The Lpt multiprotein machinery for LPS transport in Gram-negative bacteria:molecular details of the Lpt interactome

The hallmark of Gram-negative bacteria is their cell envelope, which is composed of two membranes, the inner or cytoplasmic membrane (IM), and the outer membrane (OM), separated by a compartment (the periplasm) that contains a thin peptidoglycan layer. Lipopolysaccharide (LPS) is the major component of the OM, and it acts as a selective barrier together with the OM proteins (OMPs), preventing the entry of many toxic molecules into the cell. Despite the structure and composition of OM have been elucidated in pivotal studies in the 50s and in the 70s, the factors required for the assembly of this organelle have only recently been identified. LPS, once it is synthesized in the cytoplasm, has to be translocated through out the cell envelope. Seven essential proteins cooperate in a unique fashion to extract the macromolecule from the IM and deliver it in the outer leaflet of the OM. LptBCFG form the IM complex that empowers the translocation process by ATP hydrolysis (Narita and Tokuda, 2009), LptDE constitute a complex embedded in the OM that finally flips LPS across the OM and deliver it to its final destination (Chng et al., 2010a; Freinkman et al., 2011), and LptA is a periplasmic protein that contacts both the IM and OM complexes (Sperandeo et al. 2007; Tran et al., 2008). Notably, LptC is single-pass IM protein with a large periplasm-protruding region. LptC single mutants were obtained in this work by random-mutagenesis, and used in vivo and in vitro experiments to characterize two regions of the protein that distinctly interact with LptA and the IM protein complex LptBFG, respectively. Chimera versions of LptC, either missing the transmembrane (TM) sequence, or with the IM anchor substituted by a heterologous sequence, were additionally constructed to this purpose. Moreover, Both LptA and LptC were previously demonstrated to bind LPS in vitro, here it is presented a rapid bioinformatic tool which has been implemented to discover the molecular determinants of LptA for the interaction with Lipid A, the main component of LPS. Genetic evidences previously obtained in our laboratory together with the presented data strongly support the LPS transport machinery model defined as the trans-envelope complex by Chng and coworkers (Chng et al., 2010a): indeed LptA interacts both with the IM and the OM protein complexes (LptBCFG and LptDE respectively), bridging them together. In support of this model, a phylogeny and structural motif conservation analysis of the Lpt components suggests that the unique structural domain retained in these proteins—despite the low sequence similarity—is the key to make possible the interaction between all the Lpt components.