## 3 LPS transport to the Outer Membrane
### Molecular Mechanism of Lpt Transport: Towards the Transenvelope Bridge Model
In line with this model, a recent work fromKahne's group demonstrated that the Lpt proteins co-fractionate in sucrose density gradient centrifugation in a lighter OM fraction (referred to as OM\({}_{\rm L}\)) containing IM and OM components and that these proteins all physically interact to form a transenvelope bridge [65]. The finding that mutations impairing Lpt complex assembly result in degradation of the periplasmic component LptA [74], confirms the biological relevance of the transenvelope structure _in vivo_. Interestingly, the transenvelope model of transport is reminiscent of a model postulated more than 40 years ago by [PERSON], who based on transmission electron micrographs of plasmolysed cells suggested the existence of lipid bridges connecting IM and OM with the function of facilitating the transit of hydrophobic molecules as LPS across the aqueous periplasmic compartment [75,76]. Although initially controversial, this hypothesis obtained confirmations in two studies published some years later demonstrating the existence of zones of adhesion between IM and OM where newly synthesized LPS transiently appears during cell growth [77]. These structures were then isolated as a lighter OM fraction (the same OM\({}_{\rm L}\) fraction described later by [PERSON] and co-workers) containing IM and OM components along with murein [78]. To date, the evidence of the existence of these bridges made up of proteins appears definitive. However, some issues concerning the molecular mechanism of LPS transport mediated by the Lpt system still need to be clarified: first, how do the Lpt proteins physically favor the transit of LPS across the periplasm? And second, is the ATP hydrolysis needed to assemble the bridge or is it only required to extract the LPS from the IM? Some clues about the mechanism by which the transenvelope protein machine physically favors LPS transit through the periplasm arose from recent work by [PERSON]'s group exploiting the powerful tool of the photo-crosslinking _in vivo_. This chemical approach allows the identification of protein residues involved in protein-protein or protein-ligand interactions by labeling the proteins of interest _in vivo_ with an UV reactive cross-linkable aminoacid analog [79,80]. Using this method the authors demonstrated that LptE, which was previously found to reside within the lumen of the \(\beta\)-barrel of LptD [63], interacts directly with some residues of a predicted extracellular loop of LptD adopting a \"plug and barrel\" architecture [81]. As LptE also binds LPS _in vitro_[63], this finding suggests a dual role for this protein: a structural component of the LptDE complex and a recognition site for LPS at the OM [63,81]. In a more recent work, the same group identified the regions in LptA, LptC, and in the \(N\)-terminal domain of LptD that mediate contacts between the proteins and thus are implicated in the formation of the transenvelope bridge. These studies showed that _in vivo_ LptA contacts LptC at the IM via its \(N\)-terminal region and LptD at the OM via its \(C\)-terminal region thus creating a continuous bridge of antiparallel \(\beta\)-strands between IM and OM [64]. Indeed, LptA and LptC belong to the same OstA superfamily of the \(N\)-terminal domain of LptD and, as mentioned above, share a very similar three dimensional structure [66,69]. Interestingly, the periplasmic but not the transmembrane domain of LptC appears to be also required for interaction with the IM transporter LptBFG [82]. As the periplasmic loop of LptF and LptG are predicted to assume the \(\beta\)-jellyroll structure shared by LptA, LptC and LptD it has been proposed that the transenvelope bridge is based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components [82]. These observations are in agreement with the genomic evidence that many Gram-negative organisms encode predicted fusion proteins containing as many as four OstA superfamily domains [83]. It is not yet known how many LptA molecules compose the transenvelope bridges.