Giovannercole Fabio, De Smet Tom, Vences-Guzmán Miguel Ángel, Lauber Frédéric, Dugauquier Rémy, Dieu Marc, Lizen Laura, Dehairs Jonas, Lima-Mendez Gipsi, Guan Ziqiang, Sohlenkamp Christian, Renzi Francesco
Research Unit in Biology of Microorganisms (URBM), Namur Research Institute for life Sciences (Narilis), University of Namur, Namur, Belgium.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n Col. Chamilpa, C.P. 62210 Cuernavaca, Morelos, Mexico.
J Mol Biol. 2025 May 15;437(10):169063. doi: 10.1016/j.jmb.2025.169063. Epub 2025 Mar 3.
In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidota, TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the function of TamL in Flavobacterium johnsoniae, an environmental Bacteroidota. Unlike its homologs in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we show that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe an overall decrease in the levels of β-barrel outer membrane proteins, nor substantial alterations in outer membrane lipid composition. By pull-down assays, we found TamL co-purifying with TamB in F. johnsoniae, suggesting an interaction. Furthermore, we found that while TamL and TamB monocistronic genes are conserved among Bacteroidota, only some species encode multiple TamL, TamB and TamA proteins. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria.
在变形菌门中，外膜蛋白TamA和内膜锚定蛋白TamB形成转运与组装模块（TAM）复合物，该复合物促进自转运蛋白、毒力因子以及可能的脂质跨两层膜的运输。在拟杆菌门中，TamA被TamL取代，TamL是一种N端有脂质修饰的类TamA脂蛋白，可能将其锚定在外膜上。这种结构差异表明，与TamA相比，TamL可能具有不同的功能。然而，TAM在变形菌门以外的细菌门类中的作用仍未得到探索。我们的研究旨在阐明环境拟杆菌门的约氏黄杆菌中TamL的功能。与变形菌门中的同源物不同，我们发现TamL和TamB在约氏黄杆菌中是必需的。通过遗传、表型、蛋白质组学和脂质组学分析，我们表明，TamL的缺失严重损害外膜完整性，这表现为细胞活力降低、细胞形状改变、对膜破坏剂的敏感性增加以及外膜脂蛋白水平升高。值得注意的是，我们没有观察到β-桶状外膜蛋白水平的总体下降，也没有观察到外膜脂质组成的实质性改变。通过下拉实验，我们发现TamL在约氏黄杆菌中与TamB共纯化，表明存在相互作用。此外，我们发现虽然TamL和TamB单顺反子基因在拟杆菌门中是保守的，但只有一些物种编码多种TamL、TamB和TamA蛋白。据我们所知，这项研究首次对变形菌门以外的TAM亚基提供了功能见解。
