Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168

Thiol-disulfide oxidoreductases are required for disulfide bond formation in proteins that are exported from the cytoplasm. Four enzymes of this type, termed BdbA, BdbB, BdbC, and BdbD, have been identified in the Gram-positive eubacterium Bacillus subtilis. BdbC and BdbD have been shown to be critical for the folding of a protein required for DNA uptake during natural competence. In contrast, no function has been assigned so far to the BdbA and BdbB proteins. The bdbA and bdbB genes are located in one operon that also contains the genes specifying the lantibiotic sublancin 168 and the ATP-binding cassette transporter SunT. Interestingly sublancin 168 contains two disulfide bonds. The present studies demonstrate that SunT and BdbB, but not BdbA, are required for the production of active sublancin 168. In addition, the BdbB paralogue BdbC is at least partly able to replace BdbB in sublancin 168 production. These observations show the unprecedented involvement of thiol-disulfide oxidoreductases in the synthesis of a peptide antibiotic. Notably BdbB cannot complement BdbC in competence development, showing that these two closely related thiol-disulfide oxidoreductases have different, but partly overlapping, substrate specificities.     

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Streptococcus gordonii

Disulfide bonds are important for the stability of many extracellular proteins, including bacterial virulence factors. Formation of these bonds is catalyzed by thiol-disulfide oxidoreductases (TDORs). Little is known about their formation in Gram-positive bacteria, particularly among facultative anaerobic Firmicutes, such as streptococci. To investigate disulfide bond formation in Streptococcus gordonii, we identified five putative TDORs from the sequenced genome. Each of the putative TDOR genes was insertionally inactivated with an erythromycin resistance cassette, and the mutants were analyzed for autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This analysis revealed a single TDOR, SdbA, which exhibited a pleiotropic mutant phenotype. Using an in silico analysis approach, we identified the major autolysin AtlS as a natural substrate of SdbA and showed that SdbA is critical to the formation of a disulfide bond that is required for autolytic activity. Analysis by BLAST search revealed homologs to SdbA in other Gram-positive species. This study provides the first in vivo evidence of an oxidoreductase, SdbA, that affects multiple phenotypes in a Gram-positive bacterium. SdbA shows low sequence homology to previously identified oxidoreductases, suggesting that it may belong to a different class of enzymes. Our results demonstrate that SdbA is required for disulfide bond formation in S. gordonii and indicate that this enzyme may represent a novel type of oxidoreductase in Gram-positive bacteria.     

Crystal Structure and Biophysical Properties of Bacillus subtilis BdbD: AN OXIDIZING THIOL:DISULFIDE OXIDOREDUCTASE CONTAINING A NOVEL METAL SITE*

BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca²⁺, at an inter-domain position some 14 Å away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as −75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK_a, consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca²⁺-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca²⁺-depleted BdbD was significantly less stable than reduced Ca²⁺-containing protein, and the midpoint reduction potential was shifted by approximately −20 mV, suggesting that Ca²⁺ functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.Disulfide bonds, formed upon oxidation of two cysteine residue side chain thiols, are key for the stability and/or function of many secreted and membrane-bound peptides and proteins in bacteria, and the failure to insert these correctly has wide ranging effects (1–4). To regulate the redox state of cysteine residues on the outside of the cytoplasmic membrane, intricate disulfide bond regulatory systems have evolved. These involve enzymes of the thiol:disulfide oxidoreductase (TDOR)³ family, which contain cysteine residues often arranged in a Cys-Xaa-Xaa-Cys thioredoxin motif (5, 6). These enzymes function in pathways that lead to the formation of disulfide bonds, rearrangement of incorrectly positioned disulfide bonds, or the removal of unwanted disulfide bonds, and the redox properties of the enzymes appear to correlate closely with function.The paradigm system for disulfide bond formation is the DsbA-DsbB system of Escherichia coli, which has been characterized in great detail (7–11). DsbA is a soluble periplasmic TDOR, which has a thioredoxin-like fold with an additional helical domain (8). The protein oxidizes the di-thiol motifs of a range of substrates, generating in each a disulfide bond. Reduced DsbA is rapidly re-oxidized by DsbB, a membrane-bound TDOR (12) that channels the resulting electrons into the membrane quinol pool (13, 14). Dsb-like homologues appear to be extremely widespread in Gram-negative bacteria.Gram-positive bacteria, unlike Gram-negatives, do not have an outer membrane and so have no spatially defined periplasmic compartment. This places different functional and structural demands on extra-cytoplasmic proteins, and one consequence of this is that extra-cytoplasmic TDORs are generally membrane-anchored in Gram-positive bacteria. Systems for the introduction of disulfide bonds appear to be variable in these organisms (15, 16). As an example, Mycobacterium tuberculosis does not contain close homologues of DsbA/B but contains another thioredoxin-like TDOR, DsbE, which has been shown to have redox properties similar to those of DsbA (17). Some Gram-positive bacteria, including Staphylococcus aureus, contain a DsbA homologue but no homologue of DsbB (18). The recent structural and biochemical characterization of S. aureus DsbA revealed major similarities with the E. coli protein but was also consistent with a distinct mechanism of re-oxidation (16).Some Gram-positive bacteria, however, contain clear homologues of both DsbA and DsbB. In the model organism Bacillus subtilis, BdbD and BdbC/BdbB have been identified as homologues of DsbA and DsbB, respectively, and demonstrated to be involved in processes such as natural competence development, which requires the insertion of disulfide bonds (19). BdbD and BdbC are also involved in a number of other pathways that do not require the insertion of a disulfide bond; B. subtilis contains several extra-cytoplasmic TDORs, for example ResA and StoA (required for cytochrome c maturation and endospore biogenesis, respectively), which function to specifically reduce disulfide bonds introduced by BdbD (20, 21). To understand disulfide bond management systems in Gram-positive bacteria, detailed information on each of the different systems found is required.Here we present the crystal structure of the catalytic domain of B. subtilis BdbD in both reduced and oxidized states. The structure is broadly similar to that of E. coli DsbA, but it also reveals the presence of a novel Ca²⁺-binding site remote from the CXXC active site. Using two-dimensional NMR methods and fluorescence kinetic studies of thiolate alkylation, we report the reduction potential and pK_a properties of the soluble protein, which are entirely consistent with an oxidizing function for the protein in vivo. Conformational stability studies and NMR studies showed that the occupancy of the metal site by Ca²⁺ ion is not required for folding/stability but leads to a significant increase of the midpoint reduction potential. The possibility that the principal function of the metal site is to boost the oxidizing power of the protein is discussed.     

Mutations in the thiol-disulfide oxidoreductases BdbC and BdbD can suppress cytochrome c deficiency of CcdA-defective Bacillus subtilis cells

Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.     

Applications of thiol-disulfide oxidoreductases for optimized in vivo production of functionally active proteins in <Emphasis Type="Italic">Bacillus</Emphasis>

Bacillus subtilis is a well-established cellular factory for proteins and fine chemicals. In particular, the direct secretion of proteinaceous products into the growth medium greatly facilitates their downstream processing, which is an important advantage of B. subtilis over other biotechnological production hosts, such as Escherichia coli. The application spectrum of B. subtilis is, however, often confined to proteins from Bacillus or closely related species. One of the major reasons for this (current) limitation is the inefficient formation of disulfide bonds, which are found in many, especially eukaryotic, proteins. Future exploitation of B. subtilis to fulfill the ever-growing demand for pharmaceutical and other high-value proteins will therefore depend on overcoming this particular hurdle. Recently, promising advances in this area have been achieved, which focus attention on the need to modulate the cellular levels and activity of thiol-disulfide oxidoreductases (TDORs). These TDORs are enzymes that control the cleavage or formation of disulfide bonds. This review will discuss readily applicable approaches for TDOR modulation and aims to provide leads for further improvement of the Bacillus cell factory for production of disulfide bond-containing proteins.     

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.     

Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.     

Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase

Although quinone production and melanin formation are widely recognized as an integral part of the insect defense system, experimental evidence is lacking that the proteolytic activation of prophenoloxidase participates in the direct killing of invading microbes-active phenoloxidase generates quinones that polymerize to form melanin. Here, we report the antimicrobial effect of reactive intermediates produced in phenoloxidase-catalyzed reactions. After being treated with Manduca sexta phenoloxidase and dopamine, Escherichia coli and Bacillus subtilis ceased to grow, whereas the growth of Pichia pastoris was slightly affected. Microscopic analysis showed melanin deposition on cell surface, aggregation of bacteria, and loss of cell mobility. Viability tests revealed major decreases in the bacterial colony counts and, since the decrease remained significant after dispersion of the cell clumps, the reactive compounds were surmised to have aggregated and killed E. coli and B. subtilis cells. Under the experimental conditions, 60-94% of the Gram-negative bacteria (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium) and 52-99% of the Gram-positive bacteria (Bacillus cereus, B. subtilis, Micrococcus luteus, and Staphylococcus aureus) were killed. In the presence of phenoloxidase, dopamine or 5,6-dihydroxyindole (DHI) exhibited much higher antibacterial activity than L-dopa, N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) did, suggesting that DHI and its oxidation products were cytotoxic. The antifungal activity of DHI was detected using P. pastoris, Saccharomyces cerevisiae, Candida albicans, and Beauveria bassiana. These results established that prophenoloxidase activation is an integral component of the insect defense system involving a multitude of enzymes (e.g. proteinases, oxidases, and dopachrome conversion enzyme (DCE)), which immobilizes and kills invading microorganisms.     

The phosphotransferase system (PTS) of Streptomyces coelicolor identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH.

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.     

Cadmium uptake by growing cells of gram-positive and gram-negative bacteria

The present study evaluates the effect of the cadmium (Cd2+) on the growth and protein synthesis of some Gram-positive (Staphylococcus aureus, Bacillus subtilis and Streptococcus faecium) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and the cadmium uptake by the same micro-organisms. The Gram-negative bacteria tested were less sensitive to metal ions than the Gram-positive, and P. aeruginosa was the most resistant. The Gram-negative bacteria were also able to accumulate higher amounts of cadmium during growth than the Gram-positive bacteria. The maximum values of specific metal uptake (microgram of Cd2+ incorporated per mg of protein) were: 0.52 for S. aureus, 0.65 for S. faecium, 0.79 for B. subtilis, 2.79 for E. coli and 24.15 for P. aeruginosa, respectively. The differences in the ability to accumulate metal found between Gram-negative and Gram-positive bacteria seems to account for different mechanisms of metal resistance.     

Salt sensitivity of minimal twin arginine translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.     

Inactivation of S. epidermidis, B. subtilis, and E. coli bacteria bioaerosols deposited on a filter utilizing airborne silver nanoparticles

In the present study, a control methodology utilizing airborne silver nanoparticles is suggested and tested with respect to its potential to control Gram-positive Staphylococcus epidermidis and Bacillus subtilis, and Gram-negative Escherichia coli bacteria bioaerosols deposited on filters. As it is known that the Gram-negative bacteria are sensitive to airflow exposure, the main focus of this study for testing the airborne silver nanoparticles effect was the Gram-positive Staphylococcus epidermidis and Bacillus subtilis bacteria bioaerosols whereas Escherichia coli bioaerosols were utilized for comparison. Airborne bacteria and airborne silver nanoparticles were quantitatively generated in an experimental system. Bioaerosols deposited on the filter were exposed to airborne silver nanoparticles. The physical and biological properties of the airborne bacteria and airborne silver nanoparticles were measured via aerosol measurement devices. From the experimental results, it was demonstrated that this method utilizing airborne silver nanoparticles offers potential as a bioaerosol control methodology.     

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

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.     

Extracytoplasmic processes impaired by inactivation of trxA (thioredoxin gene) in Bacillus subtilis

The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.