In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of the addition of β-(1→2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 Å and 3.4 Å resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.

Biogenesis of membrane-bound organelles involves the synthesis, remodeling, and degradation of their constituent phospholipids. How these pathways regulate organelle size remains poorly understood. 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 find that the activity of the yeast PDAT Lro1 is regulated by a membrane-proximal helical segment facing the luminal side of the ER bilayer. To reveal the biological roles of PDATs, we engineered an Lro1 variant with derepressed activity. 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.

Outer membrane proteins (OMPs) define the surface biology of Gram-negative bacteria, with roles in adhesion, transport, catalysis and signalling. Specifically, porin beta-barrels are common diffusion channels, predominantly monomeric/trimeric in nature. Here we show that the major OMP of the bacterial predator Bdellovibrio bacteriovorus, PopA, differs from this architecture, forming a pentameric porin-like superstructure. Our X-ray and cryo-EM structures reveal a bowl-shape composite outer β-wall, which houses a central chamber that encloses a section of the lipid bilayer. We demonstrate that PopA, reported to insert into prey inner membrane, causes defects when directed into Escherichia coli membranes. We discover widespread PopA homologues, including likely tetramers and hexamers, that retain the lipid chamber; a similar chamber is formed by an unrelated smaller closed-barrel family, implicating this as a general feature. Our work thus defines oligomeric OMP superfamilies, whose deviation from prior structures requires us to revisit existing membrane-interaction motifs and folding models.

MreB, a bacterial actin homolog, plays a pivotal role in defining the shape of rod-shaped bacteria by coordinating peptidoglycan synthesis during cell elongation. It forms filaments that interact with the cytosolic leaflet of the cell membrane, as well as with membrane proteins and other cytosolic proteins. In this study, molecular dynamics simulations were used to investigate the interactions between Escherichia coli (E. coli) MreB and model cytoplasmic membranes. The simulations reveal that MreB filaments recruit cardiolipin and induce membrane bending toward the periplasmic space. Cardiolipin exhibits a concentration-dependent effect on bending, highlighting its critical role in this mechanism. Simulations with MreB mutants identify residues R105 and R136 as key contributors to both cardiolipin recruitment and membrane bending. Additionally, removal of the N-terminal helix in E. coli MreB was shown to reduce membrane bending. This study concludes that MreB induces membrane bending through two distinct mechanisms: 1) recruitment of the cone-shaped lipid cardiolipin and 2) physical distortion by the N-terminal helix. These findings uncover a novel mechanism by which MreB alters membrane architecture, offering insights into how other proteins are recruited to the filament in the E. coli elongasome.

The multiple peptide resistance factor (MprF) is a bifunctional membrane protein found in many bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus. MprF modifies inner leaflet lipid headgroups through aminoacylation and translocates modified lipid to the outer leaflet. This activity provides increased resistance to antimicrobial agents. MprF presents a promising target in multiresistant pathogens, but structural information is limited and both substrate specificity and energization of MprF-mediated lipid transport are poorly understood. Here, we present the cryo-EM structure of MprF from P. aeruginosa (PaMprF) bound to a synthetic nanobody. PaMprF adopts an "open" conformation with a wide, lipid-exposed groove on the periplasmic side that induces a local membrane deformation in molecular dynamics simulations. Using an in vitro liposome transport assay, we demonstrate that PaMprF translocates a wide range of different lipids without an external energy source. This suggests that PaMprF is the first dedicated lipid scramblase to be characterized in bacteria.

The formation of specialised membrane architectures is fundamental to biological processes. Computational modelling of membrane structures offers a means to unravel complex molecular mechanisms that remain inaccessible through in vitro or in vivo approaches. Among these architectures, toroidal membranes, otherwise known as fusion pores or fission pores, occur during the final stages of cell division, exocytosis, endocytosis, and membrane fission; including during the phagocytic-like engulfment of bacterial endospores. Here we have designed ‘MemTorMD’, a computational simulation pipeline based on the TS2CG methodology, to enable coarse-grained modelling of free toroidal membranes with biologically-relevant dimensions. This approach allowed us to determine the lipid properties required for stable toroidal membrane formation, providing a biologically accurate simulation surface. Furthermore, we demonstrate the applicability of this approach to investigate the function of proteins that localise at toroidal membranes, and drive molecular events such as membrane fission by a prokaryotic fission protein. Collectively, our work provides insight into membrane and protein behaviour at toroidal membranes and provides a platform for the development of more complex simulations involving additional molecular components at membrane toroids present in a range of processes across life. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/X008533/1 Wellcome Trust, https://ror.org/029chgv08, 208361/Z/17/Z Engineering and Physical Sciences Research Council, https://ror.org/0439y7842, EP/R029407/1

Bacteria have evolved several different biochemical pathways to either export proteins of all shapes and sizes out of the cell cytoplasm, or to secrete those proteins into the extracellular environment. Many bacterial protein secretion systems have evolutionary links to systems used by bacteriophage to move macromolecules across membranes. The Type 10 Secretion System (T10SS) was identified in gram-negative bacteria and comprises genes that bear striking sequence similarities to those found within phage lysis cassettes. The minimum components of a T10SS are an integral membrane holin-like protein together with a peptidoglycan hydrolase. Here, we review recent research in Serratia spp., Salmonella spp, Yersinia spp, and gram-positive Clostridioides spp., and consider the evidence for different T10SS mechanisms ranging from a controlled release of proteins into the environment, to stochastic altruistic lysis of specialised populations of cells.

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

Lipopolysaccharide (LPS) assembly at the surfaces-exposed leaflet of the bacterial outer membrane (OM) is mediated by the OM LPS translocon. An essential transmembrane β-barrel protein, LptD, and a cognate lipoprotein, LptE, translocate LPS selectively into the OM external leaflet via a poorly understood mechanism. Here, we characterize two additional translocon subunits, the lipoproteins LptM and LptY (formerly YedD). We use single-particle cryo-EM analysis, functional assays and molecular dynamics simulations to visualize the roles of LptM and LptY at the translocon holo-complex LptDEMY, uncovering their impact on LptD conformational dynamics. Whereas LptY binds and stabilizes the periplasmic LptD β-taco domain that functions as LPS receptor, LptM intercalates the lateral gate of the β-barrel domain, promoting its opening and access by LPS. Remarkably, we demonstrate a conformational switch of the LptD β-taco/β-barrel interface alternating between contracted and extended states. β-strand 1 of LptD, which defines the mobile side of the lateral gate, binds LPS and performs a stroke movement toward the external leaflet during the contracted-to-extended state transition. Our findings support a detailed mechanistic framework explaining the selective transport of LPS to the membrane external leaflet.

Tuberculosis (TB), a leading cause of death among infectious diseases globally, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs). These glycolipids maintain the integrity of the cell envelope, regulate permeability, and mediate host-pathogen interactions. PIMs comprise a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus in its apo and product-bound form. The structures reveal a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Key residues involved in substrate coordination and catalysis were identified and validated via in vitro assays and in vivo complementation, while molecular dynamics simulations delineated access pathways and binding dynamics. Our integrated approach provides comprehensive insights into PimE function and informs potential strategies for anti-TB therapeutics.

Gram-negative bacteria are intrinsically resistant to many antibiotics in part due to the asymmetric architecture and barrier function of their outer membrane (OM). To establish proper lipid asymmetry, cells need to ensure an intricate balance of constituent OM components, especially lipids. In this regard, the conserved, trans-envelope Tol-Pal complex plays a primary role in maintaining OM lipid homeostasis, thus OM integrity, possibly via retrograde phospholipid transport. However, mechanistic details for this process are unknown, owing to the lack of evidence for direct lipid binding. In this study, we discover that the periplasmic protein TolB, a key component of the Tol-Pal system, associates directly with membranes in vitro, via specific interactions with cardiolipin (CL). Using coarse-grained molecular dynamics simulations, we identify a CL-binding site on TolB; a single amino acid mutation at this site abolishes in vitro membrane interaction, consequently impairing cellular Tol-Pal function in maintaining OM homeostasis in Escherichia coli . Curiously, we find that the functional requirement for TolB-CL interactions can be bypassed in cells lacking CL, suggesting compensatory effects through other lipids. Our findings reveal a previously unappreciated lipid-binding role for TolB, and provide novel insights into how the Tol-Pal complex may facilitate phospholipid transport across the cell envelope. Our work will inform future strategies towards developing new antibiotics against Gram-negative bacteria. Significance The integrity of a biological membrane enables the definition of cellular boundaries, and of life. In Gram-negative bacteria, the outer membrane presents a formidable barrier against antibiotics, making it a key contributor to intrinsic antimicrobial resistance. While the Tol-Pal complex is known to ensure outer membrane stability and lipid homeostasis, its precise mechanism remains elusive. This study reveals a direct lipid-binding role for TolB, a periplasmic component of the complex, and identifies cardiolipin as a critical interacting partner. Our work establishes a novel dimension of Tol-Pal function in outer membrane lipid homeostasis and transport, opening new avenues for targeting envelope maintenance systems in Gram-negative pathogens. ### Competing Interest Statement The authors have declared no competing interest. National Medical Research Council, https://ror.org/04x3cxs03, MOH-000145 National University of Singapore, https://ror.org/01tgyzw49, AcRF Tier 1 grant

The regulation of polymerisation and translocation of biomolecules is fundamental. Wzc, an integral cytoplasmic membrane tyrosine autokinase protein serves as the master regulator of the biosynthesis and export of many bacterial capsular polysaccharides and exopolysaccharides. Such polysaccharides play essential roles in infection, defence, and some are important industrial products. Wzc comprises a large periplasmic domain, two transmembrane helices and a C-terminal cytoplasmic kinase domain with a tyrosine-rich tail. Wzc regulates polymerisation functions through cycling the formation and dissociation of an octameric complex, driven by changes in the phosphorylation status of the tyrosine-rich tail. E. coli Wzc serves a model for a wider family of polysaccharide co-polymerases. Here, we determine structures of intermediate states with different extents of phosphorylation. Structural and computational data reveal the pre-ordering of the tyrosine-rich tail, the molecular basis underlying the unidirectionality of phosphorylation events, and the underlying structural dynamics on how phosphorylation status is transmitted.

Type VII ABC transporters are ATP-powered membrane protein complexes that drive key biological processes in the bacterial cell envelope. In E. coli , three of the four Type VII ABC systems have been extensively characterised 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 the long-range transmembrane conformational changes that are the hallmark of this ABC superfamily’s mechanotransmission mechanism. We further show that YbbAP-TesA can hydrolyse 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. Significance Statement Type VII ABC transporters are unique to bacteria and play important roles in bacterial physiology including cell division, antibiotic resistance, siderophore secretion, antibiotic sensing, toxin secretion, biofilm regulation and lipoprotein trafficking. Here we identify a complex that combines an atypical Type VII ABC transporter (YbbAP) with a periplasmic lipid hydrolase (TesA). The YbbAP-TesA complex is structurally and functionally distinct from all known Type VII ABC transporter systems including FtsEX-EnvC, LolCDE-LolA, BceAB-BceS and MacAB-TolC. Structures suggest that YbbAP-TesA uses ATP-driven mechanotransmission to extract substrates from the inner membrane and hydrolyse them in the periplasmic space. The discovery and characterisation of YbbAP-TesA highlights the structural and functional diversity of Type VII ABC transporter complexes and suggests a new function for these proteins in the bacterial cell envelope. ### Competing Interest Statement The authors have declared no competing interest. Medical Research Council, MR/N014294/1 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/T00746X/1 Wellcome Trust, https://ror.org/029chgv08, 208361/Z/17/Z

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

Polymyxins are "last line of defense" antibiotics used in the treatment of multi-drug resistant gram-negative infections. However, resistance to polymyxins and other natural cationic antimicrobial peptides can develop through active modifications of lipid A, the lipidic component of the bacterial lipopolysaccharide decorating the outer membrane of gram-negative bacteria. In E. coli and S. enterica, the most effective Lipid A modification leading to polymyxin resistance is the addition of an aminoarabinose sugar (L-Ara4N) at the 4′ phosphate of lipid A.

Bacterial cell division requires septal peptidoglycan (sPG) synthesis by the divisome complex. Treadmilling of the essential tubulin homologue FtsZ has been implicated in septal constriction, though its precise role remains unclear. Here we used live-cell single-molecule imaging of the divisome transpeptidase PBP2B to investigate sPG synthesis dynamics in Bacillus subtilis . In contrast to previous models, we observed a single population of processively moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, despite the asynchronous motions of PBP2B and FtsZ, a partial dependence of PBP2B processivity on FtsZ treadmilling was observed. Additionally, through single-molecule counting experiments we provide evidence that the divisome synthesis complex is multimeric. Our results support a model for B. subtilis division where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.