PubMed · 2859528
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J R Saunders. Antigenic variation in gonococcal pili explained.. https://doi.org/10.1038/315100a0
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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.
The phytopathogen Erwinia carotovora subspecies carotovora secretes pectinases and cellulase via the general secretory pathway, a process requiring at least 13 proteins encoded by the out gene cluster. By exploiting delta::Tn5, a generalised transducing phage (psi KP) and localised mutagenesis of the out gene cluster, we have produced a histidine auxotroph and 19 new secretory mutants, including two (HJN1003 and HJN1004) which were conditional (temperature sensitive) for secretion. All of the mutants accumulated pectinases and cellulase in the periplasm, but in the case of HJN1003 and HJN1004, only at the restrictive temperature. HJN1003 and HJN1004 were complemented by the outE and outL wild-type genes, respectively, and both mutant alleles were cloned and sequenced to reveal single missense substitutions. HJN1003 carries an Arg166 to His alteration in OutE and HJN1004 carries a Pro159 to Leu alteration in OutL. Topology mapping of OutL using a beta-lactamase probe confirmed that OutL is a type II bitopic trans-inner membrane protein and that the mutated Pro159 residue in HJN1004 is located in the cytoplasmic domain of OutL. Hence, the secretion of exoenzymes across the outer membrane is critically dependent on the conformation of secretory components located at the cytoplasmic face of the inner membrane.
We have identified a new folding catalyst, PpiD, in the periplasm of Escherichia coli. The gene encoding PpiD was isolated as a multicopy suppressor of surA, a mutation which severely impairs the folding of outer membrane proteins (OMPs). The ppiD gene was also identified based on its ability to be transcribed by the two-component system CpxR-CpxA. PpiD was purified to homogeneity and shown to have peptidyl-prolyl isomerase (PPIase) activity in vitro. The protein is anchored to the inner membrane via a single transmembrane segment, and its catalytic domain faces the periplasm. In addition, we have identified by site-directed mutagenesis some of the residues essential for its PPIase activity. A null mutation in ppiD leads to an overall reduction in the level and folding of OMPs and to the induction of the periplasmic stress response. The combination of ppiD and surA null mutations is lethal. This is the first time two periplasmic folding catalysts have been shown to be essential. Another unique aspect of PpiD is that its gene is regulated by both the Cpx two-component system and the sigma32 heat shock factor, known to regulate the expression of cytoplasmic chaperones.