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INTRODUCTION

Rhomboid proteases are intramembrane serine proteases that play a key role in regulating membrane proteins across all domains of life. However, their function in archaea remains poorly understood. The model halophilic archaeon Haloferax volcanii encodes two rhomboid homologs, rho1 (HVO_1474) and (HVO_0727). Previous studies indicated that the deletion of rho2 resulted in mild alterations in motility, adhesion, biofilm formation, and cell morphology, suggesting potential functional compensation by .

MATERIALS AND METHODS

To investigate the role of these proteases, we generated single (Δ) and double (Δ Δ) deletion mutants. Phenotypic characterization included viability assays, motility tests, adhesion and biofilm formation studies, as well as morphological analysis using microscopy. Functional overlap between rho1 and rho2 was evaluated through genetic complementation/overexpression experiments in which each gene was expressed in trans in the mutant backgrounds.

RESULTS

Both Δ and Δ Δrho2 mutants were viable, indicating that these genes are not essential in H. volcanii. The Δ mutant exhibited increased motility, enhanced biofilm formation, reduced adhesion to glass surfaces, and significant morphological alterations, particularly in trace element-deficient conditions. The double mutant (Δ Δ) showed increased adhesion to surfaces, mild motility reduction, and fewer morphological abnormalities compared to Δ. Complementation assays revealed that both rho1 and could restore motility in Δ and adhesion in Δrho1. However, only rho1 was able to complement the morphological defects, suggesting a degree of functional divergence between these homologs.

DISCUSSION

This work highlights the role of rhomboid proteases in regulating critical cell surface processes in , including biofilm formation, surface adhesion, and cell shape determination. The ability of rho1 and to compensate for each other in certain functions while maintaining distinct roles underscores a complex regulatory interplay. Future research will focus on identifying natural substrates and elucidating the molecular mechanisms underlying rhomboid protease function in haloarchaea.

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

引言

菱形蛋白酶是膜内丝氨酸蛋白酶，在调节生命所有领域的膜蛋白中起关键作用。然而，它们在古菌中的功能仍知之甚少。模式嗜盐古菌沃氏嗜盐栖热菌编码两种菱形同源物，rho1（HVO_1474）和rho2（HVO_0727）。先前的研究表明，rho2的缺失导致运动性、黏附性、生物膜形成和细胞形态的轻微改变，提示rho1可能具有潜在的功能补偿作用。

材料与方法

为了研究这些蛋白酶的作用，我们构建了单缺失（Δrho1）和双缺失（Δrho1Δrho2）突变体。表型特征包括活力测定、运动性测试、黏附与生物膜形成研究，以及使用显微镜进行的形态分析。通过基因互补/过表达实验评估rho1和rho2之间的功能重叠，其中每个基因在突变体背景中进行反式表达。

结果

Δrho1和Δrho1Δrho2突变体均存活，表明这些基因在沃氏嗜盐栖热菌中并非必需。Δrho1突变体表现出运动性增加、生物膜形成增强、对玻璃表面的黏附减少以及显著的形态改变，特别是在微量元素缺乏的条件下。与Δrho1相比，双突变体（Δrho1Δrho2）表现出对表面的黏附增加、运动性轻度降低以及形态异常较少。互补试验表明，rho1和rho2均可恢复Δrho1的运动性以及Δrho2的黏附性。然而，只有rho1能够弥补形态缺陷，提示这些同源物之间存在一定程度的功能差异。