Biomedical subjects

S Lory

Publications and source records attributed to S Lory.

81 records · Page 5Linked to original sources

PubMed1985

Orientation and expression of the cloned hemolysin gene of Pseudomonas aeruginosa.

The structural gene for Pseudomonas aeruginosa hemolysin, carried on recombinant plasmid pSL2 and cloned in Escherichia coli, was analyzed by insertional and deletional mutagenesis. Expression of the hemolysin was blocked by insertion of transposon Tn5 into different locations. Two of the mutants allowed detectable synthesis of truncated hemolysin polypeptides of two different sizes and thus defined the structural gene. The location of the hemolysin gene in the recombinant plasmid, and the direction of transcription, were further established by nuclease BAL 31 digestion, and by construction of gene fusions between hemolysin and beta-galactosidase. Evidently, the tet promoter contributed to the majority of the expression of cloned hemolysin gene, but the Pseudomonas promoter was present in the cloned DNA and was functional in E. coli since inactivation of the tet promoter either by Tn5 insertion or by deletion decreased synthesis of the 80-kDal hemolysin but did not fully abolish it.

Chromosome Mapping↗

PubMed1983

Characterization of the phospholipase C gene of Pseudomonas aeruginosa cloned in Escherichia coli.

We have cloned a 4.9-kb fragment of Pseudomonas aeruginosa DNA containing the structural gene of phospholipase C (PLC), by inserting it into the BamHI site of plasmid pBR322. Strains of Escherichia coli carrying this recombinant plasmid produce PLC, but expression of the gene differs from that in P. aeruginosa in two respects: (i) synthesis of the enzyme appears to be constitutive, i.e., not repressible by the presence of inorganic phosphate in the growth medium, and (ii) most of the enzyme remains associated with the outer membrane instead of being secreted. Insertion mutagenesis at a unique restriction site within the PLC gene destroyed the ability of the plasmid to code, in maxicells, for phospholipase C activity and for an Mr 80000 polypeptide.

Cloning, Molecular↗

PubMed1983

Mechanism of protein excretion by gram-negative bacteria: Pseudomonas aeruginosa exotoxin A.

Excretion of proteins by a cell with a double membrane may involve mechanisms different from secretion across a single membrane. We studied this problem with Pseudomonas aeruginosa exotoxin A. This 68,000-dalton protein was released as rapidly as it was completed; even after short pulse-labeling the cells contained neither the toxin nor a larger precursor. Excretion is evidently cotranslational, since in fractionated lysates the toxin was formed (almost entirely in the mature form) by the membrane-polysome complexes but not by the free polysomes. When the membrane was perturbed by 10% ethanol, the cells stopped excreting the toxin and they accumulated an immunoprecipitable, enzymatically active precursor of 71,000 daltons. The precursor was located entirely in the outer membrane on its outer surface. On removal of the ethanol, the cells again excreted mature toxin, but they did not process or release the previously accumulated precursor. Based on these data, a model for the excretion of exotoxin A is presented.

ADP Ribose Transferases↗

PubMed1980

Ligand interactions of diphtheria toxin. I. Binding and hydrolysis of NAD.

Prior studies showed that diphtheria toxin could be separated into ATP-binding and nonbinding fractions (Fractions II and I, respectively) by affinity chromatography on ATP-Sepharose (Lory, S., and Collier, R. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 267-271). Here we show that the two fractions also differ in their interactions with NAD. Fraction II bound a single molecule of NAD (Kd about 9 microM) as assayed by flow dialysis or fluorescence quenching and catalyzed both NAD-glycohydrolase and auto-ADP-ribosylation reactions. Fraction I was deficient in NAD-binding and NAD-related reactions. The ratio of the two fractions vried widely among toxin preparations and was independent of the proportion of toxin in the nicked state. Properties of th NAD site on Fraction II were similar to, but not identical with, those of the corresponding site on free Fragment A.

Binding Sites↗

PubMed1980

Ligand interactions of diphtheria toxin. II. Relationships between the NAD site and the P site.

Prior studies have described two functionally distinct ligand-binding sites on whole diphtheria toxin, the NAD site, which catalyzes the intracellular ADP-ribosylation reaction, and the P site, which affects toxin binding to sensitive cells. Occupancy of the P site by ATP or other phosphorylated compounds inhibits toxin attachment to cells. Here we show that binding of NAD site and P site ligands is competitive; and we characterize ligand-binding properties of two mutant forms of the toxin, CRM 45 and CRM 197. The data suggest that the NAD site, on the A moiety, lies immediately adjacent to the P site, formed by a strongly cationic region on the COOH-terminal half of B. The cationic character of the P site slightly alters the substrate specificity of the NAD site, and occupancy of either of the sites blocks ligand binding to the other. Possible roles of the P site in toxin attachment are discussed.

Adenosine Diphosphate Ribose↗

PubMed1980

Ligand interactions of diphtheria toxin. III. Direct photochemical cross-linking of ATP and NAD to toxin.

The locations of ATP- and NAD-binding sites on diphtheria toxin were investigated by ultraviolet irradiation of ligand . toxin complexes. Illumination of ATP with ultraviolet light (253.7 nm) in the presence of various proteins resulted in photoinduced cross-linking only with Fraction II of diphtheria toxin. Under the same conditions, NAD was cross-linked most effectively to Fragment A, followed by Fraction II and CRM 45. For both ATP and NAD, the degree of protein labelling correlated well with binding data, suggesting that photoinduced cross-linking ocurred only at the high affinity binding sites for these ligands. Nonspecific labeling of unrelated proteins was consistently less than 5% of that observed for Fraction II. Analysis of nicked and reduced Fraction II . ligand complexes on SDS polyacrylamide gels demonstrated that essentially all of the cross-linked label migrated with the A fragment, whether photolysis was performed with ATP or NAD.

Adenosine Triphosphate↗

PubMed1980

Diphtheria toxin: nucleotide binding and toxin heterogeneity.

We have used flow dialysis to demonstrate binding of ATP and related compounds to diphtheria toxin. The results define a new site on the toxin molecule (the P site), which has distinctly different properties from the NAD+-binding site of the fragment A moiety. The relative affinities of various compounds for the P site are similar to their capacities to inhibit toxin attachment to cell surfaces and its action on cells. This suggests that the P site may correspond to the binding site for cell surface receptors. Affinity of nucleotides for the toxin depends strongly on the number of phosphates, although both nucleoside and phosphate moieties contribute to the interaction. A substantial fraction of the toxin in any given preparation did not bind ATP in a rapidly reversible manner and was not retained on ATP-Sepharose. This fraction, which varied in magnitude from preparation to preparation, was isolated and shown to contain an endogenous, firmly bound nucleotide or nucleotide-like compound. The presence of this compound may explain some of the physical heterogeneity within individual preparations of purified toxin as well as variations in physical and biological properties among various preparations.

Adenine Nucleotides↗

PubMed1980

Expression of enzymic activity by exotoxin A from Pseudomonas aeruginosa.

Exotoxin A from Pseudomonas aeruginosa is a single polypeptide chain (M(r), 66,000) containing little if any adenosine 5'-diphosphate ribosyltransferase or oxidized nicotinamide adenine dinucleotide glycohydrolase activity. These activities have been demonstrated in the reduced intact toxin and in a peptide (M(r), 26,000) isolated from culture fluids or toxin preparations after storage. In this report we describe methods for generating enzymically active fragments by cleaving the fully or partially reduced exotoxin by proteolytic or chemical methods. Incubation of reduced toxin with chymotrypsin in the presence of oxidized nicotinamide adenine dinucleotide yielded an enzymically active peptide (M(r), 26,000) similar to the fragment characterized previously. Chemical cleavage by treatment of the reduced molecule with CNBr or 2-nitro-5-thiocyanobenzoate yielded fragments (M(r), 50,000 and 30,000, respectively) with similar activities. Also both adenosine 5'-diphosphate ribosyltransferase and oxidized nicotinamide adenine dinucleotide glycohydrolase activities were maximally expressed by the intact exotoxin after reduction of only two of its four disulfide bridges. Kinetic constants for activated whole toxin were similar to those of fragment A of diphtheria toxin. It is evident that in the native toxin the catalytic center is buried or distorted and that alterations in the covalent structure permit the center to become exposed or assume an active configuration. It is unknown whether reduction, proteolytic processing, or both occur during the course of toxin action on whole cells.

Carbon Radioisotopes↗

PubMed1979

Structure-activity relationships in diphtheria toxin and exotoxin A from Pseudomonas aeruginosa.

Diphtheria toxin and exotoxin A from Pseudomonas aeruginosa (Pseudomonas toxin) block protein synthesis in sensitive animal cells by virtually identical mechanisms. Both toxins are proenzymes that, after activation, catalyze attachment of the adenosine diphosphate ribose (ADP-ribose) moiety of NAD to elongation factor 2 (EF-2) by covalent linkage. EF-2 is thereby inactivated. In the case of diphtheria toxin (60,000 daltons) the ADP-ribosylation of EF-2 is catalyzed by a 21,000-dalton peptide (fragment A) released after mild tryptic digestion and reduction of the toxin. The complementary B moiety of the toxin (39,000 daltons) is required for toxic activity and functions by attaching the toxin to oligosaccharide-containing cell surface receptors. In the case of the Pseudomonas toxin, the ADP-ribosylation reaction may be catalyzed either by the intact 66,000-dalton chain after reduction, or by a 26,000-dalton peptide released after mild proteolysis. Current approaches to study of the mechanisms of entry of the two toxins in active form into animal cells are reviewed.

Adenosine Diphosphate Ribose↗