Amino Acid Requirements for the Production of Enterotoxin B by Staphylococcus aureus S-6 in a Chemically Defined Medium

A minimal chemically defined medium has been developed for growth (approximately 25 Klett units) and production of detectable enterotoxin B (approximately 5-6 mug/ml) by Staphylococcus aureus S-6. This medium contains monosodium glutamate as a source of carbon, nitrogen, and energy, three additional amino acids (arginine, cystine, and phenylalanine), six inorganic salts, and four vitamins. Increasing the concentrations of several amino acids in a series of defined media gave no increase in enterotoxin production. Apparently the limiting factor for growth and enterotoxin production in these media is the biosynthesis of one or more missing amino acids, rather than the concentration of the amino acids present in the media. An additional requirement for proline and valine was observed when glucose was added as the primary source of energy. When compared to complex media, our results indicated that the inhibitory effect of glucose on enterotoxin synthesis in defined media was less evident or totally absent.     

Optimum Temperature for Enterotoxin Production by Staphylococcus aureus S-6 and 137 in Liquid Medium

The temperature for maximum toxin yield by Staphylococcus aureus S-6 and 137 in liquid medium cultured under aerobic conditions was determined to be 40 C.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose.
Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose. Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Use of Staphylococcus aureus 6-P-beta-galactosidase and GFP as fusion partners for lactose-specific IIC domain from Staphylococcus aureus

The hydrophilic part of membrane proteins plays an important role in the formation of 3D crystals. The construction of fusion proteins using well crystallizing proteins as fusion partners is a possibility to increase the hydrophilic part of membrane proteins lacking large hydrophilic domains. These fusion proteins might be easier to crystallize. Two bifunctional fusion proteins containing the membrane-bound, lactose-specific enzyme IIC domain of the lactose transporter (IICB(lac)) from S. aureus as N-terminal fusion partner were constructed by gene fusion. The C-terminal fusion partners were S. aureus 6-P-beta-Galactosidase and GFP, respectively. Both proteins were overexpressed in E. coli, purified to homogeneity and kinetically characterized: In the presence of the components of the lactose phosphotransferase system of S. aureus, the hybrid proteins phosphorylated their substrates, indicating that the fusion partners are sufficiently flexibly linked to allow the interaction of the IIC(lac) domain with the IIB(lac) domain of the lactose transporter. The activity of the 6-P-beta-Galactosidase as well as the fluorescence of GFP were preserved in the fusion proteins. The Vmax values determined for the IIC domain in the fusion proteins were dramatically reduced compared with the values determined for the separate IIC(lac) domain and the complete lactose transporter (IICB(lac)). The Km values were only slightly increased indicating that the Vmax values are much more influenced by the fusion than the substrate affinities. The substrate affinity and the Vmax value determined for the GFP-fused IIC(lac) domain are higher than for the 6-P-beta-Galactosidase-fused IIC(lac). The results suggest that the fusion with GFP enables a better interaction with the IIB(lac) domain than the fusion with 6-P-beta-Galactosidase. Moreover, the GFP-fused IIC(lac) domain proved to be more stable than the 6-P-beta-Galactosidase fusion protein.     

Non-specific immunoprecipitation of rotavirus Vp6 protein with Staphylococcus aureus

The specificity of Staphylococcus aureus and protein A-Sepharose (PA-S) were compared in the radioimmunoprecipitation assay for the characterization of monoclonal antibodies (mAbs) against rotavirus proteins. Five mAbs directed against bovine rotavirus Q17 proteins Vp6 and Vp7 and one mAb directed against human rotavirus protein Vp4 were used in this study. mAbs directed against other viruses, NS-1 culture supernatant and ascitic fluid, were used as control reagents. A non-specific immunoprecipitation of the viral protein Vp6 was always found with S. aureus, but not with PA-S. mAb 74 reacted with rotavirus antigens in ELISA and in indirect immunofluorescence assay but did not immunoprecipitate a viral protein with PA-S. This mAb immunoprecipitated the viral protein Vp6 when S. aureus reagent was used. This false positive reaction was always present and could lead to confusing results in the analysis and characterization of mAbs against rotavirus.     

Nutritional Requirements of Microbacterium thermosphactum

Microbacterium thermosphactum requires cysteine, α-lipoate, nicotinate, pantothenate, p-aminobenzoate, biotin, and thiamin for aerobic growth in glucose-mineral salts medium. Glucose cannot be replaced by Casamino Acids or acids of the tricarboxylic acid cycle as sole carbon and energy sources. The organism can also grow anaerobically in the minimal synthetic medium.     

Molecular cloning and mapping of 16S-23S rRNA gene complexes of Staphylococcus aureus.

Staphylococcus aureus BB255, a derivative of NCTC8325, had six rRNA operons, and each operon contained two SmaI sites about 3 kb apart. By molecular cloning and pulsed-field gel electrophoresis, all operons were mapped at the junctions of SmaI fragments in the published map of NCTC8325 except one, which was connected to a previously unidentified 23-kb SmaI fragment.     

Vancomycin-resistant Staphylococcus aureus

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.     

金黄色葡萄球菌肠毒素

金黄色葡萄球菌是一种重要的病原体,它产生多种类型的毒素,从而引起各种类型的疾病。金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins,SEs),是一组血清学上互不相同的热稳定肠毒素,有10个血清型。由于食入了被SEs污染的食品而主要引起肠胃炎,此外,SEs还是一种强的超抗原,它可以刺激非特异性T细胞增殖。SEs各型之间有着相似的结构和功能。     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.     

Further Nutritional Requirements of Paramecium aurelia

SYNOPSIS. An acetone-insoluble yeast fraction required for axenic growth of P. aurelia , stock 51, variety 4, sensitive , after fractionation contained at least 3 essential components: (1) one soluble in perchloric acid and completely replaceable by a mixture of ribosides or ribotides; (2) one inactivated after digestion with trypsin, chymotrypsin or papain. Proteose-Peptone restored activity to this preparation, which suggests a peptide requirement; and (3) one not yet characterized.
As for the purine and pyrimidines, these combinations, in decreasing order of activity, supported growth: guanosine + cytidine, guanosine + uridine, guanylic acid + cytidylic acid, and guanylic acid + uridylic acid. Each combination was maximally effective when the molar purine: pyrimidine ratio was ∼ 0.4. On a molar basis, the minimal riboside combinations were ∼ 1.3 × more active than the ribotides.
Sparing of the purine and pyrimidine requirement was also investigated. In the presence of limiting amounts of guanylic acid, the following compounds, in decreasing order of activity, had sparing activity: deoxyguanosine, inosine, xanthosine, adenylic acid, and guanine. Adenine, adenosine, and deoxyadenosine were inhibitory under the test conditions. The requirement for cytidylic acid was spared by deoxycytidine, uridine, uridylic acid, deoxyuridine, thymidine, thymine, and uracil, in descending order.