H. Blobel

Plasmid-mediated chloramphenicol resistance in Staphylococcus hyicus.

A small plasmid of 3.95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.

Comparative studies on Actinomyces pyogenes and Arcanobacterium haemolyticum

Actinomyces pyogenes and Arcanobacterium haemolyticum were further characterized. A. haemolyticum, in contrast to A. pyogenes, gave synergistic hemolytic reactions with streptococci of serological group B and inhibited staphylococcal β-hemolysis. A. pyogenes and A. haemolyticum had a number of common, as well as distinctly different enzymatic properties, useful for their characterization. Extracts from A. pyogenes reacted with antiserum against streptococci of serological group G in contrast to those from A. haemolyticum.

Binding of human fibrinogen and its polypeptide chains to group B streptococci

Of 51 group-B streptococcal cultures, 20 bound125I-labelled human fibrinogen. In contrast to the streptococci of groups A, C and G, the group-B streptococci interacted more with theα, β-chain of fibrinogen than with whole fibrinogen. None of the streptococcal cultures reacted withγ-chain. The fibrinogen-negative group-B streptococci still bound theα, β-chain. The binding of125I-labelled,α, β-chain could be inhibited by unlabelled fibrinogen with fibrinogen-positive, but not with fibrinogen-negative streptococci of group B.

Isolation and characterization of hyaluronidase from Streptococcus uberis.

All tested cultures of Streptococcus uberis produced free hyaluronidase. Hyaluronidase could be isolated by ammonium sulfate precipitation and was further purified by chromatography on DEAE-cellulose, gelfiltration on ultragel ACA44 and isoelectric focusing. The purification factor was estimated to be 1689. The purified hyaluronidase had an isoelectric point at pH 4.9 and a molecular weight of approximately 54000 D. It showed maximal enzyme activity at pH 6.0 and 45 degrees C. The Michaelis constant was estimated to be 7.0 X 10(-2) mg/ml. Hyaluronidase activity was stimulated by Ca++, Mg++, Mn++, Co++, Li+, and K+ and inhibited by Zn++ and Cd++ at final concentrations of 10 mmol/l, respectively.

Interactions of plasma proteins with group A, B, C and G streptococci.

Streptococci of serological groups A, B, C and G displayed different binding activities for plasma proteins. Most of the streptococci studied, except those of group B, bound immunoglobulin G. All streptococci reacted with fibrinogen and, except those of group B, with fibronectin. The majority of streptococci, but none of group B, had an affinity for alpha 2-macroglobulin. Albumin was bound by all cultures of group G and a few of group C. Haptoglobulin interacted with only 1 group A culture. None of the streptococci bound transferrin. The specificity of binding sites for 125I-labelled plasma proteins was revealed in a series of inhibition experiments with the unlabelled proteins. The binding sites on streptococci of group G showed different sensitivities to trypsin and pepsin. Reactivities for immunoglobulin G, however, remained unaffected after treatments of the streptococci with trypsin. Exposure to heat (30 min, 80 degrees C) partially inactivated binding activities for the plasma proteins. Sodium dodecyl-sulphate and acetylimidazole strongly reduced binding of albumin and to a lesser extent that of alpha 2-macroglobulin. They had no or little effect on the interaction with the other plasma proteins. Dioxane decreased almost all binding activities. Ethanol partially diminished the binding of immunoglobulin G, fibrinogen, fibronectin and alpha 2-macroglobulin. Treatments of group G streptococci with guanidine, urea, formamide or methanol-HC1 did not affect their plasma protein binding activities.

Absorption of human α2-macroglobulin with selected strains of streptococci

A new interaction was observed between humanα2-macroglobulin (α2M) and selected strains of streptococci. The streptococci boundα2M. After its elution from the bacteria,α2M was demonstrated by immunoelectrophoresis against anti-humanα2M. The binding ofα2M to one streptococcal strain of serological group C and two of group G was confirmed by radial immunodiffusion. Treating the streptococci with trypsin reduced their reactivity withα2M. On the other hand, prolonged treatment with 6M guanidine-HCl had no effect on theα2M binding. Binding sites forα2M were most likely protein structures on the streptococcal surface.

Isolation and restriction endonuclease analysis of a tetracycline resistance plasmid from Staphylococcus hyicus

A plasmid of 4.550 kb, conferring resistance to tetracycline, was demonstrated in Staphylococcus hyicus cultures from piglets with exudative epidermidis. The plasmid-encoded properties were determined both by curing and interspecific protoplast transformation experiments. The tetracycline resistance (TET) plasmid, designated pST1, was characterized by restriction endonuclease analysis and a preliminary restriction map was constructed. The pST1 plasmid was demonstrated in 19 (57.6%) of 33 S. hyicus cultures by Southern blot hybridization. It was also detectable by electron microscopy.

Fibrinogen Binding Inhibits the Fixation of the Third Component of Human Complement on Surface of Groups A, B, C, and G Streptococci

Effects of fibrinogen binding to M protein‐positive and ‐negative streptococci on fixation of the third component of human complement (C3) were determined. In all test cultures of serological groups A, B, C, and G fixation of C3 was observed in normal human serum as revealed by quantitative fluorescent immunoassay. Fibrinogen binding inhibited the fixation of C3 on streptococci. The degree of inhibition was proportional to the extent of fibrinogen binding. Thus, inhibition of C3 fixation was most pronounced in strongly fibrinogen‐positive streptococci of groups A, C, and G and not demonstrable in fibrinogen‐negative cultures of groups C and G. Trypsinization of the streptococci destroyed their capacity to bind fibrinogen and consequently the inhibitory effects on C3 fixation. The carboxymethylated α and β chains of fibrinogen moderately inhibited C3 fixation whereas γ chain had no influence. These studies may indicate that fibrinogen binding structures other than M protein could also be involved in streptococcal pathogenicity.

Isolation and characterization of hyaluronidases from Streptococcus dysgalactiae, S. zooepidemicus and S. equi.

10 out of 10 cultures each of Streptococcus dysgalactiae and S. zooepidemicus and 6 out of 10 cultures of S. equi tested for hyaluronidase produced this enzyme. Hyaluronidase could be precipitated from the cell-free culture supernatant with ammonium sulphate and purified by chromatography on DEAE-cellulose, isoelectric focussing and preparative polyacrylamide gel electrophoresis. The isoelectric points of the hyaluronidases from S. dysgalactiae and S. equi were near pH 5, of that from S. zooepidemicus near pH 6. The hyaluronidases from S. dysgalactiae, S. zooepidemicus and S. equi had molecular weights of about 55,000 D. Maximal enzyme activities developed between 40 degrees C and 45 degrees C and pH 5.6 and 5.8. The Michaelis constants ranged from 7.5 x 10(-2) to 8.8 x 10(-2) mg/ml. Hyaluronidase activities were stimulated by Ca++, Mg++, Mn++, Co++, K+, and Li+ and inhibited by Zn++ and Cd++.

Guanidine extraction enhances the binding of human fibrinogen to group-B streptococci

Treatment of group-B streptococci with guanidine chloride significantly enhanced their capacity to bind125I-labelled fibrinogen. The increase in binding activity was almost proportional in the range of 0.05 M to 6 M guanidine chloride. Repeated extractions with guanidine chloride further increased the capacity of B streptococci to bind the labelled fibrinogen. On the other hand, urea, even at concentrations up to 8 M, did not alter the fibrinogen binding. The enhancement was most pronounced with B streptococci of serotypes III and III-R. Cultivation of these streptococci in Baker and Kasper medium, which is known to stimulate formation of microcapsules, reduced125I-fibrinogen binding. Subsequent treatment with guanidine increased their binding activity, presumably by facilitating the accessibility of the binding sites.