How the assembly and protection of the bacterial cell envelope depend on cysteine residues

Abstract

The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria. The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria. The cell envelope of Gram-negative bacteria is a complex macromolecular structure that consists of an inner membrane surrounding the cytoplasm and an outer membrane that separates the cell from the environment. While the inner membrane is a classic phospholipid bilayer, the outer membrane is asymmetric, with phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet (1Silhavy T.J. Kahne D. Walker S. The bacterial cell envelope.Cold Spring Harb. Perspect. Biol. 2010; 2 (20452953): a00041410.1101/cshperspect.a000414Crossref PubMed Scopus (1351) Google Scholar). The two membranes are separated by the periplasm, a viscous compartment that represents 10–20% of the total cell volume (2Vollmer W. Bertsche U. Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli.Biochim. Biophys. Acta. 2008; 1778 (17658458): 1714-173410.1016/j.bbamem.2007.06.007Crossref PubMed Scopus (260) Google Scholar) and contains a thin layer of peptidoglycan. The peptidoglycan, also referred to as the cell wall, is a polymer made of repeating units of a disaccharide (GlcNAc-N-acetylmuramic acid) cross-linked by short peptides (3Typas A. Banzhaf M. Gross C.A. Vollmer W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology.Nat. Rev. Microbiol. 2011; 10 (22203377): 123-13610.1038/nrmicro2677Crossref PubMed Scopus (700) Google Scholar, 4Egan A.J. Errington J. Vollmer W. Regulation of peptidoglycan synthesis and remodelling.Nat. Rev. Microbiol. 2020; 10.1038/s41579-020-0366-3Crossref PubMed Scopus (8) Google Scholar). 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Soluble proteins are present in the periplasm, where they engage in a variety of functions, including peptidoglycan assembly, protein folding, and nutrient import. Integral membrane proteins are present in both membranes. While inner membrane proteins cross the lipid bilayer via hydrophobic α-helices, proteins inserted in the outer membrane contain amphipathic β-strands that are arranged in a linear antiparallel β-sheet; this β-sheet folds into a barrel by establishing hydrogen bonds between the first and last β-strands (1Silhavy T.J. Kahne D. Walker S. The bacterial cell envelope.Cold Spring Harb. Perspect. Biol. 2010; 2 (20452953): a00041410.1101/cshperspect.a000414Crossref PubMed Scopus (1351) Google Scholar, 8Konovalova A. Kahne D.E. Silhavy T.J. Outer membrane biogenesis.Annu. Rev. Microbiol. 2017; 71 (28886680): 539-55610.1146/annurev-micro-090816-093754Crossref PubMed Scopus (89) Google Scholar). Some of these so-called β-barrels function as passive diffusion channels, allowing small hydrophilic molecules to enter the cell, when others, connected to energy sources in the inner membrane, actively import specific compounds (9Noinaj N. Guillier M. Barnard T.J. Buchanan S.K. TonB-dependent transporters: regulation, structure, and function.Annu. Rev. Microbiol. 2010; 64 (20420522): 43-6010.1146/annurev.micro.112408.134247Crossref PubMed Scopus (479) Google Scholar). Other important envelope proteins are the lipoproteins, globular proteins anchored to a membrane by a lipid moiety. Although some lipoproteins remain in the inner membrane, most of them are targeted to the outer membrane (10Okuda S. Tokuda H. Lipoprotein sorting in bacteria.Annu. Rev. Microbiol. 2011; 65 (21663440): 239-25910.1146/annurev-micro-090110-102859Crossref PubMed Scopus (205) Google Scholar, 11Szewczyk J. Collet J.F. The journey of lipoproteins through the cell: one birthplace, PubMed Scopus Google Scholar). envelope layer is essential for viability; the outer membrane serves as a permeability barrier against compounds present in the A. Kahne D.E. Silhavy T.J. Outer membrane biogenesis.Annu. Rev. Microbiol. 2017; 71 (28886680): 539-55610.1146/annurev-micro-090816-093754Crossref PubMed Scopus (89) Google the peptidoglycan provides shape and osmotic protection to the cell (3Typas A. Banzhaf M. Gross C.A. Vollmer W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology.Nat. Rev. Microbiol. 2011; 10 (22203377): 123-13610.1038/nrmicro2677Crossref PubMed Scopus (700) Google and the inner membrane the cytoplasm and cellular including The crucial of the envelope is by the that (3Typas A. Banzhaf M. Gross C.A. Vollmer W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology.Nat. Rev. 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