Rhomboid Proteases: Key Players at the Cell Surface within Haloarchaea

Mariana Costa Micaela Cerletti Roberto Paggi Sofia Denise Frecha Valeria Zoratti Lucas Latorre Rosana Esther De Castro Maria Ines Gimenez ^*

• Instituto de Investigaciones Biologicas, Universidad Nacional de Mar del Plata-CONICET, MAR DEL PLATA - (CP: 7600), Argentina

Rhomboids are intramembrane proteases that perform hydrolytic activity within the hydrophobic environment of the cell membrane. Although conserved across the three domains of life, knowledge about archaeal rhomboids remains limited. The model halophilic archaeon Haloferax volcanii encodes two rhomboid protease homologs in its chromosome, rho1 (HVO_1474) and rho2 (HVO_0727). Previous studies identified that a rho2 knockout mutant exhibited alterations in novobiocin sensitivity, recovery after UV irradiation, motility, adhesion to glass surfaces and glycosylation at N732 of the S-layer glycoprotein. While these phenotypes were consistent, they were relatively mild, suggesting potential compensation by Rho1. To explore this hypothesis, additional mutants were generated: a rho1 knockout and a double mutant lacking both rhomboid homologs. Both single (Δrho1) and double (Δrho1 Δrho2) mutants were viable, indicating that these genes are not essential in H. volcanii. The Δrho1 mutant showed increased motility and biofilm formation, a slight reduction in adhesion to glass surfaces and profound morphological changes, including aberrant shapes and size heterogeneity, particularly in trace element-deficient medium. In contrast, the double mutant showed enhanced adhesion to glass surfaces, a mild reduction in motility (similar to Δrho2) while biofilm formation was not affected and fewer cells evidenced morphological abnormalities. These findings suggest that compensatory mechanisms likely mitigate phenotypic effects when both rhomboid homologs are absent. Potential functional overlap between rhomboid homologs was investigated through complementation experiments. Both rho1 and rho2 could complement, when expressed in trans, the motility defect of Δrho2 and the adhesion defect of Δrho1. Conversely, while rho1 could complement the morphological defects of the Δrho1 mutant, rho2 failed to do so, highlighting a degree of functional divergence. These results advance our understanding of archaeal rhomboids, revealing their participation in the regulation of critical cell surface processes including biofilm formation, surface adhesion and cell shape modulation.