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Lun J, Zheng P, Liang X, Hu Y, An L, Xiao G, Chen X, Chen Y, Gong H, Zhong M, Zhang Y, Hu Z. Identification of a conserved cryptic epitope with cross-immunoreactivity in outer membrane protein K (OmpK) from Vibrio species. Vaccine 2025; 53:126964. [PMID: 40037129 DOI: 10.1016/j.vaccine.2025.126964] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2024] [Revised: 02/25/2025] [Accepted: 02/25/2025] [Indexed: 03/06/2025]
Abstract
Outer membrane protein K (OmpK) has been proven to be an ideal vaccine candidate for broad-spectrum cross-prevention against Vibriosis. However, due to the extensive biological and genetic diversity of Vibrio species, current OmpK subunit vaccines can only target different strains of the same bacterial species or closely related species and have difficulty providing promising cross-immunoprotection against more diverse Vibrio infections. In recent years, the development of epitope-focused vaccines has been described as the latest stage in the development of vaccine formulations, providing new ideas for the development of broad-spectrum Vibrio vaccines. Interestingly, a cryptic epitope (K7) was identified in OmpK from Vibrio species, which is itself immunogenic but is not involved in the immune response to intact OmpK. Epitope K7 is a 15-residue hairpin structure in OmpK predicted to contain a 6-residue extracellular turn region. Interestingly, unlike other highly variable extracellular long loops, epitope K7 is the only conserved extracellular short turn in OmpK, with a similarity of 33 % to 93 %. K7 homologous peptides stimulated the production of specific antibodies, confirming their high immunogenicity. Cross-immunoreactivity between K7 homologous and K7-induced antibodies was evaluated by peptide-based ELISA, western blot, and cell-based ELISA. Flow cytometry and immunofluorescence assay further confirmed that the native epitope K7 in OmpK is surface-exposed and therefore an extracellular target that binds to antibodies. Moreover, an antibody-dependent and complement-mediated serum bactericidal assay suggested that epitope K7-induced antibodies have vibriocidal activity. In conclusion, we identified a conserved cryptic epitope with cross-immunoreactivity in OmpK from Vibrio species. Our results suggest that epitope K7 could be an ideal candidate for the design of epitope-focused vaccines against diverse Vibrio infections.
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Affiliation(s)
- Jingsheng Lun
- Department of Biology, College of Science, Shantou University, Shantou 515063, China; Marine Biology Institute, Shantou University, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou 515063, China.
| | - Peng Zheng
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Xueji Liang
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Yihui Hu
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Lu An
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Guiqian Xiao
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Xinyi Chen
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Ying Chen
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Huisheng Gong
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Mingqi Zhong
- Department of Biology, College of Science, Shantou University, Shantou 515063, China
| | - Yueling Zhang
- Department of Biology, College of Science, Shantou University, Shantou 515063, China; Marine Biology Institute, Shantou University, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou 515063, China
| | - Zhong Hu
- Department of Biology, College of Science, Shantou University, Shantou 515063, China; Marine Biology Institute, Shantou University, Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou 515063, China.
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2
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Dubois L, Vettiger A, Buss JA, Bernhardt TG. Using fluorescently labeled wheat germ agglutinin to track lipopolysaccharide transport to the outer membrane in Escherichia coli. mBio 2025; 16:e0395024. [PMID: 39992125 PMCID: PMC11898776 DOI: 10.1128/mbio.03950-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2025] [Accepted: 01/24/2025] [Indexed: 02/25/2025] Open
Abstract
The cell envelope of gram-negative bacteria consists of two membranes sandwiching the peptidoglycan (PG) cell wall. The outer membrane (OM) contains integrated beta-barrel proteins and has an outer leaflet composed of lipopolysaccharide (LPS). LPS is transported from the inner membrane where it is made to the OM surface by the Lpt system. In the polarly elongating alpha-proteobacterium Brucella abortus, LPS transport has been localized to the polar growth zone and division site. However, LPS transport has not been tracked in live proteobacteria like Escherichia coli that elongate by dispersed incorporation of envelope material along their cell body. Here, we report an investigation into the binding target of fluorescently labeled wheat germ agglutinin (FL-WGA) on E. coli cells that led to the development of a method for visualizing LPS transport. We show that instead of PG or enterobacterial common antigen for which FL-WGA labeling has been used to detect in the past, this probe recognizes LPS modified with a terminal N-acetylglucosamine formed by the defective O-antigen synthesis pathway of laboratory strains of E. coli. This finding enabled the construction of mutants inducible for LPS modification that were used together with FL-WGA labeling to track LPS transport to the cell surface. We show that new LPS is inserted throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously for PG synthesis and OM protein insertion in E. coli, suggesting that LPS transport to the OM is coordinated with these processes.IMPORTANCEGram-negative bacteria like Escherichia coli are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the E. coli outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.
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Affiliation(s)
- Laurent Dubois
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Andrea Vettiger
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Jackson A. Buss
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Thomas G. Bernhardt
- Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
- Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
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3
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Kumar S, Inns PG, Ward S, Lagage V, Wang J, Kaminska R, Booth MJ, Uphoff S, Cohen EAK, Mamou G, Kleanthous C. Immobile lipopolysaccharides and outer membrane proteins differentially segregate in growing Escherichia coli. Proc Natl Acad Sci U S A 2025; 122:e2414725122. [PMID: 40030021 PMCID: PMC11912417 DOI: 10.1073/pnas.2414725122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2024] [Accepted: 01/07/2025] [Indexed: 03/19/2025] Open
Abstract
The outer membrane (OM) of gram-negative bacteria is a robust, impermeable barrier that excludes many classes of antibiotics. Contrary to the classical model of an asymmetric lipid bilayer, recent evidence suggests the OM is predominantly an asymmetric proteolipid membrane (APLM). Outer leaflet lipopolysaccharides (LPS) that surround integral β-barrel outer membrane proteins (OMPs) are shared with other OMPs to form a supramolecular network in which the levels of OMPs approach those of LPS. Some of the most abundant OMPs in the Escherichia coli OM are trimeric porins. How porins and LPS are incorporated into the OM of growing bacteria is poorly understood. Here, we use live-cell imaging and microfluidics to investigate how LPS, labeled using click chemistry, and the porin OmpF, labeled using the bacteriocin colicin N, are incorporated into the E. coli OM. Diffraction-limited fluorescence microscopy shows OmpF and LPS to be uniformly distributed and immobile. However, clustering of both macromolecules becomes evident by superresolution microscopy, which is also the case for their biogenesis proteins, BamA and LptD, respectively. Notwithstanding these common organizational features, OmpF insertion into the OM is cell-cycle-dependent leading to binary partitioning and strong polar accumulation of old OmpF. Old LPS on the other hand is diluted ~50% at each division cycle by new LPS, resulting in only mild polar accumulation of preexisting LPS. We conclude that although LPS and OMPs are destined to form the APLM their insertion dynamics are fundamentally different, which has major implications for understanding how the OM is assembled.
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Affiliation(s)
- Sandip Kumar
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
| | - Patrick G. Inns
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
| | - Scott Ward
- Department of Mathematics, Imperial College London, LondonSW7 1AZ, United Kingdom
| | - Valentine Lagage
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
| | - Jingyu Wang
- Department of Engineering Science, University of Oxford, OxfordOX1 3PJ, United Kingdom
| | - Renata Kaminska
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
| | - Martin J. Booth
- Department of Engineering Science, University of Oxford, OxfordOX1 3PJ, United Kingdom
| | - Stephan Uphoff
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
| | - Edward A. K. Cohen
- Department of Mathematics, Imperial College London, LondonSW7 1AZ, United Kingdom
| | - Gideon Mamou
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
| | - Colin Kleanthous
- Department of Biochemistry, University of Oxford, OxfordOX1 3QU, United Kingdom
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4
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Fitzmaurice DR, Amador A, Starr T, Hocky GM, Rojas ER. β-Barrel proteins dictate the effect of core oligosaccharide composition on outer membrane mechanics. Biophys J 2025; 124:765-777. [PMID: 39863924 PMCID: PMC11897544 DOI: 10.1016/j.bpj.2025.01.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 12/18/2024] [Accepted: 01/23/2025] [Indexed: 01/27/2025] Open
Abstract
The outer membrane is the defining structure of Gram-negative bacteria. We previously demonstrated that it is a major load-bearing component of the cell envelope and is therefore critical to the mechanical robustness of the bacterial cell. Here, to determine the key molecules and moieties within the outer membrane that underlie its contribution to cell envelope mechanics, we measured cell-envelope stiffness across several sets of mutants with altered outer-membrane sugar content, protein content, and electric charge. To decouple outer membrane stiffness from total cell envelope stiffness, we developed a novel microfluidics-based "osmotic force-extension" assay. In tandem, we developed a method to increase throughput of microfluidics experiments by performing them on color-coded pools of mutants. We found that truncating the core oligosaccharide, deleting the β-barrel protein OmpA, or deleting lipoprotein outer membrane-cell wall linkers all had the same modest, convergent effect on total cell-envelope stiffness in Escherichia coli. However, these mutations had large, variable effects on the ability of the cell wall to transfer tension to the outer membrane during large hyperosmotic shocks. Surprisingly, altering the electric charge of lipid A had little effect on the mechanical properties of the envelope. Finally, the presence or absence of OmpA determined whether truncating the core oligosaccharide decreased or increased envelope stiffness (respectively), revealing sign epistasis between these components. Based on these data we propose a putative structural model in which the spatial interactions between lipopolysaccharides, β-barrel proteins, and phospholipids coordinately determine cell envelope stiffness.
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Affiliation(s)
| | - Anthony Amador
- Department of Biology, New York University, New York, New York
| | - Tahj Starr
- Department of Biology, New York University, New York, New York
| | - Glen M Hocky
- Department of Chemistry and Simons Center for Computational Physical Chemistry, New York University, New York, New York
| | - Enrique R Rojas
- Department of Biology, New York University, New York, New York.
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5
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Fitzmaurice D, Amador A, Starr T, Hocky GM, Rojas ER. β-barrel proteins dictate the effect of core oligosaccharide composition on outer membrane mechanics. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.02.610904. [PMID: 39282359 PMCID: PMC11398377 DOI: 10.1101/2024.09.02.610904] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/25/2024]
Abstract
The outer membrane is the defining structure of Gram-negative bacteria. We previously demonstrated that it is critical for the mechanical integrity of the cell envelope and therefore to the robustness of the bacterial cell as a whole. Here, to determine the key molecules and moieties within the outer membrane that underlie its contribution to cell envelope mechanics, we measured cell-envelope stiffness across several sets of mutants with altered outer-membrane sugar content, protein content, and electric charge. To decouple outer membrane stiffness from total cell envelope stiffness, we developed a novel microfluidics-based "osmotic force extension" assay. In tandem, we developed a simple method to increase throughput of microfluidics experiments by performing them on color-coded pools of mutants. Using Escherichia coli as a model Gram-negative bacterium, we found that truncating the core oligosaccharide, deleting the β-barrel protein OmpA, or deleting lipoprotein outer membrane-cell wall linkers all had the same modest, convergent effect on total cell-envelope stiffness but had large, varying effects on the ability of the cell wall to transfer tension to the outer membrane during large hyperosmotic shocks. Surprisingly, altering lipid A charge had little effect on the mechanical properties of the envelope. Importantly, the presence or absence of OmpA determined whether truncating the core oligosaccharide decreased or increased envelope stiffness (respectively), revealing sign epistasis between these components. Based on these data we propose a specific structural model in which the chemical interactions between lipopolysaccharides, β-barrel proteins, and phospholipids coordinately determine cell envelope stiffness, and the ability of the outer membrane to functionally share mechanical loads with the cell wall. Statement of Significance The outer membrane is the defining cellular structure of Gram-negative bacteria, a group that contains many important pathogens like Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa. One role of the outer membrane is to block the uptake of small molecules like antibiotics. However, it is becoming increasingly clear that it also functions as a structural exoskeleton that is critical for the cell's ability to cope with internal and external mechanical forces. Here, we carefully dissect the molecular basis for the load-bearing capacity of the outer membrane by screening a set of mutants with a new cell biophysics assay.
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Affiliation(s)
- Dylan Fitzmaurice
- Department of Biology, New York University, New York, New York, 10003, USA
| | - Anthony Amador
- Department of Biology, New York University, New York, New York, 10003, USA
| | - Tahj Starr
- Department of Biology, New York University, New York, New York, 10003, USA
| | - Glen M. Hocky
- Department of Chemistry and Simons Center for Computational Physical Chemistry, New York University, New York, New York, 10003, USA
| | - Enrique R. Rojas
- Department of Biology, New York University, New York, New York, 10003, USA
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6
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Xu Y, Wang X, Zaal EA, Berkers CR, Lorent JH, Heise T, Cox R, Pieters RJ, Breukink E. Specific labeling of newly synthesized lipopolysaccharide via metabolic incorporation of azido-galactose. Biochim Biophys Acta Mol Cell Biol Lipids 2024; 1869:159467. [PMID: 38382574 DOI: 10.1016/j.bbalip.2024.159467] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 02/12/2024] [Accepted: 02/17/2024] [Indexed: 02/23/2024]
Abstract
Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.
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Affiliation(s)
- Yang Xu
- Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands
| | - Xiaoqi Wang
- Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands
| | - Esther A Zaal
- Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, the Netherlands
| | - Celia R Berkers
- Division of Cell Biology, Metabolism & Cancer, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, the Netherlands
| | - Joseph H Lorent
- Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands
| | - Torben Heise
- Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands
| | - Ruud Cox
- Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands
| | - Roland J Pieters
- Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands
| | - Eefjan Breukink
- Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands.
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Goh KJ, Stubenrauch CJ, Lithgow T. The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer. EMBO Rep 2024; 25:1711-1720. [PMID: 38467907 PMCID: PMC11014939 DOI: 10.1038/s44319-024-00111-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2023] [Revised: 02/08/2024] [Accepted: 02/20/2024] [Indexed: 03/13/2024] Open
Abstract
The assembly of β-barrel proteins into the bacterial outer membrane is an essential process enabling the colonization of new environmental niches. The TAM was discovered as a module of the β-barrel protein assembly machinery; it is a heterodimeric complex composed of an outer membrane protein (TamA) bound to an inner membrane protein (TamB). The TAM spans the periplasm, providing a scaffold through the peptidoglycan layer and catalyzing the translocation and assembly of β-barrel proteins into the outer membrane. Recently, studies on another membrane protein (YhdP) have suggested that TamB might play a role in phospholipid transport to the outer membrane. Here we review and re-evaluate the literature covering the experimental studies on the TAM over the past decade, to reconcile what appear to be conflicting claims on the function of the TAM.
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Affiliation(s)
- Kwok Jian Goh
- Centre to Impact AMR, Monash University, Melbourne, VIC, 3800, Australia
- Infection Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia
| | - Christopher J Stubenrauch
- Centre to Impact AMR, Monash University, Melbourne, VIC, 3800, Australia
- Infection Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia
| | - Trevor Lithgow
- Centre to Impact AMR, Monash University, Melbourne, VIC, 3800, Australia.
- Infection Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia.
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8
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George A, Patil AG, Mahalakshmi R. ATP-independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next-generation therapeutics. Protein Sci 2024; 33:e4896. [PMID: 38284489 PMCID: PMC10804688 DOI: 10.1002/pro.4896] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2023] [Revised: 12/28/2023] [Accepted: 12/31/2023] [Indexed: 01/30/2024]
Abstract
Diderm bacteria employ β-barrel outer membrane proteins (OMPs) as their first line of communication with their environment. These OMPs are assembled efficiently in the asymmetric outer membrane by the β-Barrel Assembly Machinery (BAM). The multi-subunit BAM complex comprises the transmembrane OMP BamA as its functional subunit, with associated lipoproteins (e.g., BamB/C/D/E/F, RmpM) varying across phyla and performing different regulatory roles. The ability of BAM complex to recognize and fold OM β-barrels of diverse sizes, and reproducibly execute their membrane insertion, is independent of electrochemical energy. Recent atomic structures, which captured BAM-substrate complexes, show the assembly function of BamA can be tailored, with different substrate types exhibiting different folding mechanisms. Here, we highlight common and unique features of its interactome. We discuss how this conserved protein complex has evolved the ability to effectively achieve the directed assembly of diverse OMPs of wide-ranging sizes (8-36 β-stranded monomers). Additionally, we discuss how darobactin-the first natural membrane protein inhibitor of Gram-negative bacteria identified in over five decades-selectively targets and specifically inhibits BamA. We conclude by deliberating how a detailed deduction of BAM complex-associated regulation of OMP biogenesis and OM remodeling will open avenues for the identification and development of effective next-generation therapeutics against Gram-negative pathogens.
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Affiliation(s)
- Anjana George
- Molecular Biophysics Laboratory, Department of Biological SciencesIndian Institute of Science Education and ResearchBhopalIndia
| | - Akanksha Gajanan Patil
- Molecular Biophysics Laboratory, Department of Biological SciencesIndian Institute of Science Education and ResearchBhopalIndia
| | - Radhakrishnan Mahalakshmi
- Molecular Biophysics Laboratory, Department of Biological SciencesIndian Institute of Science Education and ResearchBhopalIndia
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9
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Surveying membrane landscapes: a new look at the bacterial cell surface. Nat Rev Microbiol 2023:10.1038/s41579-023-00862-w. [PMID: 36828896 DOI: 10.1038/s41579-023-00862-w] [Citation(s) in RCA: 26] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/30/2023] [Indexed: 02/26/2023]
Abstract
Recent studies applying advanced imaging techniques are changing the way we understand bacterial cell surfaces, bringing new knowledge on everything from single-cell heterogeneity in bacterial populations to their drug sensitivity and mechanisms of antimicrobial resistance. In both Gram-positive and Gram-negative bacteria, the outermost surface of the bacterial cell is being imaged at nanoscale; as a result, topographical maps of bacterial cell surfaces can be constructed, revealing distinct zones and specific features that might uniquely identify each cell in a population. Functionally defined assembly precincts for protein insertion into the membrane have been mapped at nanoscale, and equivalent lipid-assembly precincts are suggested from discrete lipopolysaccharide patches. As we review here, particularly for Gram-negative bacteria, the applications of various modalities of nanoscale imaging are reawakening our curiosity about what is conceptually a 3D cell surface landscape: what it looks like, how it is made and how it provides resilience to respond to environmental impacts.
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10
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Webby MN, Oluwole AO, Pedebos C, Inns PG, Olerinyova A, Prakaash D, Housden NG, Benn G, Sun D, Hoogenboom BW, Kukura P, Mohammed S, Robinson CV, Khalid S, Kleanthous C. Lipids mediate supramolecular outer membrane protein assembly in bacteria. SCIENCE ADVANCES 2022; 8:eadc9566. [PMID: 36322653 PMCID: PMC9629720 DOI: 10.1126/sciadv.adc9566] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Accepted: 09/13/2022] [Indexed: 06/16/2023]
Abstract
β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.
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Affiliation(s)
- Melissa N. Webby
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
| | - Abraham O. Oluwole
- Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, UK
- The Kavli Institute for Nanoscience Discovery, South Parks Road, Oxford OX1 3QZ, UK
| | - Conrado Pedebos
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
| | - Patrick G. Inns
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
| | - Anna Olerinyova
- Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, UK
| | - Dheeraj Prakaash
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
| | - Nicholas G. Housden
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
| | - Georgina Benn
- London Centre for Nanotechnology, University College London, London WC1H 0AH, UK
- Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
| | - Dawei Sun
- Structural Biology, Genentech Inc., South San Francisco, USA
| | - Bart W. Hoogenboom
- London Centre for Nanotechnology, University College London, London WC1H 0AH, UK
- Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
- Department of Physics and Astronomy, University College London, WC1E 6BT London, UK
| | - Philipp Kukura
- Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, UK
| | - Shabaz Mohammed
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
- Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford OX1 3QZ, UK
- Mechanistic Proteomics, Rosalind Franklin Institute, Harwell Campus, Didcot OX11 OFA, UK
| | - Carol V. Robinson
- Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, UK
- The Kavli Institute for Nanoscience Discovery, South Parks Road, Oxford OX1 3QZ, UK
| | - Syma Khalid
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
| | - Colin Kleanthous
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK
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11
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Mamou G, Corona F, Cohen-Khait R, Housden NG, Yeung V, Sun D, Sridhar P, Pazos M, Knowles TJ, Kleanthous C, Vollmer W. Peptidoglycan maturation controls outer membrane protein assembly. Nature 2022; 606:953-959. [PMID: 35705811 PMCID: PMC9242858 DOI: 10.1038/s41586-022-04834-7] [Citation(s) in RCA: 46] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Accepted: 05/05/2022] [Indexed: 11/12/2022]
Abstract
Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1–5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the β-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design. Peptidoglycan stem peptides in the Gram-negative bacterial cell wall regulate the insertion of essential outer membrane proteins, thus representing a potential target for antibiotic design.
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Affiliation(s)
- Gideon Mamou
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford, UK
| | - Federico Corona
- Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK.,Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Ruth Cohen-Khait
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford, UK
| | - Nicholas G Housden
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford, UK
| | - Vivian Yeung
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford, UK
| | - Dawei Sun
- Structural Biology, Genentech, South San Francisco, CA, USA
| | - Pooja Sridhar
- School of Biosciences, University of Birmingham, Birmingham, UK
| | - Manuel Pazos
- Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK.,Department of Molecular Biology, Center of Molecular Biology 'Severo Ochoa' (UAM-CSIC), Autonomous University of Madrid, Madrid, Spain
| | | | - Colin Kleanthous
- Department of Biochemistry, South Parks Road, University of Oxford, Oxford, UK.
| | - Waldemar Vollmer
- Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK.
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12
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13
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Sun J, Rutherford ST, Silhavy TJ, Huang KC. Physical properties of the bacterial outer membrane. Nat Rev Microbiol 2022; 20:236-248. [PMID: 34732874 PMCID: PMC8934262 DOI: 10.1038/s41579-021-00638-0] [Citation(s) in RCA: 168] [Impact Index Per Article: 56.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/14/2021] [Indexed: 11/09/2022]
Abstract
It has long been appreciated that the Gram-negative outer membrane acts as a permeability barrier, but recent studies have uncovered a more expansive and versatile role for the outer membrane in cellular physiology and viability. Owing to recent developments in microfluidics and microscopy, the structural, rheological and mechanical properties of the outer membrane are becoming apparent across multiple scales. In this Review, we discuss experimental and computational studies that have revealed key molecular factors and interactions that give rise to the spatial organization, limited diffusivity and stress-bearing capacity of the outer membrane. These physical properties suggest broad connections between cellular structure and physiology, and we explore future prospects for further elucidation of the implications of outer membrane construction for cellular fitness and survival.
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Affiliation(s)
- Jiawei Sun
- Department of Bioengineering, Stanford University, Stanford, CA 94305, USA
| | - Steven T. Rutherford
- Department of Infectious Diseases, Genentech Inc., South San Francisco, CA 94080, USA,To whom correspondence should be addressed: , ,
| | - Thomas J. Silhavy
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA,To whom correspondence should be addressed: , ,
| | - Kerwyn Casey Huang
- Department of Bioengineering, Stanford University, Stanford, CA, USA. .,Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA. .,Chan Zuckerberg Biohub, San Francisco, CA, USA.
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14
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Doyle MT, Jimah JR, Dowdy T, Ohlemacher SI, Larion M, Hinshaw JE, Bernstein HD. Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding. Cell 2022; 185:1143-1156.e13. [PMID: 35294859 DOI: 10.1016/j.cell.2022.02.016] [Citation(s) in RCA: 61] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 12/01/2021] [Accepted: 02/13/2022] [Indexed: 02/08/2023]
Abstract
Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "β signal" motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that β sheets progressively fold toward BamA to form a β barrel. Along with in vivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding.
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Affiliation(s)
- Matthew Thomas Doyle
- Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - John R Jimah
- Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Tyrone Dowdy
- Neuro-Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Shannon I Ohlemacher
- Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Mioara Larion
- Neuro-Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Jenny E Hinshaw
- Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Harris D Bernstein
- Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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15
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Mandela E, Stubenrauch CJ, Ryoo D, Hwang H, Cohen EJ, Torres VVL, Deo P, Webb CT, Huang C, Schittenhelm RB, Beeby M, Gumbart JC, Lithgow T, Hay ID. Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability. eLife 2022; 11:73516. [PMID: 35084330 PMCID: PMC8824477 DOI: 10.7554/elife.73516] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Accepted: 01/21/2022] [Indexed: 11/17/2022] Open
Abstract
The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation
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Affiliation(s)
- Eric Mandela
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | | | - David Ryoo
- Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, United States
| | - Hyea Hwang
- School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
| | - Eli J Cohen
- Department of Life Sciences, Imperial College London, London, United Kingdom
| | | | - Pankaj Deo
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | - Chaille T Webb
- Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | - Cheng Huang
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
| | - Ralf B Schittenhelm
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
| | - Morgan Beeby
- Department of Life Sciencesa, Imperial College London, London, United Kingdom
| | - James C Gumbart
- School of Physics, Georgia Institute of Technology, Atlanta, United States
| | - Trevor Lithgow
- Department of Microbiology, Monash University, Melbourne, Australia
| | - Iain D Hay
- School of Biological Sciences, The University of Auckland, Auckland, New Zealand
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16
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The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine. Proc Natl Acad Sci U S A 2022; 119:2114997119. [PMID: 34969846 PMCID: PMC8740687 DOI: 10.1073/pnas.2114997119] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/22/2021] [Indexed: 01/25/2023] Open
Abstract
The outer membrane (OM) of gram-negative bacteria acts as a robust permeability barrier to enable cell survival in a wide variety of harsh environments. Crucial to OM integrity are β-barrel outer membrane proteins (OMPs) that are assembled into the membrane by the broadly conserved β-barrel assembly machine (Bam) complex. Here, we identify specific roles for the periplasmic chaperone Skp in functioning as a sacrificial adaptor protein to remove stalled substrates from the Bam complex, imposing an active quality control mechanism that ensures efficient assembly of nascent OMPs into the OM. This work identifies the molecular mechanism of the Skp/DegP functional relationship and clarifies the long-standing paradox of how substrate release from the high-affinity, long-lived Skp–OMP complex is achieved in vivo. The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.
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17
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Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis. mBio 2021; 12:e0309921. [PMID: 34844428 PMCID: PMC8630537 DOI: 10.1128/mbio.03099-21] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity.
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18
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Çelik P, Derkuş B, Erdoğan K, Barut D, Manga EB, Yıldırım Y, Pecha S, Çabuk A. Bacterial membrane vesicle functions, laboratory methods, and applications. Biotechnol Adv 2021; 54:107869. [PMID: 34793882 DOI: 10.1016/j.biotechadv.2021.107869] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 10/19/2021] [Accepted: 11/09/2021] [Indexed: 12/13/2022]
Abstract
Bacterial membrane vesicles are cupped-shaped structures formed by bacteria in response to environmental stress, genetic alteration, antibiotic exposure, and others. Due to the structural similarities shared with the producer organism, they can retain certain characteristics like stimulating immune responses. They are also able to carry molecules for long distances, without changes in the concentration and integrity of the molecule. Bacteria originally secrete membrane vesicles for gene transfer, excretion, cell to cell interaction, pathogenesis, and protection against phages. These functions are unique and have several innovative applications in the pharmaceutical industry that have attracted both scientific and commercial interest.This led to the development of efficient methods to artificially stimulate vesicle production, purification, and manipulation in the lab at nanoscales. Also, for specific applications, engineering methods to impart pathogen antigens against specific diseases or customization as cargo vehicles to deliver payloads to specific cells have been reported. Many applications of bacteria membrane vesicles are in cancer drugs, vaccines, and adjuvant development with several candidates in clinical trials showing promising results. Despite this, applications in therapy and commercialization stay timid probably due to some challenges one of which is the poor understanding of biogenesis mechanisms. Nevertheless, so far, bacterial membrane vesicles seem to be a reliable and cost-efficient technology with several therapeutic applications. Research toward characterizing more membrane vesicles, genetic engineering, and nanotechnology will enable the scope of applications to widen. This might include solutions to other currently faced medical and healthcare-related challenges.
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Affiliation(s)
- PınarAytar Çelik
- Environmental Protection and Control Program, Eskişehir Osmangazi University, Eskişehir 26110, Turkey; Department of Biotechnology and Biosafety, Graduate School of Natural and Applied Science, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey.
| | - Burak Derkuş
- Department of Chemistry, Faculty of Science, Ankara University, 06560 Ankara, Turkey
| | - Kübra Erdoğan
- Department of Biotechnology and Biosafety, Graduate School of Natural and Applied Science, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey
| | - Dilan Barut
- Department of Biotechnology and Biosafety, Graduate School of Natural and Applied Science, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey
| | - Enuh Blaise Manga
- Department of Biotechnology and Biosafety, Graduate School of Natural and Applied Science, Eskisehir Osmangazi University, 26040 Eskisehir, Turkey
| | - Yalın Yıldırım
- Department of Cardiovascular Surgery, University Heart & Vascular Center Hamburg, Hamburg, Germany
| | - Simon Pecha
- Department of Cardiovascular Surgery, University Heart & Vascular Center Hamburg, Hamburg, Germany
| | - Ahmet Çabuk
- Department of Biology, Faculty of Science and Letter, Eskişehir Osmangazi University, Eskişehir 26040, Turkey
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19
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Benn G, Mikheyeva IV, Inns PG, Forster JC, Ojkic N, Bortolini C, Ryadnov MG, Kleanthous C, Silhavy TJ, Hoogenboom BW. Phase separation in the outer membrane of Escherichia coli. Proc Natl Acad Sci U S A 2021; 118:e2112237118. [PMID: 34716276 PMCID: PMC8612244 DOI: 10.1073/pnas.2112237118] [Citation(s) in RCA: 62] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Accepted: 09/20/2021] [Indexed: 11/18/2022] Open
Abstract
Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.
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Affiliation(s)
- Georgina Benn
- London Centre for Nanotechnology, University College London, London WC1H 0AH, United Kingdom
- Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, United Kingdom
- National Physical Laboratory, Teddington TW11 0LW, United Kingdom
| | - Irina V Mikheyeva
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544
| | - Patrick George Inns
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
| | - Joel C Forster
- Department of Physics and Astronomy, University College London WC1E 6BT London, United Kingdom
- Institute for the Physics of Living Systems, University College London WC1E 6BT London, United Kingdom
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom
| | - Nikola Ojkic
- Department of Physics and Astronomy, University College London WC1E 6BT London, United Kingdom
| | - Christian Bortolini
- London Centre for Nanotechnology, University College London, London WC1H 0AH, United Kingdom
| | - Maxim G Ryadnov
- National Physical Laboratory, Teddington TW11 0LW, United Kingdom
- Department of Physics, King's College London, London WC2R 2LS, United Kingdom
| | - Colin Kleanthous
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
| | - Thomas J Silhavy
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544;
| | - Bart W Hoogenboom
- London Centre for Nanotechnology, University College London, London WC1H 0AH, United Kingdom;
- Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, United Kingdom
- Department of Physics and Astronomy, University College London WC1E 6BT London, United Kingdom
- Institute for the Physics of Living Systems, University College London WC1E 6BT London, United Kingdom
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20
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Cambré A, Aertsen A. Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria. Microbiol Mol Biol Rev 2020; 84:e00008-20. [PMID: 33115939 PMCID: PMC7599038 DOI: 10.1128/mmbr.00008-20] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The rise in fluorescence-based imaging techniques over the past 3 decades has improved the ability of researchers to scrutinize live cell biology at increased spatial and temporal resolution. In microbiology, these real-time vivisections structurally changed the view on the bacterial cell away from the "watery bag of enzymes" paradigm toward the perspective that these organisms are as complex as their eukaryotic counterparts. Capitalizing on the enormous potential of (time-lapse) fluorescence microscopy and the ever-extending pallet of corresponding probes, initial breakthroughs were made in unraveling the localization of proteins and monitoring real-time gene expression. However, later it became clear that the potential of this technique extends much further, paving the way for a focus-shift from observing single events within bacterial cells or populations to obtaining a more global picture at the intra- and intercellular level. In this review, we outline the current state of the art in fluorescence-based vivisection of bacteria and provide an overview of important case studies to exemplify how to use or combine different strategies to gain detailed information on the cell's physiology. The manuscript therefore consists of two separate (but interconnected) parts that can be read and consulted individually. The first part focuses on the fluorescent probe pallet and provides a perspective on modern methodologies for microscopy using these tools. The second section of the review takes the reader on a tour through the bacterial cell from cytoplasm to outer shell, describing strategies and methods to highlight architectural features and overall dynamics within cells.
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Affiliation(s)
- Alexander Cambré
- KU Leuven, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Leuven, Belgium
| | - Abram Aertsen
- KU Leuven, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Leuven, Belgium
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21
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Cao P, Wall D. The Fluidity of the Bacterial Outer Membrane Is Species Specific: Bacterial Lifestyles and the Emergence of a Fluid Outer Membrane. Bioessays 2020; 42:e1900246. [PMID: 32363627 PMCID: PMC7392792 DOI: 10.1002/bies.201900246] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Revised: 03/23/2020] [Indexed: 01/17/2023]
Abstract
The outer membrane (OM) is an essential barrier that guards Gram-negative bacteria from diverse environmental insults. Besides functioning as a chemical gatekeeper, the OM also contributes towards the strength and stiffness of cells and allows them to sustain mechanical stress. Largely influenced by studies of Escherichia coli, the OM is viewed as a rigid barrier where OM proteins and lipopolysaccharides display restricted mobility. Here the discussion is extended to other bacterial species, with a focus on Myxococcus xanthus. In contrast to the rigid OM paradigm, myxobacteria possess a relatively fluid OM. It is concluded that the fluidity of the OM varies across environmental species, which is likely linked to their evolution and adaptation to specific ecological niches. Importantly, a fluid OM can endow bacteria with distinct functions for cell-cell and cell-environment interactions.
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Affiliation(s)
| | - Daniel Wall
- Department of Molecular Biology, University of Wyoming, 1000 E University Avenue, Laramie, WY, 82071, USA
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22
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Horne JE, Brockwell DJ, Radford SE. Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria. J Biol Chem 2020; 295:10340-10367. [PMID: 32499369 PMCID: PMC7383365 DOI: 10.1074/jbc.rev120.011473] [Citation(s) in RCA: 102] [Impact Index Per Article: 20.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Revised: 06/03/2020] [Indexed: 01/09/2023] Open
Abstract
β-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.
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Affiliation(s)
- Jim E Horne
- Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
- Department of Biochemistry, University of Oxford, Oxford, United Kingdom
| | - David J Brockwell
- Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
| | - Sheena E Radford
- Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
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23
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Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS. Trends Biochem Sci 2020; 45:549-551. [DOI: 10.1016/j.tibs.2020.03.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2020] [Revised: 03/03/2020] [Accepted: 03/12/2020] [Indexed: 12/29/2022]
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24
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Gunasinghe SD, Shiota T, Stubenrauch CJ, Schulze KE, Webb CT, Fulcher AJ, Dunstan RA, Hay ID, Naderer T, Whelan DR, Bell TDM, Elgass KD, Strugnell RA, Lithgow T. The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane. Cell Rep 2019; 23:2782-2794. [PMID: 29847806 DOI: 10.1016/j.celrep.2018.04.093] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Revised: 03/05/2018] [Accepted: 04/23/2018] [Indexed: 01/28/2023] Open
Abstract
The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.
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Affiliation(s)
- Sachith D Gunasinghe
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
| | - Takuya Shiota
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia; Organization for Promotion of Tenure Track, University of Miyazaki, Miyazaki 889-1692, Japan
| | - Christopher J Stubenrauch
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
| | - Keith E Schulze
- Monash Micro Imaging, Monash University, Clayton, VIC 3800, Australia
| | - Chaille T Webb
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
| | - Alex J Fulcher
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia; Monash Micro Imaging, Monash University, Clayton, VIC 3800, Australia; Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800, Australia
| | - Rhys A Dunstan
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
| | - Iain D Hay
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
| | - Thomas Naderer
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800, Australia
| | - Donna R Whelan
- School of Chemistry, Monash University, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA
| | - Toby D M Bell
- School of Chemistry, Monash University, Clayton, VIC 3800, Australia
| | - Kirstin D Elgass
- Monash Micro Imaging, Monash University, Clayton, VIC 3800, Australia; Hudson Institute of Medical Research, Clayton, VIC 3800, Australia
| | - Richard A Strugnell
- Department of Microbiology & Immunology, University of Melbourne, Parkville, VIC 3052, Australia
| | - Trevor Lithgow
- Infection & Immunity Program, Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia.
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25
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Nilaweera TD, Nyenhuis DA, Nakamoto RK, Cafiso DS. Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter. Biophys J 2019; 117:1476-1484. [PMID: 31582182 DOI: 10.1016/j.bpj.2019.09.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2019] [Revised: 08/28/2019] [Accepted: 09/06/2019] [Indexed: 10/26/2022] Open
Abstract
Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here, we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic-facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.
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Affiliation(s)
- Thushani D Nilaweera
- Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia
| | - David A Nyenhuis
- Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia
| | - Robert K Nakamoto
- Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia
| | - David S Cafiso
- Department of Chemistry and Center for Membrane Biology, University of Virginia, Charlottesville, Virginia.
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26
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Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms. J Bacteriol 2019; 201:JB.00177-19. [PMID: 31209077 DOI: 10.1128/jb.00177-19] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2019] [Accepted: 06/12/2019] [Indexed: 11/20/2022] Open
Abstract
OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In Caulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCE Protein concentration gradients play a relevant role in the organization of the bacterial cell. The Caulobacter crescentus protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in Escherichia coli, suggesting a different organization of the outer membrane proteins.
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27
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Hatlem D, Trunk T, Linke D, Leo JC. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. Int J Mol Sci 2019; 20:E2129. [PMID: 31052154 PMCID: PMC6539128 DOI: 10.3390/ijms20092129] [Citation(s) in RCA: 86] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2019] [Revised: 04/25/2019] [Accepted: 04/26/2019] [Indexed: 01/05/2023] Open
Abstract
The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.
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Affiliation(s)
- Daniel Hatlem
- Bacterial Cell Surface Group, Section for Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
| | - Thomas Trunk
- Bacterial Cell Surface Group, Section for Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
| | - Dirk Linke
- Bacterial Cell Surface Group, Section for Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
| | - Jack C Leo
- Bacterial Cell Surface Group, Section for Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
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28
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Chavent M, Duncan AL, Rassam P, Birkholz O, Hélie J, Reddy T, Beliaev D, Hambly B, Piehler J, Kleanthous C, Sansom MSP. How nanoscale protein interactions determine the mesoscale dynamic organisation of bacterial outer membrane proteins. Nat Commun 2018; 9:2846. [PMID: 30030429 PMCID: PMC6054660 DOI: 10.1038/s41467-018-05255-9] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Accepted: 06/15/2018] [Indexed: 01/07/2023] Open
Abstract
The spatiotemporal organisation of membranes is often characterised by the formation of large protein clusters. In Escherichia coli, outer membrane protein (OMP) clustering leads to OMP islands, the formation of which underpins OMP turnover and drives organisation across the cell envelope. Modelling how OMP islands form in order to understand their origin and outer membrane behaviour has been confounded by the inherent difficulties of simulating large numbers of OMPs over meaningful timescales. Here, we overcome these problems by training a mesoscale model incorporating thousands of OMPs on coarse-grained molecular dynamics simulations. We achieve simulations over timescales that allow direct comparison to experimental data of OMP behaviour. We show that specific interaction surfaces between OMPs are key to the formation of OMP clusters, that OMP clusters present a mesh of moving barriers that confine newly inserted proteins within islands, and that mesoscale simulations recapitulate the restricted diffusion characteristics of OMPs. In Escherichia coli, outer membrane protein (OMP) cluster and form islands, but the origin and behaviour of those clusters remains poorly understood. Here authors use coarse grained molecular dynamics simulation and show that their mesoscale simulations recapitulate the restricted diffusion characteristics of OMPs.
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Affiliation(s)
- Matthieu Chavent
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK.,Institut de Pharmacologie et Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 205 Route de Narbonne, Toulouse, 31400, France
| | - Anna L Duncan
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK
| | - Patrice Rassam
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK.,Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74 Route du Rhin, 67401, Illkirch, France
| | - Oliver Birkholz
- Department of Biology, University of Osnabrück, Barbarastraße 11, 49076, Osnabrück, Germany
| | - Jean Hélie
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK.,SEMMLE, Blue Boar Court, 9 Alfred St, Oxford, OX1 4EH, UK
| | - Tyler Reddy
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK.,Theoretical Biology and Biophysics, T-6, Los Alamos National Laboratory, Los Alamos, NM, 87525, USA
| | - Dmitry Beliaev
- Mathematical Institute, University of Oxford, Andrew Wiles Building, Radcliffe Observatory Quarter (550), Woodstock Road, Oxford, OX2 6GG, UK
| | - Ben Hambly
- Mathematical Institute, University of Oxford, Andrew Wiles Building, Radcliffe Observatory Quarter (550), Woodstock Road, Oxford, OX2 6GG, UK
| | - Jacob Piehler
- Department of Biology, University of Osnabrück, Barbarastraße 11, 49076, Osnabrück, Germany
| | - Colin Kleanthous
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK.
| | - Mark S P Sansom
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 5RJ, UK.
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29
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Rojas ER, Billings G, Odermatt PD, Auer GK, Zhu L, Miguel A, Chang F, Weibel DB, Theriot JA, Huang KC. The outer membrane is an essential load-bearing element in Gram-negative bacteria. Nature 2018; 559:617-621. [PMID: 30022160 PMCID: PMC6089221 DOI: 10.1038/s41586-018-0344-3] [Citation(s) in RCA: 369] [Impact Index Per Article: 52.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2016] [Accepted: 06/05/2018] [Indexed: 12/24/2022]
Abstract
Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.
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Affiliation(s)
- Enrique R Rojas
- Department of Bioengineering, Stanford University, Stanford, CA, USA
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
| | | | - Pascal D Odermatt
- Department of Bioengineering, Stanford University, Stanford, CA, USA
- Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, USA
| | - George K Auer
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA
| | - Lillian Zhu
- Department of Bioengineering, Stanford University, Stanford, CA, USA
| | - Amanda Miguel
- Department of Bioengineering, Stanford University, Stanford, CA, USA
| | - Fred Chang
- Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, USA
| | - Douglas B Weibel
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA
- Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA
| | - Julie A Theriot
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA
- Howard Hughes Medical Institute, Stanford, CA, USA
- Biophysics Program, Stanford University, Stanford, CA, USA
| | - Kerwyn Casey Huang
- Department of Bioengineering, Stanford University, Stanford, CA, USA.
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
- Biophysics Program, Stanford University, Stanford, CA, USA.
- Chan Zuckerberg Biohub, San Francisco, CA, USA.
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30
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Abstract
Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.
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Affiliation(s)
- Samuel I. Miller
- Departments of Genome Sciences, University of Washington, Seattle, Washington, United States of America
- Department of Medicine, University of Washington, Seattle, Washington, United States of America
- Department of Microbiology, University of Washington, Seattle, Washington, United States of America
- * E-mail:
| | - Nina R. Salama
- Department of Microbiology, University of Washington, Seattle, Washington, United States of America
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America
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31
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Keeble AH, Banerjee A, Ferla MP, Reddington SC, Anuar INAK, Howarth M. Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics. Angew Chem Int Ed Engl 2017; 56:16521-16525. [PMID: 29024296 PMCID: PMC5814910 DOI: 10.1002/anie.201707623] [Citation(s) in RCA: 131] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2017] [Revised: 09/15/2017] [Indexed: 12/19/2022]
Abstract
SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram-positive bacterial adhesin. In this work, we established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide.
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Affiliation(s)
- Anthony H. Keeble
- Department of BiochemistryUniversity of OxfordSouth Parks RoadOxfordOX1 3QUUK
| | - Anusuya Banerjee
- Department of BiochemistryUniversity of OxfordSouth Parks RoadOxfordOX1 3QUUK
| | - Matteo P. Ferla
- Department of BiochemistryUniversity of OxfordSouth Parks RoadOxfordOX1 3QUUK
| | | | | | - Mark Howarth
- Department of BiochemistryUniversity of OxfordSouth Parks RoadOxfordOX1 3QUUK
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32
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Keeble AH, Banerjee A, Ferla MP, Reddington SC, Anuar INAK, Howarth M. Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics. Angew Chem Int Ed Engl 2017. [DOI: 10.1002/ange.201707623] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Affiliation(s)
- Anthony H. Keeble
- Department of Biochemistry; University of Oxford; South Parks Road Oxford OX1 3QU UK
| | - Anusuya Banerjee
- Department of Biochemistry; University of Oxford; South Parks Road Oxford OX1 3QU UK
| | - Matteo P. Ferla
- Department of Biochemistry; University of Oxford; South Parks Road Oxford OX1 3QU UK
| | - Samuel C. Reddington
- Department of Biochemistry; University of Oxford; South Parks Road Oxford OX1 3QU UK
| | | | - Mark Howarth
- Department of Biochemistry; University of Oxford; South Parks Road Oxford OX1 3QU UK
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33
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Kreft JU, Plugge CM, Prats C, Leveau JHJ, Zhang W, Hellweger FL. From Genes to Ecosystems in Microbiology: Modeling Approaches and the Importance of Individuality. Front Microbiol 2017; 8:2299. [PMID: 29230200 PMCID: PMC5711835 DOI: 10.3389/fmicb.2017.02299] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2017] [Accepted: 11/07/2017] [Indexed: 01/04/2023] Open
Abstract
Models are important tools in microbial ecology. They can be used to advance understanding by helping to interpret observations and test hypotheses, and to predict the effects of ecosystem management actions or a different climate. Over the past decades, biological knowledge and ecosystem observations have advanced to the molecular and in particular gene level. However, microbial ecology models have changed less and a current challenge is to make them utilize the knowledge and observations at the genetic level. We review published models that explicitly consider genes and make predictions at the population or ecosystem level. The models can be grouped into three general approaches, i.e., metabolic flux, gene-centric and agent-based. We describe and contrast these approaches by applying them to a hypothetical ecosystem and discuss their strengths and weaknesses. An important distinguishing feature is how variation between individual cells (individuality) is handled. In microbial ecosystems, individual heterogeneity is generated by a number of mechanisms including stochastic interactions of molecules (e.g., gene expression), stochastic and deterministic cell division asymmetry, small-scale environmental heterogeneity, and differential transport in a heterogeneous environment. This heterogeneity can then be amplified and transferred to other cell properties by several mechanisms, including nutrient uptake, metabolism and growth, cell cycle asynchronicity and the effects of age and damage. For example, stochastic gene expression may lead to heterogeneity in nutrient uptake enzyme levels, which in turn results in heterogeneity in intracellular nutrient levels. Individuality can have important ecological consequences, including division of labor, bet hedging, aging and sub-optimality. Understanding the importance of individuality and the mechanism(s) underlying it for the specific microbial system and question investigated is essential for selecting the optimal modeling strategy.
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Affiliation(s)
- Jan-Ulrich Kreft
- Centre for Computational Biology, Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom
| | - Caroline M Plugge
- Laboratory of Microbiology, Wageningen University and Research, Wageningen, Netherlands
| | - Clara Prats
- Department of Physics, School of Agricultural Engineering of Barcelona, Universitat Politècnica de Catalunya-BarcelonaTech, Castelldefels, Spain
| | - Johan H J Leveau
- Department of Plant Pathology, University of California, Davis, Davis, CA, United States
| | - Weiwen Zhang
- Laboratory of Synthetic Microbiology, Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China
| | - Ferdi L Hellweger
- Civil and Environmental Engineering Department, Marine and Environmental Sciences Department, Bioengineering Department, Northeastern University, Boston, MA, United States
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34
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Gunasinghe SD, Webb CT, Elgass KD, Hay ID, Lithgow T. Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria. Front Cell Infect Microbiol 2017; 7:220. [PMID: 28611954 PMCID: PMC5447050 DOI: 10.3389/fcimb.2017.00220] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2017] [Accepted: 05/12/2017] [Indexed: 12/28/2022] Open
Abstract
Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i) outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii) the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii) translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.
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Affiliation(s)
- Sachith D Gunasinghe
- Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash UniversityClayton, VIC, Australia
| | - Chaille T Webb
- Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash UniversityClayton, VIC, Australia
| | | | - Iain D Hay
- Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash UniversityClayton, VIC, Australia
| | - Trevor Lithgow
- Infection and Immunity Program, Department of Microbiology, Biomedicine Discovery Institute, Monash UniversityClayton, VIC, Australia
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35
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Ursell T, Lee TK, Shiomi D, Shi H, Tropini C, Monds RD, Colavin A, Billings G, Bhaya-Grossman I, Broxton M, Huang BE, Niki H, Huang KC. Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library. BMC Biol 2017; 15:17. [PMID: 28222723 PMCID: PMC5320674 DOI: 10.1186/s12915-017-0348-8] [Citation(s) in RCA: 82] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2016] [Accepted: 01/06/2017] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features. RESULTS Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics. CONCLUSIONS Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.
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Affiliation(s)
- Tristan Ursell
- Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.,Department of Physics, University of Oregon, Eugene, OR, 97403, USA
| | - Timothy K Lee
- Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA
| | - Daisuke Shiomi
- National Institute of Genetics, Shizuoka, Japan.,Current address: Department of Life Science, Rikkyo University, Tokyo, Japan
| | - Handuo Shi
- Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA
| | - Carolina Tropini
- Biophysics Program, Stanford University School of Medicine, Stanford, CA, 94305, USA.,Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Russell D Monds
- Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.,Current address: Synthetic Genomics Inc., La Jolla, CA, 92037, USA
| | - Alexandre Colavin
- Biophysics Program, Stanford University School of Medicine, Stanford, CA, 94305, USA
| | - Gabriel Billings
- Department of Physics, Stanford University, Stanford, CA, 94305, USA
| | | | - Michael Broxton
- Department of Computer Science, Stanford University, Stanford, CA, 94305, USA
| | | | | | - Kerwyn Casey Huang
- Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA. .,Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
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36
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Sisa C, Turroni S, Amici R, Brigidi P, Candela M, Cerri M. Potential role of the gut microbiota in synthetic torpor and therapeutic hypothermia. World J Gastroenterol 2017; 23:406-413. [PMID: 28210076 PMCID: PMC5291845 DOI: 10.3748/wjg.v23.i3.406] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2016] [Revised: 11/18/2016] [Accepted: 12/19/2016] [Indexed: 02/06/2023] Open
Abstract
Therapeutic hypothermia is today used in several clinical settings, among them the gut related diseases that are influenced by ischemia/reperfusion injury. This perspective paved the way to the study of hibernation physiology, in natural hibernators, highlighting an unexpected importance of the gut microbial ecosystem in hibernation and torpor. In natural hibernators, intestinal microbes adaptively reorganize their structural configuration during torpor, and maintain a mutualistic configuration regardless of long periods of fasting and cold temperatures. This allows the gut microbiome to provide the host with metabolites, which are essential to keep the host immunological and metabolic homeostasis during hibernation. The emerging role of the gut microbiota in the hibernation process suggests the importance of maintaining a mutualistic gut microbiota configuration in the application of therapeutic hypothermia as well as in the development of new strategy such as the use of synthetic torpor in humans. The possible utilization of tailored probiotics to mold the gut ecosystem during therapeutic hypothermia can also be taken into consideration as new therapeutic strategy.
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37
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Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions. mBio 2016; 7:mBio.01532-16. [PMID: 27795394 PMCID: PMC5082901 DOI: 10.1128/mbio.01532-16] [Citation(s) in RCA: 78] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell’s successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions. All Gram-negative bacteria alter the structural composition of LPS present in their OM in response to various environmental stimuli. We developed a system to track the native dynamics of lipid A change in Salmonella enterica serovar Typhimurium following an environmental shift to PhoP/Q- and PmrA/B-inducing conditions. We show that growth conditions influence OMV production, size, and lipid A content. We further demonstrate that the lipid A content of OMVs does not fit a stochastic model of content selection, revealing the significant retention of lipid A species containing covalent modifications that mask their 1- and 4′-phosphate moieties under host-like conditions. Furthermore, palmitoylation of the lipid A to form hepta-acylated species substantially increases the likelihood of its incorporation into OMVs. These results highlight a role for the OMV response in OM remodeling and maintenance processes in Gram-negative bacteria.
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Puiggalí-Jou A, Pérez-Madrigal MM, Del Valle LJ, Armelin E, Casas MT, Michaux C, Perpète EA, Estrany F, Alemán C. Confinement of a β-barrel protein in nanoperforated free-standing nanomembranes for ion transport. NANOSCALE 2016; 8:16922-16935. [PMID: 27714137 DOI: 10.1039/c6nr04948f] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Bioinspired free-standing nanomembranes (FSNMs) for selective ion transport have been tailored by immobilizing the Omp2a β-barrel membrane protein inside nanoperforations created in flexible poly(lactic acid) (PLA) nanomembranes. Perforated PLA FSNMs have been prepared by spin-coating a 99 : 1 PLA : poly(vinyl alcohol) mixture, and through a phase segregation process nanofeatures with dimensions similar to the entire nanomembrane thickness (∼110 nm) were induced. These nanofeatures have subsequently been transformed into nanoperforations (diameter: ∼51 nm) by selective solvent etching. The protein confined inside the nanopores of PLA FSNMs preserves the β-barrel structure and organizes in ovoid aggregates. The transport properties of Na+, K+, and Ca2+ across non-perforated PLA, nanoperforated PLA, and Omp2a-filled nanoperforated PLA have been monitored by measuring the nanomembrane resistance with electrochemical impedance spectroscopy (EIS). The incorporation of nanoperforations enhances the transport of ions across PLA nanomembranes, whereas the functionality of immobilized Omp2a is essential to exhibit effects similar to those observed in biological nanomembranes. Indeed, Omp2a-filled nanoperforated PLA nanomembranes exhibit stronger affinity towards Na+ and Ca2+ ions than towards K+. In summary, this work provides a novel bioinspired strategy to develop mechanically stable and flexible FSNMs with channels for ion transport, which are precisely located inside artificial nanoperforations, thus holding great potential for applications in biofiltration and biosensing.
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Affiliation(s)
- Anna Puiggalí-Jou
- Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Avda. Diagonal 647, Barcelona E-08028, Spain. and Center for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C', C/Pasqual i Vila s/n, Barcelona E-08028, Spain
| | - Maria M Pérez-Madrigal
- Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Avda. Diagonal 647, Barcelona E-08028, Spain. and Center for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C', C/Pasqual i Vila s/n, Barcelona E-08028, Spain
| | - Luis J Del Valle
- Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Avda. Diagonal 647, Barcelona E-08028, Spain. and Center for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C', C/Pasqual i Vila s/n, Barcelona E-08028, Spain
| | - Elaine Armelin
- Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Avda. Diagonal 647, Barcelona E-08028, Spain. and Center for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C', C/Pasqual i Vila s/n, Barcelona E-08028, Spain
| | - María T Casas
- Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Avda. Diagonal 647, Barcelona E-08028, Spain.
| | - Catherine Michaux
- Laboratoire de Chimie Physique des Biomolécules, Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur, Rue de Bruxelles, 61, 5000 Namur, Belgium
| | - Eric A Perpète
- Laboratoire de Chimie Physique des Biomolécules, Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur, Rue de Bruxelles, 61, 5000 Namur, Belgium
| | - Francesc Estrany
- Center for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C', C/Pasqual i Vila s/n, Barcelona E-08028, Spain and Departament d'Enginyeria Química, Escola Universitària d'Enginyeria Tècnica Industrial de Barcelona, Universitat Politècnica de Catalunya, Comte d'Urgell 187, 08036 Barcelona, Spain
| | - Carlos Alemán
- Departament d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Avda. Diagonal 647, Barcelona E-08028, Spain. and Center for Research in Nano-Engineering, Universitat Politècnica de Catalunya, Campus Sud, Edifici C', C/Pasqual i Vila s/n, Barcelona E-08028, Spain
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39
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Plummer AM, Fleming KG. From Chaperones to the Membrane with a BAM! Trends Biochem Sci 2016; 41:872-882. [PMID: 27450425 DOI: 10.1016/j.tibs.2016.06.005] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Revised: 06/13/2016] [Accepted: 06/20/2016] [Indexed: 01/17/2023]
Abstract
Outer membrane proteins (OMPs) play a central role in the integrity of the outer membrane of Gram-negative bacteria. Unfolded OMPs (uOMPs) transit across the periplasm, and subsequent folding and assembly are crucial for biogenesis. Chaperones and the essential β-barrel assembly machinery (BAM) complex facilitate these processes. In vitro studies suggest that some chaperones sequester uOMPs in internal cavities during their periplasmic transit to prevent deleterious aggregation. Upon reaching the outer membrane, the BAM complex acts catalytically to accelerate uOMP folding. Complementary in vivo experiments have revealed the localization and activity of the BAM complex in living cells. Completing an understanding of OMP biogenesis will require a holistic view of the interplay among the individual components discussed here.
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Affiliation(s)
- Ashlee M Plummer
- Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
| | - Karen G Fleming
- Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
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40
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Abstract
With the realization that bacteria achieve exquisite levels of spatiotemporal organization has come the challenge of discovering the underlying mechanisms. In this review, we describe three classes of such mechanisms, each of which has physical origins: the use of landmarks, the creation of higher-order structures that enable geometric sensing, and the emergence of length scales from systems of chemical reactions coupled to diffusion. We then examine the diversity of geometric cues that exist even in cells with relatively simple geometries, and end by discussing both new technologies that could drive further discovery and the implications of our current knowledge for the behavior, fitness, and evolution of bacteria. The organizational strategies described here are employed in a wide variety of systems and in species across all kingdoms of life; in many ways they provide a general blueprint for organizing the building blocks of life.
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Affiliation(s)
- Ned S Wingreen
- Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544;
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41
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Urfer M, Bogdanovic J, Lo Monte F, Moehle K, Zerbe K, Omasits U, Ahrens CH, Pessi G, Eberl L, Robinson JA. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli. J Biol Chem 2015; 291:1921-1932. [PMID: 26627837 DOI: 10.1074/jbc.m115.691725] [Citation(s) in RCA: 80] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2015] [Indexed: 01/05/2023] Open
Abstract
Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.
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Affiliation(s)
- Matthias Urfer
- From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich
| | - Jasmina Bogdanovic
- From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich
| | - Fabio Lo Monte
- From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich
| | - Kerstin Moehle
- From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich
| | - Katja Zerbe
- From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich
| | - Ulrich Omasits
- the Institute of Molecular Systems Biology, ETH Zurich, Auguste-Piccard Hof 1, 8093 Zurich, Switzerland
| | - Christian H Ahrens
- the Institute for Plant Production Sciences, Research Group Molecular Diagnostics, Genomics, and Bioinformatics and the Swiss Institute of Bioinformatics, Agroscope, Schloss 1, 8820 Wädenswil, Switzerland, and
| | - Gabriella Pessi
- the Department of Microbiology, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland
| | - Leo Eberl
- the Department of Microbiology, Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland
| | - John A Robinson
- From the Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich,.
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42
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Kleanthous C, Rassam P, Baumann CG. Protein-protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins. Curr Opin Struct Biol 2015; 35:109-15. [PMID: 26629934 PMCID: PMC4684144 DOI: 10.1016/j.sbi.2015.10.007] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2015] [Revised: 10/26/2015] [Accepted: 10/30/2015] [Indexed: 01/14/2023]
Abstract
We discuss spatiotemporal patterning in the bacterial outer membrane. Promiscuous interactions between outer membrane proteins govern their behaviour. Turnover and biogenesis of outer membrane proteins linked to formation of clusters. Implications of spatiotemporal patterning for bacterial physiology discussed. It has until recently been unclear whether outer membrane proteins (OMPs) of Gram-negative bacteria are organized or distributed randomly. Studies now suggest promiscuous protein–protein interactions (PPIs) between β-barrel OMPs in Escherichia coli govern their local and global dynamics, engender spatiotemporal patterning of the outer membrane into micro-domains and are the basis of β-barrel protein turnover. We contextualize these latest advances, speculate on areas of bacterial cell biology that might be influenced by the organization of OMPs into supramolecular assemblies, and highlight the new questions and controversies this revised view of the bacterial outer membrane raises.
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Affiliation(s)
- Colin Kleanthous
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
| | - Patrice Rassam
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
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43
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Rassam P, Copeland NA, Birkholz O, Tóth C, Chavent M, Duncan AL, Cross SJ, Housden NG, Kaminska R, Seger U, Quinn DM, Garrod TJ, Sansom MSP, Piehler J, Baumann CG, Kleanthous C. Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria. Nature 2015; 523:333-6. [PMID: 26061769 PMCID: PMC4905513 DOI: 10.1038/nature14461] [Citation(s) in RCA: 147] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2014] [Accepted: 04/08/2015] [Indexed: 12/24/2022]
Abstract
Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen. Intricate regulatory mechanisms ensure that bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat. Yet no mechanism is known for replacing OMPs in the outer membrane, an issue that is further confounded by the lack of an energy source and the high stability and abundance of OMPs. Here we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form ∼0.5-μm diameter islands, where their diffusion is restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the outer membrane. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence, the outer membrane of a Gram-negative bacterium is a spatially and temporally organized structure, and this organization lies at the heart of how OMPs are turned over in the membrane.
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Affiliation(s)
- Patrice Rassam
- 1] Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK [2] Department of Biology, University of York, York YO10 5DD, UK
| | | | - Oliver Birkholz
- Department of Biology, University of Osnabrück, Barbarastraße 11, 49076 Osnabrück, Germany
| | - Csaba Tóth
- Department of Biology, University of York, York YO10 5DD, UK
| | - Matthieu Chavent
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Anna L Duncan
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Stephen J Cross
- Department of Biology, University of York, York YO10 5DD, UK
| | - Nicholas G Housden
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Renata Kaminska
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Urban Seger
- Department of Biology, University of York, York YO10 5DD, UK
| | - Diana M Quinn
- Department of Biology, University of York, York YO10 5DD, UK
| | - Tamsin J Garrod
- Department of Biology, University of York, York YO10 5DD, UK
| | - Mark S P Sansom
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Jacob Piehler
- Department of Biology, University of Osnabrück, Barbarastraße 11, 49076 Osnabrück, Germany
| | | | - Colin Kleanthous
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
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44
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Misra R. Entry and exit of bacterial outer membrane proteins. Trends Microbiol 2015; 23:452-4. [PMID: 26169444 DOI: 10.1016/j.tim.2015.07.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Accepted: 07/02/2015] [Indexed: 01/29/2023]
Abstract
The sites of new outer membrane protein (OMP) deposition and the fate of pre-existing OMPs are still enigmatic despite numerous concerted efforts. Rassam et al. identified mid-cell regions as the primary entry points for new OMP insertion in clusters, driving the pre-existing OMP clusters towards cell poles for long-term storage.
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Affiliation(s)
- Rajeev Misra
- School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.
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45
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Siegrist MS, Swarts BM, Fox DM, Lim SA, Bertozzi CR. Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface. FEMS Microbiol Rev 2015; 39:184-202. [PMID: 25725012 DOI: 10.1093/femsre/fuu012] [Citation(s) in RCA: 100] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology.
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Affiliation(s)
- M Sloan Siegrist
- Department of Chemistry, University of California, Berkeley, CA 94720, USA
| | - Benjamin M Swarts
- Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859, USA
| | - Douglas M Fox
- Department of Chemistry, University of California, Berkeley, CA 94720, USA
| | - Shion An Lim
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
| | - Carolyn R Bertozzi
- Department of Chemistry, University of California, Berkeley, CA 94720, USA Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA
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46
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Abstract
The rod is a ubiquitous shape adopted by walled cells from diverse organisms ranging from bacteria to fungi to plants. Although rod-like shapes are found in cells of vastly different sizes and are constructed by diverse mechanisms, the geometric similarities among these shapes across kingdoms suggest that there are common evolutionary advantages, which may result from simple physical principles in combination with chemical and physiological constraints. Here, we review mechanisms of constructing rod-shaped cells and the bases of different biophysical models of morphogenesis, comparing and contrasting model organisms in different kingdoms. We then speculate on possible advantages of the rod shape, and suggest strategies for elucidating the relative importance of each of these advantages.
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47
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Bonnington KE, Kuehn MJ. Protein selection and export via outer membrane vesicles. BIOCHIMICA ET BIOPHYSICA ACTA 2014; 1843:1612-9. [PMID: 24370777 PMCID: PMC4317292 DOI: 10.1016/j.bbamcr.2013.12.011] [Citation(s) in RCA: 215] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/01/2013] [Revised: 12/13/2013] [Accepted: 12/17/2013] [Indexed: 12/24/2022]
Abstract
Outer membrane vesicles (OMVs) are constitutively produced by all Gram-negative bacteria. OMVs form when buds from the outer membrane (OM) of cells encapsulate periplasmic material and pinch off from the OM to form spheroid particles approximately 10 to 300nm in diameter. OMVs accomplish a diversity of functional roles yet the OMV's utility is ultimately determined by its unique composition. Inclusion into OMVs may impart a variety of benefits to the protein cargo, including: protection from proteolytic degradation, enhancement of long-distance delivery, specificity in host-cell targeting, modulation of the immune response, coordinated secretion with other bacterial effectors, and/or exposure to a unique function-promoting environment. Many enriched OMV-associated components are virulence factors, aiding in host cell destruction, immune system evasion, host cell invasion, or antibiotic resistance. Although the mechanistic details of how proteins become enriched as OMV cargo remain elusive, recent data on OM biogenesis and relationships between LPS structure and OMV-cargo inclusion rates shed light on potential models for OM organization and consequent OMV budding. In this review, mechanisms based on pre-existing OM microdomains are proposed to explain how cargo may experience differing levels of enrichment in OMVs and degrees of association with OMVs during extracellular export. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
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Affiliation(s)
- K E Bonnington
- Department of Biochemistry, Duke University Medical Center, USA
| | - M J Kuehn
- Department of Biochemistry, Duke University Medical Center, USA.
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48
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Ursell TS, Nguyen J, Monds RD, Colavin A, Billings G, Ouzounov N, Gitai Z, Shaevitz JW, Huang KC. Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization. Proc Natl Acad Sci U S A 2014; 111:E1025-34. [PMID: 24550515 PMCID: PMC3964057 DOI: 10.1073/pnas.1317174111] [Citation(s) in RCA: 177] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.
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Affiliation(s)
- Tristan S. Ursell
- Department of Bioengineering, Stanford University, Stanford, CA 94305
| | - Jeffrey Nguyen
- Department of Physics, Princeton University, Princeton, NJ 08544
| | - Russell D. Monds
- Department of Bioengineering, Stanford University, Stanford, CA 94305
| | | | | | - Nikolay Ouzounov
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544
| | - Zemer Gitai
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544
| | - Joshua W. Shaevitz
- Department of Physics, Princeton University, Princeton, NJ 08544
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; and
| | - Kerwyn Casey Huang
- Department of Bioengineering, Stanford University, Stanford, CA 94305
- Biophysics Program, Stanford University, Stanford, CA 94305
- Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
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49
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Lee TK, Huang KC. The role of hydrolases in bacterial cell-wall growth. Curr Opin Microbiol 2013; 16:760-6. [PMID: 24035761 DOI: 10.1016/j.mib.2013.08.005] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2013] [Accepted: 08/18/2013] [Indexed: 01/18/2023]
Abstract
Although hydrolysis is known to be as important as synthesis in the growth and development of the bacterial cell wall, the coupling between these processes is not well understood. Bond cleavage can generate deleterious pores, but may also be required for the incorporation of new material and for the expansion of the wall, highlighting the importance of mechanical forces in interpreting the consequences of hydrolysis in models of growth. Critically, minimal essential subsets of hydrolases have now been identified in several model organisms, enabling the reduction of genetic complexity. Recent studies in Bacillus subtilis have provided evidence for both the presence and absence of coupling between synthesis and hydrolysis during sporulation and elongation, respectively. In this review, we discuss strategies for dissecting the relationship between synthesis and hydrolysis using time-lapse imaging, biophysical measurements of cell-wall architecture, and computational modeling.
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Affiliation(s)
- Timothy K Lee
- Department of Bioengineering, Stanford University, Stanford, CA, USA
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Tran ENH, Doyle MT, Morona R. LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles. PLoS One 2013; 8:e70508. [PMID: 23936222 PMCID: PMC3723647 DOI: 10.1371/journal.pone.0070508] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2013] [Accepted: 06/18/2013] [Indexed: 11/29/2022] Open
Abstract
The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP(HA) was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP(HA) was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP(HA) and IcsA showed that IcsP(HA) preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP(HA) in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP(HA) was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP(HA) detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.
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Affiliation(s)
- Elizabeth Ngoc Hoa Tran
- Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
| | - Matthew Thomas Doyle
- Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
| | - Renato Morona
- Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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