Promote Protein Folding Research Articles

Transcriptional profiling of the model Archaeon Halobacteriumsp. NRC-1: responses to changes in salinity and temperature

BackgroundThe model halophile Halobacterium sp. NRC-1 was among the first Archaea to be completely sequenced and many post-genomic tools, including whole genome DNA microarrays are now being applied to its analysis. This extremophile displays tolerance to multiple stresses, including high salinity, extreme (non-mesophilic) temperatures, lack of oxygen, and ultraviolet and ionizing radiation.ResultsIn order to study the response of Halobacterium sp. NRC-1 to two common stressors, salinity and temperature, we used whole genome DNA microarrays to assay for changes in gene expression under differential growth conditions. Cultures grown aerobically in rich medium at 42°C were compared to cultures grown at elevated or reduced temperature and high or low salinity. The results obtained were analyzed using a custom database and microarray analysis tools. Growth under salt stress conditions resulted in the modulation of genes coding for many ion transporters, including potassium, phosphate, and iron transporters, as well as some peptide transporters and stress proteins. Growth at cold temperature altered the expression of genes involved in lipid metabolism, buoyant gas vesicles, and cold shock proteins. Heat shock showed induction of several known chaperone genes. The results showed that Halobacterium sp. NRC-1 cells are highly responsive to environmental changes at the level of gene expression.ConclusionTranscriptional profiling showed that Halobacterium sp. NRC-1 is highly responsive to its environment and provided insights into some of the specific responses at the level of gene expression. Responses to changes in salt conditions appear to be designed to minimize the loss of essential ionic species and abate possible toxic effects of others, while exposure to temperature extremes elicit responses to promote protein folding and limit factors responsible for growth inhibition. This work lays the foundation for further bioinformatic and genetic studies which will lead to a more comprehensive understanding of the biology of a model halophilic Archaeon.

Effects of chemical chaperones on oxidative stress and detergent-insoluble species formation following conditional expression of amyloid precursor protein carboxy-terminal fragment

Oxidative stress, protein misfolding, protein complex formation, and detergent insolubility are biochemical features of Alzheimer's disease (AD). We tested the cause-and-effect relationships among these using MC65 human neuroblastoma cells that exhibit toxicity upon conditional expression of carboxy-terminal fragments (CTFs) of the human amyloid precursor protein (APP). Treatments with three different antioxidants (alpha-tocopherol, N-acetyl cysteine, and alpha-lipoic acid) or three different compounds (glycerol, trimethylamine-N-oxide, and 4-phenylbutyric acid) that have been described to have a "chemical chaperone" function in promoting protein folding all had a protective effect on MC65 cells and decreased markers of oxidative damage and accumulation of high molecular weight amyloid (A) beta-immunoreactive (IR) species. However, chaperones partially reduced detergent insolubility of the remaining Abeta-IR species, while antioxidants did not. These results suggest that protein misfolding associated with overexpression of APP CTFs promotes oxidative stress and cytotoxicity and contributes to formation of detergent-insoluble species that appear unrelated to cytotoxicity.

Tissue and allelic-specific expression of hsp70 gene in chickens: basal and heat-stress-induced mRNA level quantified with real-time reverse transcriptase polymerase chain reaction

1. The 70 kDa heat shock proteins (hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The objective of the current research was to investigate the relationship between tissue or allele and the expression of chicken hsp70 under normal growth conditions and during acute heat stress (44°C for 4 h). 2. A total of 279 individuals were genotyped for two single nucleotide polymorphisms (A258G and C276G) in chicken hsp70 gene by single-strand conformation polymorphism (SSCP) analysis. 3. The mRNA abundance of chicken hsp70 genes was evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The expression of hsp70 gene in the liver (9·83 ± 0·84, 107) was significantly higher than that in the muscle (4·42 ± 0·36, 107) under normal growth conditions. However, during acute heat stress, the expression of hsp70 gene in the brain (1·82 ± 0·25, 109) was the highest and was significantly different from those in the liver (1·08 ± 0·16, 109) and muscle (1·08 ± 0·13, 109). 4. The expression of hsp70 among different genotypes or haplotype combinations is quite different under normal and heat-stress conditions. The haplotype H3 (GC) is probably advantageous to improving heat resistance of chickens. 5. The results from the present study indicate that the expression of hsp70 in chickens is affected by heat stress, and is tissue- and allele-dependent.

The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.

Saccharomyces cerevisiae Rot1p Is an ER-Localized Membrane Protein That May Function with BiP/Kar2p in Protein Folding

The 70-kDa heat shock protein (Hsp70) family of molecular chaperones cooperates with cofactors to promote protein folding, assembly of protein complexes and translocation of proteins across membranes. Although many cofactors of cytosolic Hsp70s have been identified, knowledge about cofactors of BiP/Kar2p, an endoplasmic reticulum (ER)-resident Hsp70, is still poor. Here we propose the Saccharomyces cerevisiae protein Rot1p as a possible cofactor of BiP/Kar2p involved in protein folding. Rot1p was found to be an essential, ER-localized membrane protein facing the lumen. ROT1 genetically interacted with several ER chaperone genes including KAR2, and the rot1-2 mutation triggered the unfolded protein response. Rot1p associated with Kar2p, especially under conditions of ER stress, and maturation of a model protein, a reduced form of carboxypeptidaseY, was impaired in a kar2-1 rot1-2 double mutant. These findings suggest that Rot1p participates in protein folding with Kar2p. Morphological analysis of rot1-2 cells revealed cell wall defects and accumulation of autophagic bodies in the vacuole. This implies that the protein folding machinery in which Rot1p is involved chaperones proteins acting in various physiological processes including cell wall synthesis and lysis of autophagic bodies.

Stabilization of free and immobilized enzymes using hyperthermophilic chaperonin

Chaperonins suppress the denaturation of proteins and promote protein folding in vivo. Because hyperthermophilic chaperonins are expected to be used as a stabilizer for proteins, the effects of a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1 (T. KS-1 cpn), on the stabilization of mesophilic and thermophilic free enzymes and an enzyme co-immobilized with T. KS-1 cpn were studied. T. KS-1 cpn prevented the thermal inactivation of yeast alcohol dehydrogenase (ADH), jack bean urease, and Thermus flavus malate dehydrogenase (MDH) at high temperatures. T. KS-1 cpn also improved the long-term stability of ADH at lower temperatures. Moreover, the residual ADH activity of ADH co-entrapped with T. KS-1 cpn was improved and maintained at a higher level than that of the entrapped ADH without chaperonin. T. KS-1 cpn is useful for the stabilization of free and immobilized enzymes and applicable to various fields of biotechnology.

Hsp90: a chaperone for protein folding and gene regulation

Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins, or they can lead to the degradation of misfolded and destabilized proteins. Hsp90 is a key member of this machinery. It is a ubiquitous molecular chaperone that is found in eubacteria and all branches of eukarya. It plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins, including steroid hormone receptors and protein kinases. Hsp90 is currently a novel anticancer drug target since it is overexpressed in some cancer cells. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort to elucidate the Hsp90 chaperone network, we carried out a large-scale proteomics study to identify physical and genetic interactors of the chaperone. We identified 2 highly conserved novel Hsp90 cofactors, termed Tah1 and Pih1, that bind to the chaperone and that also associate physically and functionally with the essential DNA helicases Rvb1 and Rvb2. These helicases are key components of the chromatin remodeling complexes Ino80 and SWR-C. Tah1 and Pih1 seem to represent a novel class of Hsp90 cofactors that allow the chaperone to indirectly affect gene regulation in the cell in addition to its ability to directly promote protein folding. In this review, we provide an overview of Hsp90 structure and function, and we discuss the literature that links the chaperone activity to gene regulation.

Glycoproteomics: protein modifications for versatile functions

This satellite meeting of the Thirtieth FEBS Congress and the Ninth IUBMB Conference was held in Dubrovnik, Croatia, between 28 and 30 June 2005. The conference was organized by G. Lauc, J. Dumic and M. Flogel from the University of Zagreb, Croatia. ![][1] Glycosylation is perhaps the most common and versatile post‐translational modification of proteins. It is estimated that well over one‐half of all mammalian proteins are glycosylated (Apweiler et al , 1999). The roles of protein glycans, whether N ‐ or O ‐linked forms, are varied. Glycan is required for the biological function of certain proteins, such as the Fc‐effector function of immunoglobulin G (IgG), the regulated clearance of the glycohormone lutropin (LH), the targeting of lysosomal enzymes and controlling the circulation lifetime of glycoprotein drugs (for example, erythropoietin). Glycans also serve as recognition targets for their complementary binding proteins, the lectins. This family includes self proteins that are involved in such diverse functions as fertilization, development, differentiation and lymphocyte circulation, as well as in the development of pathological conditions. Glycans are also used as sites of attachment by microbes and toxins, and often constitute the first contact point between pathogen and host. In addition, glycans serve subtle functions, such as protecting proteins from proteolytic enzymes, promoting protein folding into proper tertiary and quaternary structures, and modulating the biological activities of proteins. Other forms of glycans, such as glycolipids and proteoglycans, also have important structural roles, as well as acting as recognition targets. Glycomics is the investigation and documentation of the large collection of glycan structures. The function of a glycan can vary depending on the protein to which it is attached and the precise site of attachment. It is imperative, therefore, to document the structure and function of the entire glycoprotein. This integrated approach of compiling glycoprotein structure and function … [1]: /embed/graphic-1.gif

Proteome-wide Analysis of Chaperonin-Dependent Protein Folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.

Conditional mutations in the mitotic chromosome binding function of the bovine papillomavirus type 1 E2 protein.

The papillomavirus E2 protein is required for viral transcriptional regulation, DNA replication and genome segregation. We have previously shown that the E2 transactivator protein and BPV1 genomes are associated with mitotic chromosomes; E2 links the genomes to cellular chromosomes to ensure efficient segregation to daughter nuclei. The transactivation domain of the E2 protein is necessary and sufficient for association of the E2 protein with mitotic chromosomes. To determine which residues of this 200-amino-acid domain are important for chromosomal interaction, E2 proteins with amino acid substitutions in each conserved residue of the transactivation domain were tested for their ability to associate with mitotic chromosomes. Chromatin binding was assessed by using immunofluorescence on both spread and directly fixed mitotic chromosomes. E2 proteins defective in the transactivation and replication functions were unable to associate with chromosomes, and those that were competent in these functions were attached to mitotic chromosomes. However, several mutated proteins that were defective for chromosomal interaction could associate with chromosomes after treatment with agents that promote protein folding or when cells were incubated at lower temperatures. These results indicate that precise folding of the E2 transactivation domain is crucial for its interaction with mitotic chromosomes and that this association can be modulated.

Delineation of the lectin site of the molecular chaperone calreticulin

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.

Recombinant protein folding and misfolding in Escherichia coli

The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.

A long and winding road: symposium on membrane trafficking in plants.

The Society for Experimental Biology symposium on Membrane Trafficking in Plants took place in Glasgow between 23 and 26 August 2003 and was organized by M. Blatt and G. Thiel. ![][1] In August 2003, the plant endomembrane community gathered under the auspices of the Society for Experimental Biology to consider the latest developments in the rapidly growing field of the plant secretory pathway. The meeting was preceded by the two‐day annual gathering of the European Plant Secretory Pathway Group, during which postgraduates and postdocs presented their latest data. Plants have homologues of the majority of the vertebrate and yeast genes that encode both the structural and the regulatory proteins of the secretory pathway. However, it is clear that plant cells are different in (1) the organization and operation of the components of the pathway, such as highly motile Golgi stacks, (2) the necessity to sort proteins into different vacuoles and (3) the need to organize a precisely orientated apparatus for cell division (Fig 1). Here we consider the highlights of the meeting and the controversies surrounding the mechanisms by which the plant cell exports material from the site of synthesis to the site of action and imports material from the external environment. Figure 1. A simplified map of the secretory pathway of higher plants. Cargo synthesized and processed in the endoplasmic reticulum (ER) is transferred directly to the closely apposed and mobile Golgi apparatus (G). However, direct transfer of some storage proteins to the vacuole may occur (route 1). Proteins destined for the protein storage vacuole may leave the Golgi at the cis ‐ and trans ‐compartments (route 2), whilst exocytotic cargo predominantly leaves at the trans‐ face (route 3). Product destined for the lytic vacuole is sorted at the trans ‐Golgi and routed in a clathrin‐ and receptor‐dependent fashion through a prevacuolar … [1]: /embed/graphic-1.gif

Overexpression of HDJ-2 protects astrocytes from ischemia-like injury and reduces redistribution of ubiquitin staining in vitro.

HDJ-2, a member of the HSP40 family, functions as a cochaperone to promote protein folding both by binding to unfolded polypeptides and by regulating the activity of HSP70. This study tested whether HDJ-2 overexpression alone could provide significant protection from ischemia-like injury. Primary mouse astrocyte cultures were infected with an HDJ-2 encoding retroviral vector or control retrovirus lacking HDJ-2. Expression of HDJ-2 was confirmed by immunohistochemical staining and immunoblotting. Injury paradigms to mimic ischemia were glucose deprivation (GD) for 24 hours and oxygen-glucose deprivation (OGD) for 8 hours. Cell death was determined by trypan blue exclusion and cell counting. Overexpression of HDJ-2 alone significantly reduced astrocyte injury after both GD and OGD, independent of an elevation in HSP70. To further search for the mechanism of HDJ-2 protection, cultured astrocytes allowed to recover 16 hours after 8 hours GD were labeled with a monoclonal antiubiquitin antibody that recognizes both free ubiquitin and ubiquitinated proteins. The immunolabeling pattern changed from a relatively even distribution in both nuclei and cytoplasm in control cells to heterogeneous aggregates and marked reduction of nuclear staining in most of the cells after GD. When HDJ-2 was overexpressed, the number of cells with evidence of protein aggregation was significantly reduced. Thus, blocking protein aggregation may be an important mechanism by which HDJ-2 protects cells from damage.

Trigger Factor Retards Protein Export in Escherichia coli

Trigger factor (TF) is a ribosome-associated protein that interacts with a wide variety of nascent polypeptides in Escherichia coli. Previous studies have indicated that TF cooperates with DnaK to facilitate protein folding, but the basis of this cooperation is unclear. In this study we monitored protein export in E. coli that lack or overproduce TF to obtain further insights into its function. Whereas inactivation of genes encoding most molecular chaperones (including dnaK) impairs protein export, inactivation of the TF gene accelerated protein export and suppressed the need for targeting factors to maintain the translocation competence of presecretory proteins. Furthermore, overproduction of TF (but not DnaK) markedly retarded protein export. Manipulation of TF levels produced similar effects on the export of a cytosolic enzyme fused to a signal peptide. The data strongly suggest that TF has a unique ability to sequester nascent polypeptides for a relatively prolonged period. Based on our results, we propose that TF and DnaK promote protein folding by distinct (but complementary) mechanisms.

Chaperone-like functions of high-mannose type and complex-type N-glycans and their molecular basis.

It has recently become apparent that high-mannose type N-glycans directly promote protein folding, whereas complex-type ones play a crucial role in the stabilization of protein functional conformations through hydrophobic interactions with the hydrophobic protein surfaces. Here an attempt was made to understand more deeply the molecular basis of these chaperone-like functions with the aid of information obtained from spacefill models of N-glycans. The promotion of protein folding by high-mannose N-glycans seemed to be based on their unique structure, which includes a hydrophobic region similar to the cyclodextrin cavity. The promotive features of high-mannose N-glycans newly observed under various conditions furnished strong support for the view that both intra- and extramolecular high-mannose N-glycans are directly involved in the promotion of protein folding in the endoplasmic reticulum. Further, it was revealed that the N-acetyllactosamine units in complex-type N-glycans have an amphiphilic structure and greatly contribute to the formation of extensive hydrophobic surfaces and, consequently, to the N-glycan-protein hydrophobic interactions. The processing of high-mannose type N-glycans to complex-type ones seems to be an ingenious device to enable the N-glycans to perform these two chaperone-like functions.

Chaperone-Like Functions of N-Glycans in the Formation and Stabilization of Protein Conformation.

This article deals with the chaperone-like functions of N-glycans, laying stress on the fact that the information accumulated mostly by in vitro studies has clarified the general relationship between the functions and structures of N-glycans. High-mannose N-glycans, especially of large size, directly promote protein folding and subunit assembly as their major functions, and they also stabilize the resulting protein conformation in some degree. On the other hand, complex-type N-glycans fulfil the serious requirements for the stabilization of the functional conformation of parent proteins through their hydrophobic interactions with the hydrophobic protein surface unfavorable for protein stability. Newly acquired knowledge about the molecular bases underlying these functions confirms the view that a full understanding of N-glycan-protein interactions is essential for solid progress in medical science and protein engineering. Lastly, and most important, the possible function of high-mannose N-glycans in the direct promotion of the folding of nascent polypeptides in endoplasmic reticulum is discussed in connection with the calnexin and calreticulin functions widely accepted.

A 11.5 Å single particle reconstruction of GroEL using EMAN

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to ∼11.5 Å performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.

The Molecular Chaperone DnaJ Is Required for the Degradation of a Soluble Abnormal Protein in Escherichia coli

In addition to promoting protein folding and translocation, molecular chaperones of Hsp70/DnaJ families are essential for the selective breakdown of many unfolded proteins. It has been proposed that chaperones function in degradation to maintain the substrates in a soluble form. In Escherichia coli, a nonsecreted alkaline phosphatase mutant that lacks its signal sequence (PhoADelta2-22) fails to fold in the cytosol and is rapidly degraded at 37 degrees C. We show that PhoADelta2-22 is degraded by two ATP-dependent proteases, La (Lon) and ClpAP, and breakdown by both is blocked in a dnaJ259-ts mutant at 37 degrees C. Both proteases could be immunoprecipitated with PhoA, but to a much lesser extent in the dnaJ mutant. Therefore, DnaJ appears to promote formation of protease-substrate complexes. DnaJ could be coimmunoprecipitated with PhoA, and the extent of this association directly correlated with its rate of degradation. Although PhoA was not degraded when DnaJ was inactivated, 50% or more of the PhoA remained soluble. PhoA breakdown and solubility did not require ClpB. PhoA degradation was reduced in a thioredoxin-reductase mutant (trxB), which allowed PhoADelta2-22 to fold into an active form in the cytosol. Introduction of the dnaJ mutation into trxB cells further stabilized PhoA, increased enzyme activity, and left PhoA completely soluble. Thus, DnaJ, although not necessary for folding (or preventing PhoA aggregation), is required for PhoA degradation and must play an active role in this process beyond maintaining the substrate in a soluble form.

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Bacillus subtilis

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.