The N-Terminal Domain of LIC12756 Is the Key Determinant of This Protein’s Anti-Sigma Activity Toward LIC12757 in Pathogenic Leptospira interrogans

Extracytoplasmic function (ECF) σ factors are central regulators of bacterial adaptation to environmental changes. The genome of Leptospira interrogans encodes 11 such factors, including LIC12757. Previous studies have shown that LIC12757 is regulated by the FecR-like protein LIC12756, forming a regulatory system similar to the Escherichia coli FecI-FecR system. Here, the domain-specific regulatory role of LIC12756 was investigated. Interactions between LIC12757 and several LIC12756 variants, including the N-terminal domain (NTD) alone, NTD with half or full transmembrane domain (NTD-TMD), and full-length LIC12756 (FL, control), were analyzed using the BACTH system. During logarithmic growth, interactions were detected only with FL and NTD-TMD, whereas in the stationary phase, all variants interacted with varying strengths. Pull-down assays using His₆-tagged NTD confirmed its direct binding to LIC12757. Promoter activity analysis revealed that the NTD alone functions as an anti-σ factor in the logarithmic stage of growth. However, it is insufficient for full activation of LIC12757-dependent transcription during the stationary phase, as observed with FL protein. The NTD-TMD variant caused only minor stimulation compared to FL. These results indicate that NTD is a key determinant of LIC12756’s anti-σ activity toward LIC12757, whereas full activation of LIC12757 requires additional extrinsic signals, which are likely sensed by the C-terminal extracytoplasmic region (ECR). These findings provide mechanistic insight into ECF σ factor regulation in L. interrogans.