLinked to original sources

bioRxiv2026

Host exonuclease SbcB and a phage-encoded SSB-like protein control activation of the DRT10 reverse transcriptase defense system

Defense-associated reverse transcriptases (DRTs) employ diverse mechanisms of cDNA synthesis to protect bacteria against phage infection, yet their full diversity and regulatory logic remain poorly understood. Here we provide a mechanistic characterization of UG17 (DRT10), a class-2 system within the UG/Abi reverse transcriptase lineage, classifying it into three phylogenetically and architecturally distinct subtypes with subtype-specific ncRNAs and experimentally validating its role in phage defense. DRT10 operates as a tripartite module, comprising a structured ncRNA, a SLATT effector, and an RT that catalyzes processive synthesis of DNA containing 7 nt tandem repeats. The tandem-repeat cDNA intermediate accumulates as both first- and second-strand species. SbcB suppresses DRT10-mediated toxicity and appears to selectively degrade the second strand under basal conditions, while phage infection correlates with enhanced first-strand accumulation. A phage-encoded protein with predicted SSB-like architecture and a conserved C-terminal tip motif is required for efficient DRT10 activation during infection. Together, these findings establish DRT10 as a surveillance system whose activation threshold is jointly controlled by constitutive cDNA synthesis, host exonuclease activity, and a phage-encoded SSB-like trigger that perturbs host ssDNA metabolism. We propose a model in which accumulation of DRT10-derived cDNA triggers activation of the SLATT transmembrane effector to initiate immune defense.

microbiology↗

bioRxiv2025

A UG5 reverse transcriptase-nitrilase antiviral module confers phage immunity in the plant symbiont Sinorhizobium meliloti

Bacteriophages exert strong selective pressure on soil- and rhizosphere-associated bacteria, including plant-associated symbionts. Reverse transcriptase-associated defense systems of the UG family are widespread across bacterial lineages, yet their ecological roles remain largely undefined. Within this family, UG5 systems are distinguished by reverse transcriptases fused to or associated with a nitrilase domain. Here, we combine phylogenetic, metagenomic, and functional analyses to investigate the evolutionary context and antiviral activity of UG5-associated systems. Phylogenetic analysis of 728 nitrilase domains places UG5-associated nitrilases within a well-supported UG-related radiation encompassing the UG1, UG5, and UG6 families, with UG1 nested within a broader UG5 lineage. Metagenomic analysis further revealed UG5-associated reverse transcriptases in soil- and rhizosphere-derived metagenomes. Based on this observation, we characterized a UG5-large RT-associated system, here designated DRT11, encoded on the pSymA megaplasmid of Sinorhizobium meliloti RMO17, a nitrogen-fixing symbiont of Medicago sativa. Despite lacking the transmembrane protein typical of canonical UG5-large architectures, DRT11 confers protection against naturally occurring M. sativa rhizosphere phages with Podoviridae-like morphology. Phage infection assays reveal protection at low multiplicities of infection, consistent with an abortive-infection-like mechanism. Moreover, mutational analyses demonstrate that antiviral activity requires only the RT and its fused C-terminal nitrilase domain, establishing DRT11 as a minimal UG5-associated antiviral system.

microbiology↗

bioRxiv2024

Spacer acquisition in type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins

In prokaryotes, CRISPR-Cas systems store memories of past infections in the form of spacers integrated into CRISPR arrays. When associated with type III CRISPR-Cas systems, Reverse transcriptase-Cas1 fusion proteins (RT-Cas1) enable these defense systems to acquire spacers from RNA sources. However, despite the specific targeting of RNA by the Cas13-containing type VI CRISPR-Cas systems, there is no evidence of RNA-origin spacer acquisition. Using computational analyses, we recently reported the association of RT-Cas1 fusion proteins with type VI-A systems. In this study, we found that RT-Cas1 fusion proteins were also associated with complete type VI-B systems in bacteria from gut metagenomes, constituting a variant system that harbors a linked CorA-encoding locus in addition to the CRISPR array and adaptation RT-Cas1/Cas2 module. By combining in vitro and in vivo experiments, we demonstrated that type VI RT-CRISPR systems are functional for spacer acquisition and CRISPR array processing, and that the associated RT enables spacer acquisition from RNA molecules, thus demonstrating that the system is capable of functioning independently of other in-trans systems. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, suggesting a potential defense mechanism against RNA-based invaders in specific environments.

microbiology↗