A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization...

Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and cellular processes in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free H. volcanii strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel plasmid system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins-from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1-while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal Cas1 colocalizing with DNA-dense cellular regions and significant, dose-dependent changes in Cas1 mobility following UV-light-induced damage, providing evidence for its possible involvement in DNA damage response processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.