Biology subjects

Chong, Z.-S.

Publications and source records attributed to Chong, Z.-S..

8 recordsLinked to original sources

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↗

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↗

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

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↗

bioRxiv2018

Structure of the tripartite motif of KAP1/TRIM28 identifies molecular interfaces required for transcriptional silencing of retrotransposons

Transcription of transposable elements is tightly regulated to prevent damage to the genome. The family of KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, which controls the assembly of an epigenetic silencing complex including histone H3K9 methyltransferase SETDB1. The chromatin remodeling activities of this complex repress transcription of the targeted transposable element and any adjacent genes. Here, we use biophysical and structural approaches to show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevented higher-order oligomerization without a significant loss of retrotransposon silencing activity in a cell-based assay, indicating that, in contrast to other TRIM family members, self-assembly is not essential for the function of KAP1. The crystal structure of the KAP1 RBCC dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 RBCC motif responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.

biochemistry↗

bioRxiv2017

Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults. In Escherichia coli, the OmpC-MlaA complex is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How it performs this function is unknown. Here, we define the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. We establish that MlaA sits entirely within the bilayer in complex with OmpC and provides a hydrophilic channel possibly for PL translocation across the OM. Furthermore, we show that flexibility in a hairpin loop adjacent to the channel modulates MlaA activity. Finally, we demonstrate that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antimicrobial drug development.

biochemistry↗

bioRxiv2017

MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

microbiology↗