Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.     

Heme binding properties of Staphylococcus aureus IsdE

Staphylococcus aureus is the source of a large number of hospital-acquired infections, of which many are serious and can lead to death. Iron is critically important to the survival and growth of the bacterium, and complex, multistep mechanisms are present to fulfill the necessary iron requirement. Isd proteins located on the wall and membrane of S. aureus have been proposed to function in heme acquisition. We report characterization of the S. aureus heme-binding protein IsdE, the lipoprotein component of a membrane-localized ABC transporter that is believed key to receiving heme from cell wall-anchored Isd proteins. Magnetic circular dichroism (MCD) data, which greatly extend the results from our initial study of IsdE in bacterial cell lysates (Mack, J., Vermeiren, C., Heinrichs, D. E., and Stillman, M. J. (2004) Biochem. Biophys. Res. Commun. 320, 781-788), probe the ligand and redox properties of the bound heme. The MCD data show that IsdE, when overexpressed in E. coli, binds either ferric or ferrous heme but that the largest fraction is low spin ferrous heme. Studies of mutants allowed identification and characterization of the ligands in the fifth and sixth position on the heme iron as histidine, proximally, and methionine, distally. This histidine-methionine heme-iron ligation is unique to heme transport proteins. The smaller fraction of ferric heme in the protein is not bound by methionine, allowing for access by strong field ligands, such as cyanide. Electrospray ionization mass spectral data are reported for the first time and show that only one heme ligand binds per IsdE protein molecule. These data also show there is little change in the conformation of the protein between the heme-bound and heme-free species, indicating that the heme-free IsdE adopts a structure essentially independent of the heme. The mass spectral data clearly show that IsdE reversibly unwinds under denaturing conditions to form at least two distinct, heme-free conformations.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.     

Effect of milk on surface properties of Staphylococcus aureus from bovine mastitis

Abstract The surface hydrophobicity of cells of Staphylococcus aureus strains isolated from bovine mastitis grown on conventional agar and broth media was drastically reduced after incubation with bovine milk. Strains grown in high carbohydrate-high salt media yielded cells with reduced surface hydrophobicity compared to cells grown in conventional media, and adding bovine milk to minimal medium also yielded cells with reduced surface hydrophobicity, as determined by hydrophobic interaction chromatography and the salt aggregation test. Incubation of strains in milk and growth in a medium supplemented with bovine milk also significantly changed bacterial surface charge as determined by free-zone electrophoresis. Strains with high or with decreased adsorptive and aggregating properties did not produce surface capsule or slime. Heat treatment (60° C or 80° C) of the bacterial suspensions did not significantly change their adsorptive and aggregating properties.     

Spectral properties of Achromobacter xylosoxidans cytochromes c' and their NO complexes.

Cytochromes c' have been isolated from six strains of Achromobacter xylosoxidans: NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, 1048, 1051, 1055 and 1764. They are dimeric proteins with more positive redox potentials than those of cytochromes c' from phototrophic bacteria at neutral pH. The electronic absorption, EPR and MCD spectra on NO-ferrous cytochromes c' at physiological pH showed that the major part of the heme-iron of nitrosylheme was penta-coordinated. The EPR spectral results indicated that the ground state of the heme-iron of ferric cytochromes c' appears to be in an admixed spin states which consists of predominant high-spin with a slight intermediate-spin character at pH 7.2. These spectra were compared with those for cytochromes c' from phototrophic bacteria and the other hemoproteins.     

Spectral analysis of cytochromes inAquaspirillum magnetotacticum

The respiratory chain ofAquaspirillum magnetotacticum strain MS-1 cells denitrifying microaerobically included a-, a₁-, b-, c-, cd₁-, and o-type hemes. More than 85% of the total cytochromes detected were of the c type. Virtually all of the a and b types were detected in cell membranes, whereas 70% of the c-type hemes were soluble. Large quantities of soluble c-type hemes were released with periplasm by freezing and thawing cells. Soluble c₅₅₁ occurred in two forms: as a single compound of apparent molecular weight of 17,000 daltons, which bound CO, and, together with d₁ heme, as a component of nitrite reductase. Both a₁-type hemes (which usually comprise part of the low aeration cytochrome oxidase) and o types (usually part of the high aeration oxidase) were simultaneously expressed in microaerobically grown denitrifying cells ofA. magnetotacticum; this indicated branching of the respiratory chain.