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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 (
) 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 (
). 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 (
). 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 (
). 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 (
), and the inner membrane delimits the cytoplasm and hosts many vital cellular processes, including respiratory systems. The crucial importance of the envelope is nicely illustrated by the fact that several antibiotics (
) target the mechanisms of peptidoglycan assembly while others, such as colistin (a last resort antibiotic), destabilize the outer membrane. Assembling the envelope properly is challenging, in part because the complex machineries involved in the biogenesis of its different layers need to coordinate and to adapt their activities to the growth rate. In addition, most of the envelope building blocks necessary for synthesis, being produced in the cytoplasm or in the inner membrane, need to be transported to their final destination in an assembly-competent state and correctly integrated in the construction despite the lack of an obvious energy source (there is no ATP in the periplasm (
), correct folding often involves the formation of one or more disulfide bonds.
The understanding of the mechanisms of envelope assembly and maintenance has increased tremendously during the past two decades. For instance, the machineries involved in the biogenesis of the outer membrane have been identified and their characterization has been initiated. Further, elegant mechanisms used by cells to monitor the integrity of their envelopes have started to be elucidated. These major achievements are reviewed here; however, they are discussed from an unusual perspective. Indeed, we have chosen to give central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties make it often critical in biological processes. Putting cysteine residues under the spotlight brings to the surface often overlooked connections between essential cellular processes; it also highlights the important role played by redox-dependent mechanisms in cell envelope homeostasis. In the envelope, cysteine residues have two major functions that are reviewed here. First, they are involved in the formation of disulfide bonds that give envelope proteins further stability. Second, through their ability to function as nucleophiles in enzymatic reactions, cysteine residues are central to the activity of enzymes that are required for proper envelope assembly. Using nucleophilic cysteines comes with a price, however; because cysteines are highly vulnerable to oxidizing molecules that target bacteria during infection, cysteine-based enzymes are susceptible to irreversible inactivation and therefore need specific protection mechanisms. This is true both in the cytoplasm and in the periplasm. However, the reducing equivalents used by rescuing systems often originate from the cytoplasmic pool of NADPH (
). As a result, the protection of cysteine-based enzymes functioning in the envelope offers an additional challenge, as it involves transporting electrons across the inner membrane. Here, after briefly introducing the pathways of disulfide formation, we discuss the importance of cysteine residues in the folding of three essential assembly factors (FtsN, LptD, and BamA) and two proteins that cells use to monitor envelope integrity (RcsF and NlpE). Next, we focus on enzymes that utilize cysteine side chains as part of their catalytic machinery, and we address how these proteins are maintained as active in the oxidizing environment of the periplasm.
How disulfides are formed in the periplasm: a brief overview
The formation of a disulfide bond between two cysteine residues stabilizes a protein structure, mainly by decreasing the conformational entropy of the denatured state. This stabilizing effect can be up to ∼4 kcal/mol per disulfide formed (
). Disulfide bond formation is vital for the stability of many secreted proteins, both in bacteria and in eukaryotes. Proteins that are secreted to extracytoplasmic compartments such as the cell envelope or to the extracellular milieu benefit from stabilizing disulfides to remain folded in environments that lack ATP-dependent chaperones and often are rich in proteases and destabilizing compounds. Although disulfide bonds can form spontaneously in the presence of molecular oxygen, the process is rather slow and needs to be catalyzed in vivo. The first catalyst of disulfide bond formation identified in bacteria was E. coli DsbA (for Disulfide bond), a small (23-kDa) soluble periplasmic protein with a thioredoxin (Trx) fold. The biochemical characterization of DsbA established this protein as a highly oxidizing oxidoreductase (redox potential of −119 mV) (
) with a CXXC catalytic motif, mostly found oxidized in vivo. The oxidizing power of DsbA comes from the fact that reduction of the CXXC motif increases the stability of DsbA, thereby favoring the transfer of its catalytic disulfide to newly synthesized proteins entering the periplasm (
Outer membrane lipoprotein monitoring the integrity of the peptidoglycan and of the outer membrane; two nonconsecutive disulfides required for folding; induces the Rcs phosphorelay pathway under stress
Outer membrane lipoprotein monitoring lipoprotein trafficking to the outer membrane; two consecutive disulfides required for folding; induces the Cpx system when lipoprotein transport is perturbed
Envelope assembly enzymes with a catalytic cysteine residue
LdtA, LdtB, and LdtC
l,d-Transpeptidases catalyzing the attachment of the Braun lipoprotein Lpp to the peptidoglycan
LdtD and LdtE
l,d-Transpeptidases catalyzing the formation of 3-3 cross-links between two meso-diaminopimelic acid residues of adjacent stem peptides
DsbA catalyzes disulfide bond formation as cysteines in its substrates enter the periplasm. Therefore, when disulfides need to be formed between cysteine residues that are nonconsecutive in the substrate sequence, DsbA often catalyzes the formation of nonnative disulfides, causing protein misfolding, aggregation, and/or degradation. DsbC, a V-shaped dimeric (47-kDa) oxidoreductase of the Trx family, was identified as a disulfide isomerase that corrects the errors of DsbA (
). These two features allow DsbC to recognize misfolded substrates; first, the N-terminal cysteine of the DsbC active site attacks a nonnative disulfide in the substrate, which results in the formation of an unstable mixed-disulfide complex. Next, the mixed disulfide is resolved either by the attack of another cysteine from the misfolded protein or by the C-terminal cysteine of DsbC itself. In the first case, DsbC acts as an isomerase that catalyzes the reshuffling of the disulfide in the substrate. In the second case, DsbC functions as a reductase, giving DsbA another chance to oxidize the substrate protein. Either way, the active site of DsbC needs to be kept reduced and active, which is the function of DsbD (Fig. 1 and Table 1) (
), a 59-kDa protein with three domains; two domains, DsbDα and DsbDγ, are located in the periplasm, and the third domain, DsbDβ, is embedded in the inner membrane. DsbD uniquely transfers electrons across the membrane, from the cytoplasmic Trx system and NADPH (
). The actual mechanism by which DsbD transfers electrons between cytoplasmic and periplasmic oxidoreductases is not fully understood but likely involves major conformational changes within DsbDβ, as suggested by structural studies with CcdA, a DsbDβ homolog (
). In the same line, deleting dsbA decreases virulence in pathogenic strains of E. coli and other Gram-negative bacteria due to the misfolding of virulence factors involved in adhesion, secretion, and toxicity (
Strikingly, of the ∼40 essential proteins belonging to the machineries that assemble the peptidoglycan and the outer membrane, only 3 contain cysteine residues in their sequences (or in their periplasmic segments, in the case of inner membrane proteins). In all 3 cases, these cysteines are involved in disulfide formation. This remarkably small number of cysteine groups in the components of the assembly complexes suggests a negative selection for cysteine residues (
). Mutation of the two cysteines of the SPOR domain decreases intracellular FtsN levels and causes cells to grow as filaments, a phenotype indicative of an impaired cell division process. Thus, the disulfide bond of the SPOR domain of FtsN stabilizes the structure of this protein and is important for function (
A second essential assembly protein with cysteine residues is LptD (Fig. 1 and Table 1), one of the components of the Lpt (Lipopolysaccharide transport) system that transports lipopolysaccharide molecules across the cell envelope (
)), takes place in the cytoplasm and in the inner membrane; lipopolysaccharide molecules are then extracted from the inner membrane by the LptB2FGC ABC transporter, transferred to the periplasmic protein LptA, and finally delivered to the outer membrane translocon, made of the large β-barrel LptD and the lipoprotein LptE. Together, the Lpt proteins form a membrane-to-membrane bridge for the unidirectional transport of lipopolysaccharides (
). The folding pathway of LptD is particularly complex; following translocation of the nascent protein into the periplasm, a first disulfide is introduced by DsbA between the first and second cysteines. Subsequent rearrangement of this disulfide into a Cys2-Cys4 bridge involves LptE (
). Formation of the second disulfide by DsbA can then occur. Given the essential function of LptD, one would expect ΔdsbA cells not to be viable. It is indeed the case, but only under anaerobic conditions in which LptD accumulates in a reduced, inactive form (
). In the presence of oxygen, however, ΔdsbA cells grow like WT cells, presumably because oxygen-dependent background oxidation, catalyzed by low-molecular-weight thiol-oxidizing compounds present in the periplasm, is sufficient for survival.
The third assembly protein with two cysteines forming a disulfide is the outer membrane protein BamA, the core component of the β-barrel assembly machinery (BAM) that assembles β-barrel proteins in the outer membrane (Fig. 1 and Table 1). β-Barrel precursors are synthesized in the cytoplasm with an N-terminal signal peptide that targets them to the Sec translocon for transport across the inner membrane. Note that it was recently shown that the mature domains of the proteins destined for secretion contain multiple, degenerate, interchangeable hydrophobic stretches that also play a role in targeting to the translocase machinery (
). Upon emerging from the translocon on the periplasmic side of the inner membrane, the signal peptide is cleaved off and unfolded β-barrels interact with periplasmic chaperones for transport across the periplasm and delivery to BAM (
), the two cysteine residues are not well conserved among BamA homologs (Iorga B. I., unpublished results), calling into question their functional importance. Accordingly, their mutation has no impact on BAM function (
). For instance, CirA, the outer membrane colicin 1 receptor protein, and FhuA, a β-barrel that functions as a ferrichrome iron receptor, contain one and two disulfide bonds, respectively. When and where these disulfides are formed (before or after BAM folding) remain to be established.
Disulfide bond formation is required for the folding of two important envelope stress sensors
Bacteria evolve in always-changing environments in which they can be exposed to molecules or conditions that alter envelope integrity. Given the vital importance of this compartment, bacteria rely on stress sensor proteins to detect perturbations in their envelope and to respond in a fast and adequate manner to inflicted damage. In E. coli, two major envelope stress sensors, the outer membrane lipoproteins RcsF and NlpE (Fig. 4 and Table 1), both contain disulfide-linked cysteine residues in their native conformations.
RcsF is a small (11-kDa) surface-exposed lipoprotein that monitors the integrity of the outer part of the envelope, i.e. the outer membrane and the peptidoglycan, in enterobacteria (
), a β-lactam that interferes with peptidoglycan synthesis by inhibiting the essential transpeptidase penicillin-binding protein 2 (PBP2). As a result, RcsF triggers a complex signaling cascade known as the Rcs phosphorelay pathway, which tries to contain the inflicted damage by modulating the expression of dozens of genes, including those producing capsular oligosaccharides (
). It is remarkable that the folding of RcsF, a protein required to sense most Rcs-inducing cues, critically depends on two nonconsecutive disulfide bonds (Fig. 4); in cells impaired in disulfide formation (ΔdsbA or ΔdsbB) or disulfide isomerization (ΔdsbC or ΔdsbD), RcsF does not fold (
), it is clear that the ability of RcsF to sense stress is linked to the unusual presence of this protein on the cell surface (the general view is that E. coli outer membrane lipoproteins face the periplasm). Interestingly, the export of RcsF to the surface is mediated by BAM via the assembly of complexes between RcsF and abundant β-barrel proteins, such as OmpC and OmpF (Fig. 4). The structure of a BamA-RcsF complex, which forms as an intermediate in the assembly of the complexes between RcsF and its β-barrel partners, was solved recently. In this complex, RcsF is lodged deep inside the lumen of the BamA barrel, which is observed in the inward-open conformation (
A second envelope stress sensor with disulfide-bonded cysteines in its native conformation is the outer membrane lipoprotein NlpE (Fig. 4), which activates the Cpx stress response when lipoprotein trafficking to the outer membrane is perturbed (
), thus suggesting that the C-terminal disulfide functions as a molecular sensor for redox perturbations. Because dsbA is a Cpx regulon member, this sensing would establish a neat feedback loop. The molecular mechanism of this redox-regulated Cpx induction remains to be determined, however. It is noteworthy that, whereas RcsF occupies a critical position in Rcs, NlpE is not central for Cpx function. Indeed, most Cpx-inducing cues, such as accumulation of misfolded proteins in the periplasm, inner membrane stress, and cell wall perturbations (
The activity of a family of enzymes important for envelope integrity depends on a single reduced cysteine residue
In E. coli, most envelope proteins either do not have any cysteine residues or have cysteine residues that are involved in disulfides. In the previous paragraphs, we discussed the importance of forming correct disulfide bonds in proteins that are required for envelope biogenesis and protection. In the following, we focus instead on the important role played by cysteine residues that are part of catalytic machineries and therefore need to remain reduced in the envelope. In fact, only a small group of enzymes use cysteine-based chemistry in the E. coli envelope, and these enzymes all belong to the l,d-transpeptidase family.
E. coli expresses six l,d-transpeptidases, but other bacteria, such as Bdellovibrio bacteriovorus, express more than 20 (
). Three of the E. colil,d-transpeptidases, i.e. LdtA, LdtB, and LdtC, attach the C-terminal lysine residue of the outer membrane lipoprotein Lpp, the numerically most abundant protein in E. coli (also known as the Braun lipoprotein), to a diaminopimelic acid residue in the peptide stems of the peptidoglycan (
). The other l,d-transpeptidases expressed by E. coli have a different function. LdtD and LdtE catalyze the formation of 3-3 cross-links between two meso-diaminopimelic acid residues of adjacent stem peptides during peptidoglycan synthesis (
) (Fig. 5 and Table 1), while the enzymatic activity of LdtF (YafK) remains unknown. In E. coli, there are only 2–10% 3-3 cross-links, with the majority of cross-links being between d-Ala and meso-diaminopimelic acid residues (4-3 cross-links) (
The functional importance of l,d-transpeptidases in the assembly of the envelope is beginning to be fully appreciated, not only in E. coli but also in a large number of bacteria, including mycobacteria, where they play a major role in peptidoglycan assembly by catalyzing abundant 3-3 cross-links (
), the thiol side chains of cysteine residues are indeed oxidized to sulfenic acids (-SOH), which are highly reactive and can be irreversibly oxidized to sulfinic acids (-SO2H) and sulfonic acids (-SO3H) (
). These latter two modifications are often detrimental for protein function and inactivate l,d-transpeptidases. Accordingly, it was recently shown that exposure of E. coli to copper, a redox-active metal that is able to catalyze cysteine oxidation in the presence of oxygen (
) (Fig. 5). Thus, intracellular metabolism (DsbD is recycled at the expense of NADPH) provides the reducing equivalents to keep cysteine-based envelope enzymes functional, thus maintaining envelope integrity.
Conclusions and perspectives
Since the discovery of DsbA in 1991, an impressive body of research has revealed the critical role played by cysteine residues in the biogenesis and maintenance of the bacterial cell envelope. In the previous sections, we discussed the importance of disulfide bond formation for the folding and stability of envelope proteins, including crucial assembly factors and stress sensors, and we highlighted the functional relevance of enzymes that use cysteine-based chemistry to build the cell envelope properly. It is likely that additional examples of envelope assembly factors with cysteine residues important for folding and/or activity will be identified in the future, in E. coli or in other Gram-negative bacteria. In addition, future research will probably identify novel antioxidant factors protecting envelope proteins from oxidation, an area that has been less well explored than that of oxidative protein folding.
As we reach the end of this review, we would like to suggest the hypothesis that cysteine residues may play an additional role in the envelope by serving as regulatory switches controlling processes necessary for envelope homeostasis. Given their ability to undergo reversible redox modifications, cysteine residues indeed often act as powerful molecular switches allowing organisms to adapt to changes in the environment, as has been extensively described for the bacterial cytoplasm and for higher organisms (
). To our knowledge, no such example has been described so far for the mechanisms that participate in envelope biogenesis. However, several envelope proteins display features that hint at potential redox regulation. For instance, as discussed above, the uncommon presence of a disulfide bond between two adjacent β-strands in RcsF (
) is intriguing and suggests that a layer of redox regulation remains to be discovered for this protein. In addition, both the stress sensor NlpE and PBP1a (an enzyme required for peptidoglycan synthesis) have a disulfide bond between two cysteine residues that are found in a CXXC motif. The fact that CXXC motifs can function as redox switches (
). Another intriguing case is the abundant outer membrane protein OmpA, which is important for envelope integrity. OmpA is composed of an N-terminal 8-stranded β-barrel and a C-terminal periplasmic domain binding to the peptidoglycan (
). Interestingly, a disulfide bond present in the C-terminal domain might function as a redox switch controlling the OmpA conformation, as suggested by work in Salmonella enterica serovar Typhymurium (
) will facilitate further exploration of the versatile function and crucial roles of the amino acid cysteine in the bacterial cell envelope. In the same line, the fact that the cell envelope is an environment in which disulfides can be formed will prove very useful in studying the mechanism of crucial assembly and surveillance processes. Indeed, in addition to their native roles, disulfides can be artificially introduced into proteins to affect their structures, allowing inference of their function. In the case of BamA, for instance, using artificial disulfides has led to major mechanistic insights by demonstrating the importance of BamA cycling between an outward-open conformation and an inward-open conformation (
Author contributions—J.-F. C. and C. V. G. writing-original draft; J.-F. C., S.-H. C., B. I. I., and C. V. G. writing-review and editing.
Funding and additional information—This work was supported, in part, by grants from the Fonds de la Recherche Scientifique, from the CNRS, from the FRFS-WELBIO (Grant WELBIO-CR-2019-03), from the EOS Excellence in Research Program of the FWO and FRS-FNRS (Grant G0G0818N), and from the Fédération Wallonie-Bruxelles (Grant ARC 17/22-087).
Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.