Protein disulfide bond formation in Escherichia coli requires the periplasmic protein DsbA. We describe here mutations in the gene for a second protein, DsbB, which is also necessary for disulfide bond formation. Evidence suggests that DsbB may act by reoxidizing DsbA, thereby regenerating its ability to donate its disulde bond to target proteins. We propose that DsbB, an integral membrane protein, may be involved in transducing redox potential across the cytoplasmic membrane.

Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

Disulfide bond formation is catalyzed in the periplasm ofEscherichia coi. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. {Kishigami et al.

DsbB is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of Escherichia coli. Previous studies have led to the hypothesis that DsbB oxidizes the periplasmic protein DsbA, which in turn oxidizes the cysteines in other periplasmic proteins to make disulfide bonds. Gene fusion approaches were used to show that (i) DsbB is a membrane protein which spans the membrane four times and (ii) both the N‐ and C‐termini of the protein are in the cytoplasm. Mutational analysis shows that of the six cysteines in DsbB, four are necessary for proper DsbB function in vivo. Each of the periplasmic domains of the protein has two essential cysteines. The two cysteines in the first periplasmic domain are in a Cys‐X‐Y‐Cys configuration that is characteristic of the active site of other proteins involved in disulfide bond formation, including DsbA and protein disulfide isomerase.

In this review, we describe the outer membrane proteins of Pseudomonas aeruginosa and related strains from the Pseudomonas fluorescens rRNA homology group of the Pseudomonadaceae, with emphasis on the physiological function and biochemical characteristics of these proteins. The use of opr (for outer membrane protein) is proposed as the genetic designation for the P. aeruginosa outer membrane proteins and letters are assigned, in conjunction with this designation, to known outer membrane proteins. Proteins whose primary functions involve pore formation, transport of specific substrates, cell structure determination and membrane stabilization are discussed. The conservation of selected proteins in the above Pseudomonas species is also examined.

Gentanticin, an atninoglycoside antibiotic known to inhibit protein synthesis, had a detrimental effect on the integrity of the Cell wall of Pseudomonas aeruginosa ATCC 9027 (a susceptible strain) as shown by electron microscopy using negative-staining, thin-sectioning, and freeze-fracture techniques. The disruption occurred in a sequential manner, moving from the outer membrane to the inner membrane, and could result in lysis of the cell. During this process the outer membrane lost 34% of its total protein and 30% of its lipopolysaccharide (measured as 2-keto-3-deoxyoctonate) upon exposure to 25 ,ug of gentamicin per ml for 15 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the outer membrane proteins showed altered banding patterns after exposure to gentamicin. Atomic absorption spectrophotometry revealed a decrease in magnesium and calcium content (18 and 38%, respectively) in the cell envelopes after gentamicin treatment. It is proposed that gentamicin displaces essential metal cations within the outer membrane, consequently destabilizing and extracting organic constituents. Small transient holes are thereby produced which make the outer membrane more permeable to the antibiotic and which expose the protoplast to high concentrations of gentamicin. This membrane effect may contribute to the effects of protein synthesis inhibition during the killing process.The accepted mechanism of action of the aminoglycoside antibiotics on sensitive strains of Pseudomonas aeruginosa requires that the antibiotic gain access to the cytoplasm by an energy-requiring process (2). Once inside, it is believed that these drugs work at the ribosomal level by causing either misreading or suppression of protein synthesis (4). There are three mechanisms by which bacteria may circumvent the action of aminoglycoside antibiotics. First, organisms may have altered ribosomes to which the antibiotics cannot bind. Because of the rarity of this occurrence, such organisms are not clinically significant (8). A second mechanism, which is of major clinical importance, is the inactivation of aminoglycosides by plasmid-coded enzymes (4). A third mechanism involves protection against antibiotic penetration by a permeability barrier (26). Studies with hypersensitive mutants have indicated that this resistance is at a structural level in the organism (23).Permeability resistance may be enhanced in normally sensitive strains of P. aeruginosa by the presence of divalent cations. Studies originally undertaken to improve the testing methods for gentamicin resistance in P. aeruginosa have shown that concentrations of calcium and magnesium in the medium greatly influence susceptibility of P. aeruginosa to antibiotics (31); some strains of P. aeruginosa are mnore resistant to aminoglycosides, polymyxins, and colistin in the presence of increased amounts of magnesium or calcium. This phenomenon is a particular property of P. aeruginosa and is not due to modification of the antibiotics by divalent cations (31). The divalent cation-modified res...

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmidencoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.The ability of the bacterial cell to synthesize and secrete specific proteins is dependent upon several aspects of protein secretory pathways. While the passage of proteins through the secretory apparatus in the inner membrane has been relatively well studied (reviewed in references 18, 42, and 60), the events occurring once proteins reach the periplasm of gram-negative cells are less well understood. Even though the periplasm may not be their final destination, proteins begin the process of conformational folding once they reach the periplasm (74). This folding is accompanied by proline isomerization and disulfide bond formation, both necessary to attaining and maintaining native structure (23). These processes are mediated by peptidyl-prolyl isomerases (45) and disulfide oxidoreductases (2, 50, 56) that are present in the periplasm.Many proteinaceous structures, such as fimbriae (39, 78), flagella (14), and several bacterial toxins (46,53,65,77), either contain disulfide bonds or require disulfide bonds in some component of their assembly...

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.