Biology subjects

Chng, S.-S.

Publications and source records attributed to Chng, S.-S..

At least 19 recordsLinked to original sources

bioRxiv2026

Discovery of a regulatory node that coordinates cell envelope assembly in mycobacteria

Mycobacteria possess a complex double-membrane cell envelope critical for survival and pathogenesis. Proper assembly of this architecture requires the biosynthesis and transport of major components, including arabinogalactan (AG) polysaccharides and mycolic acids (MAs), but how these processes are effectively coordinated is unknown. Here, we discover an essential membrane complex that serves as a regulatory node in mycobacterial envelope biogenesis. The acyltransferase TmaT and the arabinofuranosyltransferase AftD physically interact; cryo-EM structures reveal a 1:1 stoichiometry, and present a novel fold for TmaT, featuring a central channel that binds co-factor for acetylation in the periplasm. We establish that the TmaT-AftD interaction, and the catalytic activities of both enzymes, are required for MA transport across the cell envelope, as well as AG ligation to the cell wall, the final stage of AG biosynthesis. The TmaT-AftD complex coordinates the two major envelope assembly pathways, presenting a structural vulnerability for future anti-mycobacterial drug development.

biochemistry↗

bioRxiv2026

Cardiolipin modulation of MmpL3 in mycobacteria

The complex cell envelope of mycobacteria is characterized by the presence of a unique outer membrane (OM) rich in mycolic acids (MAs). These long-chain, branched fatty acids confer extreme hydrophobicity to the OM, in part rendering the mycobacterial envelope impermeable to antibiotics and host defences. How MAs are transported from the inner membrane (IM) to the OM is largely unknown. The integral membrane protein MmpL3 plays an essential role in this process, but the mechanism(s) by which it exploits the proton motive force to flip and/or release MAs at the IM, in the form of trehalose monomycolates (TMMs), remains elusive. Here, we reconstitute and quantify the proton translocation activity of MmpL3 in artificial lipid bilayers, and discover a novel role for the phospholipid species, cardiolipin (CL), in regulating MmpL3 function. We find that mutations in conserved residues, or binding of known inhibitors in the central channel of MmpL3 do not diminish proton translocation activity. Instead, the specific presence of CL abolishes proton translocation by MmpL3 in vitro. Furthermore, we establish that an MmpL3 variant containing substitutions in a CL-binding site predicted in silico is no longer modulated by CL in vitro, and is unable to support growth of Mycobacterium smegmatis. Our work provides previously unappreciated insights into lipid regulation of MmpL3 activity in mycobacteria, and expands the guiding principles for the development of anti-mycobacterial inhibitors targeting this essential transporter. SignificanceMycobacterial species, including the human pathogen Mycobacterium tuberculosis, are surrounded by a double-membrane cell envelope that makes them intrinsically resistant to many antibiotics. Specifically, the outer membrane (OM) contains unique lipids called mycolic acids (MAs), whose transport pathway across the envelope is poorly understood. In this study, we characterized the biochemical activity of the essential MA transporter MmpL3, and uncovered a novel mechanism for lipid-mediated functional regulation. Our work highlights the importance of protein-lipid interactions in defining transporter activity, provides insights into MA transport and OM assembly in mycobacteria, and sets the stage for the development of anti-mycobacterial strategies.

biochemistry↗

bioRxiv2026

A recipient-based anti-conjugation factor triggers an abortive mechanism by targeting the Type IV secretion system

Many bacterial defense (immune) systems prevent the entry of foreign DNA by directly recognizing and targeting nucleic acids, effectively blocking all mechanisms of horizontal gene transfer1. However, systems defending specifically against conjugation, a major route for gene dissemination, have heretofore not been reported. We have discovered a novel defense factor, which we name AbjA (Abortive conjugation protein A), that specifically limits successful plasmid conjugation into a recipient bacterium. AbjA interacts directly with and targets the ATPase component TrbE of the Type IV secretion system (T4SS)2,3 to induce cell death; this contrasts with most other defense systems that act at the nucleic acid level. AbjA therefore represents the first member of a new class of bacterial defense factors that trigger what we term abortive conjugation. Previously, recipient bacteria were viewed largely as defenseless against the mechanism of conjugation4-6; our discovery and characterization of AbjA demonstrates that recipient bacteria can block conjugation to limit the transfer (and thus spread) of plasmids. Discovery of this class of defense systems thus has implications for bacterial defense, plasmid evolution, and possible strategic alternatives to rationally target plasmid spread, particularly with respect to virulence and antibiotic resistance.

microbiology↗

bioRxiv2025

A two-component system signaling hub controls enterococcal membrane remodeling in response to daptomycin

Daptomycin is a last resort antibiotic used to treat vancomycin-resistant enterococcal infections, but daptomycin resistance (DAPR) arises quickly during treatment. Resistance is due to sequential acquisition of point mutations in the two-component system LiaFSR and in cardiolipin synthases and is associated with alteration of phospholipid and glycolipid membrane composition. The molecular mechanisms underlying these lipid changes are currently unknown. Similarly, it is unclear why mutations in liaFSR occur prior to mutations in cls. We discovered that Enterococcus faecalis remodels membrane composition as a phenotypic response to daptomycin that parallels the membrane composition of DAPR strains. The enrichment in glycolipids that follows antibiotic exposure is due to LtaS1, the main LTA synthase of E. faecalis. Moreover, LtaS1 activity is governed by a network of two-component systems formed by LiaFSR, SapRS, and BsrRS that couple antibiotic sensing with membrane lipid remodeling. Together, our results provide a unifying mechanism that drives phenotypic membrane fortification in a Gram-positive pathogen which simultaneously predisposes the cell to acquire genetic high-level daptomycin resistance. Significance StatementDaptomycin is the preferred alternative to treat vancomycin-resistant Enterococcus infections. However, the efficacy of daptomycin is limited by the acquisition of daptomycin resistance. Alterations in lipid membrane composition represent a conserved strategy to fortify the Gram-positive membrane. We discovered that E. faecalis phenotypically remodels membrane composition in response to daptomycin and that the enrichment in glycolipids is dependent on LTA synthesis. We expand our limited knowledge of enterococcal LTA synthases, confirming LtaS1 as the main LTA synthase and placing LTA biogenesis under control of a network of antibiotic-responsive two-component systems. Our work provides a molecular explanation for the sequential evolution of daptomycin resistance and may support the use of existing LtaS inhibitors to prevent acquisition of daptomycin resistance.

microbiology↗

bioRxiv2025

Lipid interactions are important for the Tol-Pal complex in maintaining outer membrane lipid homeostasis

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 partially bypassed in cells lacking CL, suggesting compensatory effects through other lipids only when CL is absent. 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.

biochemistry↗

bioRxiv2025

Polar localization of putative phospholipid transporters in Escherichia coli

The Gram-negative bacterial cell envelope comprises an outer membrane (OM) with an asymmetric arrangement of lipopolysaccharides (LPS) and phospholipids (PLs), protecting them from both physical and chemical threats. To build the OM, PLs must be transported across the cell envelope; this process has remained elusive until recently, where three collectively essential AsmA-superfamily proteins - YhdP, TamB, and YdbH - are proposed to function as anterograde PL transporters in Escherichia coli. Here, we identify the cell wall-binding protein DedD as a novel interacting partner of YhdP, and discover that all three AsmA-superfamily proteins are recruited to, and strongly enriched at the cell poles. Our observation raises the possibility that anterograde PL transport could be spatially restricted to the cell poles, and highlights the importance of understanding the spatial-temporal regulation of OM biogenesis in coordination with cell growth and division.

microbiology↗

bioRxiv2025

Exquisite specificity of Pks13 defines the essentiality of trehalose in mycobacteria

Mycolic acids (MAs) are the major component of the mycobacterial outer membrane, a key contributor to the intrinsic resistance of mycobacteria to external insults including multiple antibiotics. After being synthesized in the cytoplasm and before reaching the outer membrane, MAs are transported across the inner membrane in the form of an acylated sugar, generally believed to be trehalose monomycolates (TMMs). Whether trehalose is the only mycolate carrier during transport is under debate, and why this highly abundant disaccharide is essential for mycobacterial growth is unclear. To address these questions, we leveraged a trehalose auxotrophic Mycobacterium smegmatis strain to investigate the biosynthetic steps affording TMMs. We show that in addition to TMMs, MAs are also not produced in the absence of trehalose. This is likely due to a product inhibition mechanism where un-reduced MA precursors accumulate on Pks13, the protein catalyzing the ligation of mycolic acids and the sugar head group. We establish that the un-reduced mycolates could only be released by trehalose, revealing exquisite Pks13 specificity, and subsequently reduced by CmrA in vitro. Furthermore, only trehalose and its analogs can reactivate MA biosynthesis in cells. Finally, by replacing trehalose with a 6-deoxy analog in cells, we demonstrate that the cord factor trehalose dimycolate is dispensable for M. smegmatis growth in vitro. Our work gives a clear depiction of how TMMs are formed and provides a compelling reason for the essentiality of trehalose, shedding light on the development of future anti-mycobacterial strategies. Significance StatementTuberculosis and other diseases caused by mycobacteria pose a persistent threat to human health and well-being. The mycobacterial outer membrane is a highly impermeable barrier, conferring intrinsic resistance to multiple antibiotics. How the biosynthesis of mycolic acids, the major component of the mycobacterial outer membrane, is driven by trehalose is unknown. In this report, we study the molecular basis for the essentiality of trehalose. We define the order of two critical steps in the mycolic acid biosynthetic pathway involving trehalose and demonstrate the highly specific role that trehalose plays therein. This work answers key questions in terms of outer membrane biogenesis in mycobacteria, providing a venue for the development of novel anti-mycobacterial strategies.

biochemistry↗

bioRxiv2024

Structural insights into the force-transducing mechanism of a motor-stator complex important for bacterial outer membrane lipid homeostasis

Gram-negative bacteria assemble an asymmetric outer membrane (OM) that functions as an effective barrier against antibiotics. Building a stable and functional OM requires assembly and maintenance of balanced levels of proteins, lipopolysaccharides, and phospholipids into the bilayer. In Escherichia coli, the trans-envelope Tol-Pal complex has recently been established to play a primary role in maintaining OM lipid homeostasis. It is believed that the motor-stator complex TolQR exploits the proton motive force in the inner membrane to induce conformational changes in the TolA effector, ultimately generating a force across the cell envelope to activate processes at the OM. Molecular details of how such force transduction occurs via the TolQRA complex is unknown. Here, we solve structures of the E. coli TolQRA complex using single particle cryo-EM, capturing the transmembrane (TM) regions of the purified complex in two distinct states at [~]3.6 [A] and [~]4.2 [A] nominal resolutions. We define how the TolA N-terminal TM helix interacts with an asymmetric TolQ5R2 sub-complex in two different positions, revealing how the two TolQRA states are related by rotation of the TolQ pentamer. By considering structural prediction of the periplasmic domains of the complex, we propose a working model for how proton passage through the complex induces rotary movement that can be coupled to TolA for force transduction across the cell envelope.

biophysics↗

bioRxiv2024

Primary role of the Tol-Pal complex in bacterial outer membrane lipid homeostasis

Gram-negative bacteria are defined by an outer membrane (OM) that contributes to envelope integrity and barrier function. Building this bilayer require proper assembly of lipopolysaccharides, proteins, and phospholipids, yet how the balance of these components is achieved is unclear. One system long known for ensuring OM stability is the Tol-Pal complex, which has been implicated in maintaining OM lipid homeostasis. However, assignment of Tol-Pal function has been challenging, owing to its septal localization and associated role(s) during division. Here, we uncouple the function of Tol-Pal in OM lipid homeostasis from its impact on cell division in Escherichia coli, by engineering a chimeric complex that loses septal enrichment. We demonstrate that this peripherally-localized Tol-Pal complex is fully capable of maintaining lipid balance in the OM, thus restoring OM integrity and barrier. Our work establishes the primary function of the Tol-Pal complex in OM lipid homeostasis, independent of its role during division.

microbiology↗

bioRxiv2023

Deciphering the role of the OmpC-Mla system in bile salt resistance

The outer membrane (OM) of Gram-negative bacteria presents a formidable barrier against external insults, in part contributing to the survival of Enterobacteriaceae in the mammalian gut. The lipid asymmetry of the OM, where lipopolysaccharides (LPS) form a tight outer layer of low permeability, effectively restricts the passage of toxic substances across the bilayer. In the gut, however, bile salts pose a unique challenge to the bacterial cell due to their ability to form micelles and solubilize membranes; yet, mechanisms to prevent dissolution of the OM by such detergents are not well understood. In this study, we define a distinct role in bile salt resistance for the OmpC-Mla system in Escherichia coli, which is better known for its function in maintaining OM lipid asymmetry. We show that cells lacking a functional OmpC-Mla system are sensitive to bile salts, but only at or above critical micellar concentrations. Furthermore, we observe that these cells still exhibit bile salt sensitivity even when defects in OM lipid asymmetry have been corrected, suggesting that the OmpC-Mla system contributes to bile salt resistance independent of its role in lipid asymmetry. Finally, we demonstrate that MlaD, one of the key lipid-binding components of the system, displays specific binding to bile salts in vitro. Since the OmpC-Mla system maintains OM lipid asymmetry by transporting mislocalized PLs, our findings here support a model where this system also additionally removes bile salts that have intercalated into the OM, to ultimately prevent dissolution and disruption of this important barrier. IMPORTANCEBile salts are important components secreted into the human gut to help solubilize fats from our diet, yet they also possess anti-microbial properties due to their corresponding ability to dissolve bacterial membranes. For Enterobacteriaceae to survive in the gut environment, these bacterial cells must prevent intracellular build-up of bile salts by either restricting entry or by pumping out these molecules. Ultimately, they must resist bile salt-mediated dissolution of their membranes, particularly their outer membranes, which serve as a protective barrier against toxic substances. In this study, we reveal that a known lipid transport system in Escherichia coli has a distinct role in bile salt resistance independent of its role in maintaining outer membrane lipid asymmetry; it does so likely by removing bile salts from the outer membrane, thus preventing dissolution. Our work highlights the possibility of targeting this lipid transport system for the treatment of Enterobacteriaceae infections.

microbiology↗

bioRxiv2023

Defining a critical role of an essential membrane protein in mycolic acid transport in mycobacteria

A major feature of the mycobacterial outer membrane (OM) is the presence of long, branched chain mycolic acids (MAs), which render the OM hydrophobic and impervious against various noxious substances, including antibiotics. While the biosynthesis of MA is well studied, the mechanisms governing its transport from the inner membrane to the OM remain largely elusive. In this study, we characterized the function of MSMEG_0317 in Mycobacterium smegmatis, a membrane protein encoded within a conserved genetic locus that has been implicated in MA metabolism and/or transport. Using a conditional knockout mutant, we demonstrate that msmeg_0317 is essential for mycobacterial growth. Depleting msmeg_0317 from cells blocks the formation of MA species found at the OM, establishing a critical function in MA transport across the cell envelope. We further reveal that MSMEG_0317 exists as stable dimers in vitro that require the presence of its N- and C-terminal transmembrane helices, both of which are important for functionality in cells. Our work defines the essential role of MSMEG_0317 in MA metabolism and/or transport, and offers new insights into cell envelope biogenesis in mycobacteria.

biochemistry↗

bioRxiv2022

Depleting cationic lipids involved in antimicrobial resistance drives adaptive lipid remodeling in Enterococcus faecalis

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human {beta}-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild type and mprF mutant strains and show that while {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the {triangleup}mprF1 mutant synthesized [~]70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2. In the mprF mutants, particularly {triangleup}mprF1 {triangleup}mprF2, the decrease in L-PG and PG is compensated by an increase in the phosphorus-containing lipid, GPDGDAG, and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the {triangleup}mprF1 {triangleup}mprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2, compared to wild type and {triangleup}mprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. Significance StatementThe cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

microbiology↗

bioRxiv2022

A conserved membrane protein negatively regulates Mce1 complexes in mycobacteria

Tuberculosis continues to pose a serious threat to global health. Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host. Among their many virulence factors, mycobacteria encode Mce systems. Some of these systems are implicated in lipid uptake, but the molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. We show that the Mce1 system in Mycobacterium smegmatis comprises a canonical ATP-binding cassette transporter, associated with functionally redundant heterohexameric assemblies of substrate-binding proteins. Furthermore, we establish that the conserved membrane protein Mce1N negatively regulates Mce1 function via a unique mechanism involving blocking transporter assembly. Our work offers molecular understanding of Mce complexes, sheds light on mycobacterial lipid metabolism and its regulation, and informs future anti-mycobacterial strategies.

biochemistry↗

bioRxiv2022

Conformational changes induced by chaperone interactions facilitate lipid transport critical for bacterial outer membrane asymmetry

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer with outer leaflet lipopolysaccharides (LPS) exposed to extracellular milieu and inner leaflet phospholipids (PLs) facing the periplasm. This unique lipid asymmetry is the key to its innate drug resistance, rendering the OM impermeable to external insults, including antibiotics and bile salts. To maintain this OM barrier, the OmpC-Mla system removes mislocalized PLs from the OM outer leaflet, and transports them back to the inner membrane (IM); in the first step, the OM OmpC-MlaA complex transfers PLs to the periplasmic chaperone MlaC. This process likely occurs via a hydrophilic channel in MlaA, yet mechanistic details have remained elusive. Here, we biochemically and structurally characterize the architecture of the MlaA-MlaC transient complex. We map the interaction surfaces between MlaA and MlaC in Escherichia coli, revealing that MlaC binds MlaA at the periplasmic face in a manner that possibly juxtaposes the MlaA channel and the MlaC lipid binding cavity. In addition, we show that electrostatic interactions between the putative C-terminal tail helix of MlaA and a surface patch on MlaC are important for recruitment of the latter to the OM. We further provide biochemical evidence for conformational changes in the MlaA channel that correlate with interactions with MlaC and OM porins, as well as functional states of MlaA. Finally, we solve a 2.9-[A] cryo-EM structure of OmpC-MlaA in nanodiscs in a disulfide-trapped complex with MlaC, reinforcing the mechanism of MlaC recruitment, and highlighting membrane thinning as a plausible strategy for directing lipids into the MlaA channel. Our work offers critical insights into how the OmpC-MlaA complex catalyzes retrograde transport of PLs to the IM to maintain OM lipid asymmetry.

biochemistry↗

bioRxiv2021

Genetic interaction mapping highlights key roles of the Tol-Pal complex

The conserved Tol-Pal trans-envelope complex is important for outer membrane (OM) stability and cell division in Gram-negative bacteria. It has been proposed to mediate OM constriction during cell division via tethering to the cell wall. Yet, recent studies suggest that the complex has additional roles in OM lipid homeostasis and septal cell wall separation. How the Tol-Pal complex functions to facilitate these many processes is unclear. To gain insights into its role(s), we applied transposon insertion sequencing, and report here a detailed network of genetic interactions with the tol-pal locus in Escherichia coli. We found one positive and >20 negative strong interactions based on fitness. Disruption of genes responsible for osmoregulated periplasmic glucan biosynthesis restores fitness and OM barrier function, but not cell division defects, in tol-pal mutants. In contrast, deletions of genes involved in OM homeostasis and cell wall remodelling give rise to synthetic growth defects in strains lacking Tol-Pal, especially exacerbating OM barrier and/or cell division defects. Notably, the {Delta}tolA mutant having additional defects in OM protein assembly ({Delta}bamB) exhibited severe division phenotypes, even under conditions where the single mutants divide normally; this highlights the possibility for OM phenotypes to indirectly influence the cell division process. Overall, our work provides insights into the intricate nature of Tol-Pal function, and reinforces the model that this complex plays crucial roles in cell wall-OM tethering, cell wall remodelling, and in particular, OM homeostasis.

microbiology↗

bioRxiv2021

ATP disrupts lipid binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry

The hallmark of the Gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ABC transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [14C]-labelled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts towards discovering new antibiotics against Gram-negative pathogens. Significance StatementBiological membranes define cellular boundaries, allow compartmentalization, and represent a prerequisite for life. In Gram-negative bacteria, the outer membrane (OM) prevents entry of toxic substances, conferring intrinsic resistance against many antibiotics. This barrier function requires unequal distribution of lipids across the OM bilayer, yet how such lipid asymmetry is maintained is not well understood. In this study, we established the directionality of lipid transport for a conserved membrane protein complex, and uncovered mechanistic insights into how ATP powers such transport from the OM to the inner membrane. Our work provides fundamental understanding of lipid trafficking within the Gram-negative double-membrane envelope in the context of OM lipid asymmetry, and highlights the importance of targeting lipid transport processes for future antibiotics development.

biochemistry↗

bioRxiv2020

Structure-function characterization of the conserved regulatory mechanism of the Escherichia coli M48 metalloprotease BepA

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 [A], in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. ImportanceM48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the LPS biogenesis machinery. Here we present the structure of BepA and the results of a structure guided mutagenesis strategy, which reveal the key residues required for activity.

microbiology↗

bioRxiv2019

Structure-function analysis of the Escherichia coli β-barrel assembly enhancing protease BepA suggests a role for a self-inhibitory state

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for the bacteria against environmental insults and attack by antimicrobials. The key component of the OM barrier is the surface exposed lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system OM component, LptD, is essential and is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA, solved to a resolution of 1.9 [A]. Our structure comprises the zinc-bound m48 protease domain and a tetratricopeptide repeat (TPR) domain, consisting of four 2-helix TPR motifs and four non-TPR helices, leading to a nautilus-like shape in which the TPR repeats cup the protease domain. Using targeted mutagenesis approaches, we demonstrate that the protein is auto-regulated by the active-site plug. Further to this we reveal that mutation of a negative pocket, formed at the interface between the m48 and TPR domains, impairs BepA activity suggesting the pocket as a possible substrate binding site. We also identify a potential protein interaction site within the TPR cavity as being important for BepA function. Lastly, we provide evidence to show that increased antibiotic susceptibility in the absence of correctly functioning BepA occurs through disruption of OM lipid asymmetry, leading to reduced barrier function and increased cell permeability.

microbiology↗