Foo, S.;Baum, B.

Sulfolobus acidocaldarius is a thermoacidophilic archaeon used as a model system for studying fundamental cellular processes and for emerging biotechnological applications. However, the limited availability of selectable markers restricts advanced genetic manipulation in this organism. Here, we report the development of a tryptophan auxotrophy-based selection system in S. acidocaldarius . A Δ trpBA mutant was constructed in the Δ pyrE background strain using a classical pop-in/pop-out recombination strategy. The resulting mutant exhibited little growth defects in rich medium, likely due to exogenous tryptophan supplied by complex nutrients, but failed to grow in a newly developed defined Brock-based amino acid dropout medium lacking tryptophan. Exploiting both uracil and tryptophan auxotrophies, we achieved dual-plasmid co-transformation and co-expression of the surface layer proteins and a dominant-negative mutant of the AAA-ATPase Vps4, revealing that the accumulation of surface layer lattice forming protein SlaA at the midzone of division-arrested cells together with its membrane anchor SlaB. Together, these results provide evidence for spatial regulation of S-layer assembly during archaeal cytokinesis while expanding the genetic toolkit available for S. acidocaldarius . Importance Sulfolobus acidocaldarius is a key archaeal model organism for studying cellular processes shared with more complex life and is increasingly used for biotechnological applications. Here, we establish tryptophan auxotrophy as a new selectable marker in S. acidocaldarius , expanding the range of genetic selection systems available in this organism. By developing a defined Brock-based dropout medium, we enable stringent amino acid auxotrophy selection and precise control over nutrient composition. This system can be combined with existing uracil-based selection to support dual auxotrophy workflows, enabling co-transformation, simultaneous expression of multiple proteins, and more sophisticated genetic manipulation strategies. Using both markers, we show that S-layer proteins are localised to the division bridge in cytokinesis-arrested cells. This exemplifies ways in which the expanding molecular genetic tool kit available for Sulfolobus acidocaldarius is furthering our understanding of archaeal cell biology.