Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography (ECT) and immunofluorescence microscopy (IF) to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. ECT mapped the location of the core and accessory components of the Legionella core-transmembrane subcomplex revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. IF deciphered an early-stage assembly process that begins with targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that interestingly are homologs of the T6SS membrane complex components TssL and TssM, suggesting the Dot/Icm T4BSS is a hybrid system. Together these results revealed that the Dot/Icm complex assembles in an “axial-to-peripheral” pattern.

Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a crystallographically identified binding site, but an additional nonspecific secondary anionic phospholipid (PL(−)) is required to generate high PIP2 sensitivity of Kir2 channel gating. The PL(−)-binding site and mechanism are yet to be elucidated. Here we report docking simulations that identify a putative PL(−)-binding site, adjacent to the PIP2-binding site, generated by two lysine residues from neighbouring subunits. When either lysine is mutated to cysteine (K64C and K219C), channel activity is significantly decreased in cells and in reconstituted liposomes. Directly tethering K64C to the membrane by modification with decyl-MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(−)s. The results provide a coherent molecular mechanism whereby PL(−) interaction with a discrete binding site results in a conformational change that stabilizes the high-affinity PIP2 activatory site.

Background: Chitin synthases are stimulated by N-acetylglucosamine (GlcNAc).Results: GlcNAc and 2-acylamido analogues of GlcNAc stimulate formation of chitin oligosaccharides by yeast chitin synthase, and GlcNAc is transferred to the 2-acylamido analogues.Conclusion: Chitin synthases use GlcNAc analogues as primers and transfer one GlcNAc at a time.Significance: Results are new insights into polysaccharide synthase mechanism and suggest ways of synthesizing novel modified polysaccharides.

Legionella pneumophila, the causative agent of Legionnaires' disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells' cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of the Legionella containing vacuole (LCV). Here we show that polar targeting of the Dot/Icm coretransmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles of L. pneumophila by themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose that Legionella co-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of the Legionella T4BSS apparatus is mediated by an innovative "outside-inside" mechanism.

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