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McAndrew, Martin B. L., Cook, Jonathan P., Gill, Amy, Sahoo, Kavya, Thomas, Clare, Stansfeld, Phillip J. and Crow, Allister

Type VII ABC transporters are ATP-powered membrane protein complexes that drive key biological processes in the bacterial cell envelope. In Escherichia coli, three of the four Type VII ABC systems have been extensively characterized, including: the FtsEX-EnvC cell division complex, the LolCDE-LolA lipoprotein trafficking machinery, and the MacAB-TolC efflux pump. Here we describe a fourth E. coli Type VII ABC system, YbbAP-TesA, which combines a Type VII ABC transporter with a multifunctional hydrolytic enzyme. Structures of the complete YbbAP-TesA complex, and of YbbAP with and without bound ATP analogues, capture implied long-range transmembrane conformational changes that are the hallmark of this ABC superfamily鈥檚 mechanotransmission mechanism. We further show that YbbAP-TesA can hydrolyze a variety of ester and thioester substrates and experimentally confirm a constellation of active site residues in TesA. Our data suggests YbbAP has a role in extracting hydrophobic molecules from the inner membrane and presenting these to TesA for hydrolysis. The work extends collective knowledge of the remarkable diversity of the ABC superfamily and establishes a new function for Type VII ABC transporters in bacterial cells.

Will Scott, Esther Ivorra-Molla, Dipayan Akhuli, Teresa Massam-Wu, Pawel K. Lysyganicz, Rylie Walsh, Matthew Parent, Jonathan Cook, Lijiang Song, Abhishek Kumar, Falk Schneider, Masanori Mishima, Allister Crow, Mohan K. Balasubramanian

Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric GFP recently monomerized through sequence engineering, addresses this challenge with its high photostability. There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here, we apply the same strategy to redshift StayGold through substitution of tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favorable photostability in vivo in Escherichia coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step toward developing redshifted StayGold derivatives.

Julia A. Fairbairn, Rachel V. Kerr, Nika-Kare A. Pierre-White, Anthony Jacovides, Becca W. A. Baileeves, Phillip J. Stansfeld, Gerhard Bringmann, Andrew T. Merritt and Timothy D. H. Bugg

Escherichia coli translocase MraY is the target for bacteriolytic protein E from bacteriophage fX174, interacting at a site close to Phe-288 on helix 9, on the extracellular face of the protein. A peptide motif Arg-Trp-x-x-Trp from protein E was used to design a set of triazinedione peptidomimetics, which inhibit particulate MraY (6d IC50 48 碌M), and show antimicrobial activity against Gram-negative and Gram-positive antibiotic-resistant clinical strains (7j MIC Acinetobacter baumannii 16 碌g/mL, Staphyloccoccus aureus MRSA 2-4 碌g/mL). Docking against a predicted structure for E. coli MraY revealed two possible binding sites close to helix 9, the binding site for protein E. Antimicrobial activity of analogue 6j was found to be synergistic with bacitracin in Micrococcus flavus, consistent with a link between this inhibition site and undecaprenyl phosphate uptake. Alkaloid michellamine B, also predicted to bind in the cleft adjacent to helix 9, was also found to be synergistic with bacitracin. These data provide experimental evidence that the unusual hydrophobic cleft adjacent to helix 9 in MraY is involved in uptake of undecaprenyl phosphate, in addition to recently identified transporters UptA and PopT, and that this process can be targetted by small molecules as a novel antibacterial mechanism.

Pawel K Lysyganicz, Antonio D Barbosa, Shoily Khondker, Nicolas A Stewart, George M Carman, Phillip J Stansfeld, Marcus K Dymond, Symeon Siniossoglou

Here we demonstrate that a lipid-degradation pathway inhibits expansion of the endoplasmic reticulum (ER) membrane. Phospholipid diacylglycerol acyltransferases (PDATs) use endogenous phospholipids as fatty-acyl donors to generate triglyceride stored in lipid droplets. The significance of this non-canonical triglyceride biosynthesis pathway has remained elusive. We show that active Lro1 mediates retraction of ER membrane expansion driven by phospholipid synthesis. Furthermore, subcellular distribution and membrane turnover activity of Lro1 are controlled by diacylglycerol produced by the activity of Pah1, a conserved member of the lipin family. Collectively, our findings reveal a lipid-metabolic network that regulates endoplasmic reticulum biogenesis by converting phospholipids into storage lipids.

Georgina Benn, Christian Bortolini, David M Roberts, Alice L B Pyne, Seamus Holden, Bart W Hoogenboom

Complement proteins eliminate Gram-negative bacteria in the blood via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, leading to inner membrane permeation and cell death.

Peter Wotherspoon, Hannah Johnston, David J. Hardy, Rachel Holyfield, Soi Bui, Giedr臈 Ratkevi膷i奴t臈, Pooja Sridhar, Jonathan Colburn, Charlotte B. Wilson, Adam Colyer, Benjamin F. Cooper, Jack A. Bryant, Gareth W. Hughes, Phillip J. Stansfeld, Julien R. C. Bergeron & Timothy J. Knowles

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD 尾6-尾7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.