Optimal conditions for culturing Staphylococcus aureus strain A-676 to obtain biologically active protein A

Peptone-yeast and casein-salt culture media have been shown to be equally effective when used for the cultivation of S. aureus strain A-676 with a view to obtaining protein A. The seed dose has been determined and the optimum conditions for growing the producer strain have been established.     

Dialysis Culture for the Production of Extracellular Protein A from Staphylococcus aureus A676

Different nutrient media have been tested for the production of extracellular protein A from Staphylococcus aureus A676. High yields were obtained in trypticase soy broth with glucose as the main energy source. In aerated fermenter cultures the amount of protein A obtained was 0·2–0·3 gl⁻¹, as compared to reported yields of 0·03 gl⁻¹. Yields were further increased in dialysis cultures, where protein A concentrations of 2 gl⁻¹. were achieved.     

A note on the isolation of Staphylococcus aureus from raw minced meat

Conventional Baird-Parker medium (BP) and three modified BP-type media were tested for their suitability to detect and to enumerate Staphylococcus aureus in raw minced meat. BP with pig plasma gave the most rapid results without the need for extensive confirmatory tests. A method which combined a liquid modification of BP medium with BP agar supplemented with acriflavine, polymyxin and sulphamezathine, was cheaper, more sensitive, and did not necessitate additional identification. Conventional BP incubated at 37 degrees and 43 degrees C yielded less satisfactory results.     

A note on the isolation of Staphylococcus aureus from raw minced meat

Conventional Baird-Parker medium (BP) and three modified BP-type media were tested for their suitability to detect and to enumerate Staphylococcus aureus in raw minced meat. BP with pig plasma gave the most rapid results without the need for extensive confirmatory tests. A method which combined a liquid modification of BP medium with BP agar supplemented with acriflavine, polymyxin and sulphamezathine, was cheaper, more sensitive, and did not necessitate additional identification. Conventional BP incubated at 37 and 43C yielded less satisfactory results.     

Extracellular protein A from a methicillin-resistant strain of Staphylococcus aureus

Protein A has been isolated from a methicillin-resistant strain of Staphylococcus aureus, A676, which produces only extracellular protein A. The material is homogeneous after a one-step separation and has a molecular weight of 41000. Its physicochemical and immunochemical properties have been studied.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

The SarA protein family of Staphylococcus aureus

Staphylococcus aureus is widely appreciated as an opportunistic pathogen, primarily in hospital-related infections. However, recent reports indicate that S. aureus infections can now occur in other wise healthy individuals in the community setting. The success of this organism can be attributed to the large array of regulatory proteins, including the SarA protein family, used to respond to changing microenvironments. Sequence alignment and structural data reveal that the SarA protein family can be divided into three subfamilies: (1) single domain proteins; (2) double domain proteins; (3) MarR homologs. Structural studies have also demonstrated that SarA, SarR, SarS, MgrA and thus possibly all members of this protein family are winged helix proteins with minor variations. Mutagenesis studies of SarA disclose that the winged helix motifs are important for DNA binding and function. Recent progress concerning the functions and plausible mechanisms of regulation of SarA and its homologs are discussed.     

Isolation of the fifth IgG-binding domain from Staphylococcus aureus protein A

Using affinity chromatography on IgG-Sepharose at pH 5.0, a new fragment capable of binding to IgG (domain E) was isolated from trypsin hydrolysate of protein A. Trypsinolysis of protein A was performed at low temperatures. Thus, the intact structure of protein A was found to include six domains, of which five interact with IgG.     

Distribution of protein A on the surface of Staphylococcus aureus

Surface proteins of Staphylococcus aureus fulfill many important roles during the pathogenesis of human infections and are anchored to the cell wall envelope by sortases. Although the chemical linkage of proteins to cell wall cross bridges is known, the mechanisms whereby polypeptides are distributed on the staphylococcal surface have not been revealed. We show here that protein A, the ligand of immunoglobulin, is unevenly distributed over the staphylococcal surface. Upon removal with trypsin, newly synthesized polypeptide is deposited at two to four discrete foci. During subsequent growth, protein A appears to be slowly distributed from these sites. When viewed through multiple focal planes by laser scanning microscopy, protein A foci are arranged in a circle surrounding the bacterial cell. This pattern of distribution requires the LPXTG sorting signal of protein A as well as sortase A, the transpeptidase that anchors polypeptides to cell wall cross bridges. A model is presented whereby protein A deposition at discrete sites coupled with cell wall synthesis enables distribution of protein A on the staphylococcal surface.     

Immunologically active and structurally similar fragments of protein A from Staphylococcus aureus.

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc-part of human IgG were isolated by affinity chromatography on IgG-Sepharose 4B and purified by ion-exchange chromatography on phosphocellulose. From a partial tryptic digest of pure protein A at 30 degrees C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein-A-containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A. All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc-reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.     

The survival of Staphylococcus aureus in raw milk with different cell count

The antibacterial activity of the individual udder quarter milk sample cannot be evaluated by the number of somatic cells. Milk with the highest number of cells is not always the most bactericidal. However, when the data were obtained from a material large enough for statistical calculations, negative and highly significant correlation coefficient (-0.465, P less than or equal to 0.01) between number of somatic cells in milk and number of bacteria surviving in it, and negative and significant correlation coefficient (-0.203, P 0.05) between number of somatic cells in milk and number of bacteria surviving in whey, were found. Results of the analysis of variance demonstrated highly significant differences as regards number of cells and bacteria surviving in milk and in whey between cows, quarters and stimulation time. Nevertheless, among ten investigated cows, three cows with high cell count and high antibacterial activity in milk and whey, highly reacting to stimulant, were found.