TamL is a key player of the outer membrane homeostasis in Bacteroidetes.

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 Bacteroidetes 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 functional importance of TamL in Flavobacterium johnsoniae, an environmental Bacteroidetes. Unlike its homologues in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we discovered 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 any impact on outer membrane lipid composition. Via pull-down protein assays, we confirmed that TamL interacts with TamB in F. johnsoniae, likely forming the TAM complex. Furthermore, our in silico analysis revealed that the presence of TamL and TamB monocistronic genes is a shared genetic feature among Bacteroidetes members, including the human pathogen Capnocytophaga canimorsus where we also confirmed the essentiality of the TamL and TamB homologs. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria. SignificanceIn Proteobacteria, the outer membrane (OM) protein TamA forms with the inner membrane (IM)-anchored protein TamB the Translocation and Assembly Module Complex (TAM). which contributes to efficient biogenesis of the OM. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein of unknown role. In this work, we studied TamL in the Bacteroidetes Flavobacterium johnsoniae. We found that TamL and TamB are essential for cell viability, and that TamL depletion disrupts outer membrane stability, increases outer membrane vesicle size, and lead to higher sensitivity to OM stressors. These findings highlight TamL critical role in maintaining OM structure in Bacteroidetes. To our surprise, we also identified multiple TamL, TamB and TamA homologs in Bacteroidetes. Altogether, our findings extend the current knowledge on TAM and provide novel insights into a field of research barely investigated outside Proteobacteria.