Jon Beckwith

Identification of a protein required for disulfide bond formation in vivo

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.

THE ROLE OF THE THIOREDOXIN AND GLUTAREDOXIN PATHWAYS IN REDUCING PROTEIN DISULFIDE BONDS IN THE ESCHERICHIA COLI CYTOPLASM

In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each otherin vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.

Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins.

Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide‐reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.

Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and φ80 transducing phages

Abstract φ80 transducing phages for the proC † , phoA and phoB genes of Escherichia coli have been obtained. Two mutants have been isolated, in which the brnQ, phoA, proC, phoB (and possibly phoR ) genes have been deleted. Derivatives of a phoA, phoB deletion strain which are lysogenic for a φ80 phoA transducing phage make only very low levels of alkaline phosphatase activity. These results are in agreement with a positive control mechanism for the regulation of alkaline phosphatase synthesis.

Diverse Paths to Midcell: Assembly of the Bacterial Cell Division Machinery

At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.

E. coli mutant pleiotropically defective in the export of secreted proteins

A hybrid beta-galactosidase molecule containing a substantial portion of the amino-terminal sequence of the maltose-binding protein is inserted in the cytoplasmic membrane of E. coli; in this location, the protein has very low enzymatic activity. The strain producing it is, therefore, Lac-. Selection for derivatives of the fusion strain that are able to grow on lactose yields mutants in which the hybrid protein has become cytoplasmic, and thus has higher enzymatic activity. Among such derivatives, we have isolated a temperature-sensitive conditional lethal mutant that accumulates the precursor of the maltose-binding protein in the cytoplasm, and also accumulates precursors of alkaline phosphatase, lambda receptor protein and the ompF gene gene product. A number of periplasmic proteins are, however, properly localized at the nonpermissive temperature. The temperature-sensitive lesion has been genetically mapped to 2.5 min on the E. coli map, within or near a cluster of genes responsible for cell division and septation. The principle behind the genetic selection employed here should be useful in obtaining other secretion mutants to characterize the cells secretion machinery.

Genetic Analysis of Protein Export in Escherichia Coli

Genetic studies on the secretion process in gram-negative bacteria have made considerable progress. Within the near future, such studies should lead to a detailed understanding of the important features of signal sequences and how they function. The cloning of the structural gene for an enzyme that cleaves signal sequences from precursors of secreted proteins will allow the genetic characterization of this locus and its function. Finally, the isolation and characterization of mutants that affect components of the cells secretory apparatus are also under way. These mutants permit the detection of genes and their products that are involved in secretion. A combination of the genetic approaches and in vitro studies should lead to a picture of the details of passage of proteins through a membrane.

Bridge over Troubled Waters: Sensing Stress by Disulfide Bond Formation

are members of the thioredoxin superfamily and exert their action by a disulfide exchange reaction utilizing a Cys-X1-X2-Cys active site. Other members of the thioredoxin superfamily are responsible for the introduction and isomerization of disulfide bonds that are often presFredrik Åslund† and Jon Beckwith* Department of Microbiology and Molecular Genetics Harvard Medical School 200 Longwood Avenue ent in secreted proteins. All evidence points to a situaBoston, Massachusetts 02115 tion in which cytosolic proteins have evolved to maintain their cysteines reduced in the native form, whereas many secreted proteins have evolved to be more stable The regulation of protein activity is a major factor in when their cysteines are joined in disulfide bonds. Thus, the cellular response to a changing environment. Wellchanges in the reducing environment of the cytosol can established mechanisms for such regulation include have profound effects on protein folding and activity. protein–protein interactions, allosteric changes generPerturbations of the cellular redox conditions can be ated by ligand binding, and chemical modifications such achieved either by mutations that eliminate components as phosphorylation. It has long been postulated that of the thioredoxin and glutaredoxin systems or by envidisulfide bond formation represents a covalent modifironmental oxidative stress. Mutant analysis shows that cation that can regulate protein activity. This proposal a simultaneous block of both disulfide-reducing pathhas come from studies in which enzymes or transcripways is incompatible with growth under aerobic condition factors were shown to lose activity after oxidation tions (Prinz et al., 1997). Functional overlap between the of cysteine residues in vitro, but to regain activity when two pathways is indicated by the finding that, in the exposed to the disulfide reductant thioredoxin. Howabsence of only one of the pathways, strains can survive ever, such reports may often be based on the fact that and grow reasonably well. Nevertheless, the oxidizing the proteins studied usually exist in the reducing enviconditions in the cytosol of such strains allows for the ronment of the cytosol; the oxidative inactivation obformation of disulfide bonds in some proteins. This disulserved may simply reflect the unnatural oxidizing condifide bond–forming activity can be monitored by expresstions of the in vitro system and not reflect the in vivo ing in the cytosol a normally exported protein, such as state of affairs. alkaline phosphatase, which requires disulfide bonds for Thus, while it is not clear that loss of function by its enzymatic activity. Although it has yet to be directly disulfide bond formation has been established as a regudemonstrated, it seems likely that unwanted disulfide latory mechanism, recent results suggest that the rebonds are also generated in the normal resident proteins verse process, gain of function by disulfide bond formaof the cytosol during oxidative stress—a situation we tion, may be a common way of responding to cellular refer to as “disulfide stress.” stress. The utilization of improved techniques for asBacteria encounter oxidative stresses in environsessing the disulfide-bonded states of proteins in vivo ments with high levels of hydrogen peroxide or other has allowed a reexamination of the role of these bonds in reactive oxygen species. One example of such stress regulating protein activity. These studies provide strong occurs when pathogenic bacteria confront oxidative evidence that two bacterial proteins, the transcription bursts upon invasion of eukaryotic host cells. These factor OxyR and the chaperone heat shock protein 33 encounters, in addition to damaging other cellular mole(Hsp33), are activated by the oxidation of cysteine resicules, may also cause the introduction of deleterious dues to disulfide bonds (Zheng et al., 1998; Jakob et al., disulfide bonds into proteins. Furthermore, studies us1999). These findings and the approaches used should ing cytosolic alkaline phosphatase suggest that E. coli provide impetus to a search for what is likely to be in the stationary phase is subject to disulfide stress a more widespread occurrence of this mechanism for (Dukan and Nyström, 1998). regulating protein activity. How does the bacterial cell respond to the stress The two most important means of maintaining the that disulfide bond formation in the cytosol poses? Two reducing thiol-disulfide status of the cytosol involve the features common to this response are the restoration thioredoxin-thioredoxin reductase pathway and the gluof the redox homeostasis in the cytosol and the eliminatathione-glutaredoxin pathway (Prinz et al., 1997). Thiotion of the harmful oxidant (Figure 1). Such responses redoxins and glutaredoxins were first detected by their are a requirement for life in an aerobic environment that ability to reduce a disulfide bond in the active site of is accompanied by exposure to reactive oxygen species. ribonucleotide reductase, as part of the reduction pathIn E. coli, the exposure to reactive oxygen species way converting ribonucleotides to deoxyribonucleo(ROS) such as O2· and hydrogen peroxide activates the tides. The reduced form of thioredoxin is regenerated transcription factors SoxR/S and OxyR (Hidalgo et al., by thioredoxin reductase, whereas glutaredoxin is kept 1997; Zheng et al., 1998). These factors trigger the exreduced by glutathione. Thioredoxin and glutaredoxin pression of defense activities including superoxide dismutase and peroxidases. Recently, it was discovered * To whom correspondence should be addressed (e-mail: jbeck that the response to peroxides and disulfide stress is with@hms.harvard.edu). due to the formation of a disulfide bond within the OxyR † Present address: Department of Biology, Massachusetts Institute protein, thus converting it to a transcriptional activator of Technology, Cambridge, Massachusetts 02139 (as of April 1,

SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF.

The SecA subunit of E. coli preprotein translocase promotes protein secretion during cycles of membrane insertion and deinsertion at SecYEG. This process is regulated both by nucleotide binding and hydrolysis and by the SecD and SecF proteins. In the presence of associated preprotein, the energy of ATP binding at nucleotide-binding domain 1 (NBD1) drives membrane insertion of a 30 kDa domain of SecA, while deinsertion of SecA requires the hydrolysis of this ATP. SecD and SecF stabilize the inserted state of SecA. ATP binding at NBD2, though needed for preprotein translocation, is not needed for SecA insertion or deinsertion.

Secretion and Membrane Localization of Proteins in Escherichia Coli

The envelope of Escherichia coli consists of two distinct membranes, the outer membrane and the cytoplasmic membrane. The space between the two membranes is called the periplasmic space, and each fraction contains its own specific proteins. In this review, it is discussed how proteins are localized in their final locations in the envelope. Proteins localized in the outer membrane and the periplasmic space as well as transmembranous proteins in the cytoplasmic membranes appear to be produced from their precursors which have peptide extensions of about 20 amino acid residues at the amino terminal ends. General features for the peptide extension are deduced from the known sequences of the peptide extensions, and, based on their known properties, a hypothesis (loop model) is proposed to explain the possible functions of the peptide extension during the mechanism of secretion across the cytoplasmic membrane.