Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme.

Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues. PhoA synthesized in the mutants indeed lacked disulfide bridges. These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.     

Folding of a bacterial outer membrane protein during passage through the periplasm.

The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.     

In vivo oxidative protein folding can be facilitated by oxidation–reduction cycling

Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis , a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region; that it is a very active reductase but a poor isomerase; and that it fully complements dsbC . These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.     

Site-directed disulfide cross-linking shows that cleft flexibility in the periplasmic domain is needed for the multidrug efflux pump AcrB of Escherichia coli

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.     

New Insights into the Assembly of Bacterial Secretins: STRUCTURAL STUDIES OF THE PERIPLASMIC DOMAIN OF XcpQ FROM PSEUDOMONAS AERUGINOSA*

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.     

A bacterial glutathione transporter (Escherichia coli CydDC) exports reductant to the periplasm

Glutathione (GSH), a major biological antioxidant, maintains redox balance in prokaryotes and eukaryotic cells and forms exportable conjugates with compounds of pharmacological and agronomic importance. However, no GSH transporter has been characterized in a prokaryote. We show here that a heterodimeric ATP-binding cassette-type transporter, CydDC, mediates GSH transport across the Escherichia coli cytoplasmic membrane. In everted membrane vesicles, GSH is imported via an ATP-driven, protonophore-insensitive, orthovanadate-sensitive mechanism, equating with export to the periplasm in intact cells. GSH transport and cytochrome bd quinol oxidase assembly are abolished in the cydD1 mutant. Glutathione disulfide (GSSG) was not transported in either Cyd(+) or Cyd(-) strains. Exogenous GSH restores defective swarming motility and benzylpenicillin sensitivity in a cydD mutant and also benzylpenicillin sensitivity in a gshA mutant defective in GSH synthesis. Overexpression of the cydDC operon in dsbD mutants defective in disulfide bond formation restores dithiothreitol tolerance and periplasmic cytochrome b assembly, revealing redundant pathways for reductant export to the periplasm. These results identify the first prokaryotic GSH transporter and indicate a key role for GSH in periplasmic redox homeostasis.     

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.     

Laboratory evolution of Escherichia coli thioredoxin for enhanced catalysis of protein oxidation in the periplasm reveals a phylogenetically conserved substrate specificity determinant

Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA(-) cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA(-) cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.     

MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II

The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.     

Folding LacZ in the Periplasm of Escherichia coli

Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic β-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.     

Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan.

The periplasmic cyclic beta-1,2-glucan of Agrobacterium tumefaciens is believed to maintain high osmolarity in the periplasm during growth of the bacteria on low-osmotic-strength media. Strains with mutations in the chvA or chvB gene do not accumulate beta-1,2-glucan in their periplasm and exhibit pleiotropic phenotypes, including inability to form crown gall tumors on plants. We examined the effects of medium osmolarity to determine whether some or all of these phenotypes result from suboptimal periplasmic osmolarity. The mutants grew more slowly than wild-type cells and exhibited altered periplasmic and cytoplasmic protein content when cultured in low-osmotic-strength media, but not when cultured in high-osmotic-strength media. These observations support a role for periplasmic glucan in osmoadaptation. However, the mutants were avirulent and exhibited reduced motility regardless of the osmolarity of the medium. Therefore, beta-1,2-glucan may play roles in virulence and motility that are unrelated to its role in osmoadaptation.     

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.     

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook‐associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non‐motile mutant cells that are unable to assemble flagellar filaments and pentagon‐shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella‐deficient mutants (i.e., in the hook, rod, or MS‐ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.     

Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli

Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.     

Disulfide-dependent folding and export of Escherichia coli DsbC

DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone. Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied. The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities. Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein. Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing. DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants. The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.     

Structural and mechanistic studies of pesticin, a bacterial homolog of phage lysozymes

Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme.     

Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group

S protein, an inhibitor to the membrane attack complex of complement, was purified from human plasma. The procedure involved barium citrate adsorption and fractionation by poly(ethylene glycol) 4000 precipitation, followed by chromatography on DEAE-Sephacel, Blue Sepharose, Sephacryl S-200, and finally anti-albumin-Sepharose. Reduced glutathione was added throughout to inhibit spontaneous formation of disulfide-linked S-protein dimers. The recovery was 7%, resulting in approximately 10 mg of pure S protein from 1 L of starting plasma. S protein is a single-chain molecule; sedimentation equilibrium ultracentrifugation yielded a molecular weight of 83 000; the s020,W value was estimated to be 4.0 S. The purified protein contained a free, reactive thiol group causing spontaneous formation of disulfide-linked S-protein dimers. Alkylated and nonalkylated S proteins were equally active in inhibiting C9 polymerization, catalyzed by the C5b-8 complex. In parallel with the inhibition of C9 polymerization, nonalkylated S protein catalyzed the formation of disulfide-linked C9 dimers, presumably through disulfide interchanges.     

Insight into disulfide bond catalysis in Chlamydia from the structure and function of DsbH, a novel oxidoreductase

The Chlamydia family of human pathogens uses outer envelope proteins that are highly cross-linked by disulfide bonds but nevertheless keeps an unusually high number of unpaired cysteines in its secreted proteins. To gain insight into chlamydial disulfide bond catalysis, the structure, function, and substrate interaction of a novel periplasmic oxidoreductase, termed DsbH, were determined. The structure of DsbH, its redox potential of -269 mV, and its functional properties are similar to thioredoxin and the C-terminal domain of DsbD, i.e. characteristic of a disulfide reductase. As compared with these proteins, the two central residues of the DsbH catalytic motif (CMWC) shield the catalytic disulfide bond and are selectively perturbed by a peptide ligand. This shows that these oxidoreductase family characteristic residues are not only important in determining the redox potential of the catalytic disulfide bond but also in influencing substrate interactions. For DsbH, three functional roles are conceivable; that is, reducing intermolecular disulfides between proteins and small molecules, keeping a specific subset of exported proteins reduced, or maintaining the periplasm of Chlamydia in a generally reducing state.     

The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.