A protease-precursor system drives synergistic antagonism in haloarchaea

Antagonistic competition is a crucial survival strategy for microorganisms sharing ecological niches, playing a key role in shaping microbial communities and influencing biogeochemical cycles. Here, we report the first extracellular serine protease-dependent synergistic antagonism in archaea: a collaboration between an extracellular protease-producing strain and a precursor protein-producing strain. The serine protease secreted by the former cleaves the precursor protein released by the latter, generating an antibacterial effector molecule. This synergistic antagonism also occurs across domains (between halophilic bacteria and archaea), indicating broad ecological relevance. Using mass spectrometry and inhibition assays, we identified HFX_0892-from the model haloarchaeon <i>Haloferax mediterranei</i> ATCC 33500-as a key mediator of this process. Precursor protein HFX_0892 was cleaved by HlyR4 or other extracellular serine proteases, releasing the N-terminus of HFX_0892 (0892N), which displayed antagonistic activity against haloarchaea and bacteria. Disruption of the α-helical structure in 00892 via point mutations abolished the antagonistic activity. Furthermore, fusing the 0892N to HlyR4 did not interfere with HlyR4's proteolytic function but conferred antibacterial activity. Gene knockout experiments revealed that HFX_0892 is not the sole antagonistic precursor protein in <i>H. mediterranei</i> ATCC 33500. This study uncovers a modular proteolytic activation mechanism that can be harnessed for antimicrobial agent development. The potential prevalence of HFX_0892-like precursors among extremophiles provides a feasible strategy for exploring structurally novel antimicrobial agents.IMPORTANCEAntagonistic interactions are key drivers of microbial community dynamics in hypersaline environments. Here, we report, for the first time, a fan-shaped growth inhibition zone-an atypical phenotypic signature-resulting from synergistic antagonism between two halophilic archaeal species against a sensitive haloarchaeal strain. Using the model haloarchaeon <i>Haloferax mediterranei</i>, we identified a secreted precursor protein (HFX_0892) that is cleaved by a serine protease (such as HlyR4) to release an active antagonistic peptide (0892N). This novel form of archaeal interaction is defined as synergistic antagonism. The antagonistic activity of HFX_0892 is mediated by two α-helical motifs in its N-terminus, and this region can confer antimicrobial function when fused to other proteins. Notably, <i>H. mediterranei</i> encodes additional precursor proteins with potential antagonistic functions beyond HFX_0892. Our work identifies and elucidates a previously uncharacterized antagonistic interaction among archaea, providing critical insights into the complex interspecific interactions and microbial community assembly in hypersaline ecosystems.