Exploring the eco-evolutionary role of plasmids and defense systems in 'Fervidacidithiobacillus caldus' extreme acidophile

Sebastián Pacheco-Acosta^1,2Gustavo Castro-Toro^1,2Camila Rojas-Villalobos^1,3Cesar Valenzuela¹Haristoy Juan José¹Abraham Zapata-Araya^1,2Ana Rosa Moya-Beltrán⁴Pedro Sepúlveda-Rebolledo¹Ernesto Perez-Rueda⁵Ricardo Ulloa⁶Alejandra Giaveno⁶Francisco Luciano Issotta^1,10,7,8,9Beatriz Diez^1,7,8,9Simón Beard^1,2Raquel Quatrini^1,2*

• ¹Centro Científico y Tecnológico de Excelencia Ciencia & Vida, Santiago, Santiago Metropolitan Region (RM), Chile
• ²Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago, Santiago Metropolitan Region (RM), Chile
• ³Facultad de Ingeniería, Arquitectura y Diseño, Universidad San Sebastián, Santiago, Santiago Metropolitan Region (RM), Chile
• ⁴Departamento de Informática y Computación, Facultad de Ingeniería, Universidad Tecnológica Metropolitana, Santiago, Chile
• ⁵Instituto de Investigaciones en Matemáticas Aplicadas y en Sistemas, Universidad Nacional Autónoma de México, Unidad Académica del Estado de Yucatán,, Yucatán, Mexico
• ⁶PROBIEN (CCT Patagonia Confluencia-CONICET, UNCo), Facultad de Ingeniería, Departamento de Química, Universidad Nacional del Comahue, Neuquén, Argentina
• ⁷Centro de Genómica, Ecología y Medio Ambiente, Universidad Mayor, Santiago, Santiago Metropolitan Region (RM), Chile
• ⁸Millennium Institute Center for Genome Regulation (CGR), Santiago, Santiago Metropolitan Region (RM), Chile
• ⁹Center for Climate and Resilience Research (CR), Santiago, Santiago Metropolitan Region (RM), Chile
• ¹⁰Departamento Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica, Santiago, Chile

Plasmids are major drivers of microbial evolution, enabling horizontal gene transfer (HGT) and facilitating adaptation through the dissemination of relevant functional genes and traits. However, little is known about plasmid diversity and function in extremophiles.'Fervidacidithiobacillus caldus', a meso-thermo-acidophilic sulfur oxidizer, is a key player in sulfur cycling in natural and industrially engineered acidic environments. Here, we present a comprehensive analysis of the plasmidome, and associated anti-mobile genetic element (anti-MGE) defense systems (defensome), across genomes of this species and metagenomes from diverse natural and industrial settings harboring 'F. caldus'. We identified >30 distinct plasmids, representing five consistent replication-mobilization families. Plasmids ranged in size between 2.5-65 kb, with gene content and plasmid modularity scaling with element size and copy numbers inversely correlating with size. Plasmids carried variable numbers of hypothetical proteins and transposases, with annotated cargo genes reflecting functional differentiation by habitat. Defensome profiling revealed over 50 anti-MGE systems in sequenced 'F. caldus' isolates, including diverse restriction-modification systems, CRISPR-Cas types IV-A and V-F, and widespread abortive infection and composite defense systems such as Wadjet, Gabija, and Zorya. In environmental populations, an inverse relationship was observed between defensome complexity and plasmidome abundance and diversity, underscoring a pivotal role of the host defensome in modulating persistence, compatibility, and overall plasmid diversity across 'F. caldus' populations. Yet, other plasmids appeared decoupled from both host abundance and defensome complexity, suggesting potential host shifts, environmental persistence, or differential replication under suboptimal growth conditions for the host. Altogether, these findings point to a modular, functionally diverse adaptive plasmidome shaped by environmental pressures, by the interplay with the host´s defensome, and likely also by other eco-evolutionary processes at play in natural environments.While these associations are compelling, causal relationships remain to be experimentally validated. These insights broaden our understanding of mobile genetic elements in extreme environments and provide a foundation for plasmid-based vector design and synthetic biology applications in acidophiles, with direct implications to biomining and environmental remediation.