PubMed · 7538310
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R MacKinnon. 1995. Pore loops: an emerging theme in ion channel structure.. https://doi.org/10.1016/0896-6273(95)90327-5
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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.
Under iron-limiting conditions, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin (PaA), which, after complexing iron, is transported back into the cells via its outer membrane receptor FpvA. The recent finding that all FpvA receptors on the bacterial cell surface are loaded with iron-free PaA under iron limiting conditions has raised questions about the mechanism by which P. aeruginosa transports efficiently iron. We used [(3)H]PaA' [(55)Fe]PaA-Fe, and a kinetically stable chromium-PaA complex to show that iron loading of the receptor occurs through a siderophore displacement mechanism in vivo. Moreover, the fluorescence properties of iron-free PaA revealed that, after PaA-Fe uptake and dissociation, the PaA molecule is recycled into the extracellular medium. We used fluorescence resonance energy transfer (FRET) between the PaA chromophore and the FpvA tryptophans in vivo to monitor the kinetics of PaA displacement by PaA-Fe at the cell surface, the dissociation of iron from the siderophore, and the recycling of PaA back to the receptor on the outer membrane of the bacteria in real time. The loading status of FpvA (PaA versus PaA-Fe) was shown to depend on the relative concentration of the two forms of pyoverdin in the growth medium.
Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake. A ferripyoverdine receptor-deficient mutant of the P. aeruginosa strain was unable to use any of the three pyoverdines. Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern. Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine-peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines. Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site.