Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Reconstitution of phospholipid transport important for the maintenance of outer membrane lipid asymmetry in Escherichia coli (#125)

Bilge Ercan 1 , Wen Yi Low 1 , Shu Sin Chng 1
  1. NUS, Singapore, SINGAPORE

The cell envelope of Gram-negative bacteria consists of three compartments: inner membrane (IM), periplasm and outer membrane (OM). The IM is a typical phospholipid (PL) bilayer whereas the OM, which surrounds the peptidoglycan, is an asymmetrical lipid bilayer. In this membrane, lipopolysaccharides face extracellular milieu and PLs face the periplasm. This striking lipid asymmetry is the key to its effective barrier function, and therefore, renders Gram-negative bacteria more resistant to antibiotics than Gram-positive bacteria. Because of its importance, there are systems that regulate lipid asymmetry at the OM. The OmpC-Mla system is one of such systems and has been proposed to maintain lipid asymmetry by removing aberrantly localised PLs and shuttling them across the periplasm back to the IM1,2. In this system, the OmpC-MlaA complex extracts PLs from the OM and transfers them to the periplasmic substrate binding protein MlaC, which then transfers these PLs to the MlaFEDB2 complex at the IM. This scenario is based on bioinformatic and genetic analyses, and therefore direct evidence for PL transport and interactions between Mla proteins are still lacking. In this study, we set out to characterize and reconstitute putative PL transport activity between MlaC and the IM complex. We have successfully overexpressed and purified both MlaC and the soluble domain of MlaD (sMlaD), the second substrate binding protein of this system. We show that sMlaD forms SDS-resistant hexamers3. Furthermore, both MlaC and sMlaD co-purify with endogenous PLs, with preference for phosphatidylglycerol. We successfully over-expressed and purified MlaC variants with residues mutated to cross-linkable amino acid. MlaC variants with mutated surface residues cross-link to sMlaD, indicating these proteins interact in vitro. Using purified MlaC and sMlaD, as well as PL-free forms of these proteins, we demonstrate that the PL transfer occurs from sMlaD to MlaC but not the other way.

  1. 1. Malinverni, J. C. & Silhavy, T. J. An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane. PNAS 106, 8009-8014 (2009). 2. Chong, Z. S., Woo, W. F., Chng, S. S. Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli. Mol. Microbiol. 98, 1133-1146 (2015). 3. Thong, S., Ercan, B., Torta, F., Fong, Z. Y., Wong, H. A., Wenk, M. R., Chng, S. S. Defining key roles for the auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry. eLife 5:e19042 (2016).