Ferric-reductase activities in whole cells and cell fractions of Vibrio (Listonella) anguillarum

Abstract

The ability of Wibrio (Listonella) anguillamm strains from serotype groups 01 and 02 to reduce Fe3+ in the form of different chelates was investigated. All strains, grown in M9 minimal medium supplemented with 0.2?40 Casamino acids, reduced Fe3+ complexed by citrate, nitrilotriacetic acid and EDTA. In whole cells, the degree of reduction was dependent on the Fe3+ ligand and on the strain, with the greatest values corresponding to ferric dicitrate and serotype group 01 strains, respectively. The ferric-reductase activity increased, over the basal levels, when the cells were grown with iron added as ferric dicitrate, haemin or haemoglobin. All strains also reduced ferricyanide, a compound that is not transported into the bacterial cells. Ferricyanide reduction was also increased when the cells were grown in the presence of an iron source. All of the cell fractions (periplasm, membranes and cytoplasm) showed Fe3+-reducing activity, with the highest values observed in the presence of Mgz+, NADH and FAD in the assay buffer. Cytoplasmic ferricreductase could be visualized using native polyacrylamide or starch gel electrophoresis, whereas the periplasmic and membrane reductase(s) could only be detected on starch gels. The results indicate the presence of different ferric-reductase activities in V. anguillarum, which could be involved in the different iron-acquisition systems present in this micro-organism, i.e. siderophore-mediated systems and siderophore-independent mechanisms.

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high‐affinity ferrous iron transporter

“…Secreted Fe 31 reductants have been described for several bacteria (Adams et al, 1990;Homuth et al, 1998) and for yeasts (Dancis et al, 1990;Nyhus et al, 1997). Precedence for membrane-associated Fe 31 reductase activities also exists (Dailey and Lascelles, 1977;Fischer et al, 1990;Deneer et al, 1995;Mazoy and Lemos, 1996). It was postulated in the study of Worst et al (1998) (Fig.…”

Characterization of a Major Ferric Reductase from the Soluble Fraction of Vibrio anguillarum E6-5.

“…Except for the assay of the activity at different temperatures, ferric reductase activity was assayed as described by Mazoy and Lemos 15) with some modification. Standard assay mixtures (1 ml) contained 10 mM Tris-HCl (pH 7.4), 100 mM ferric citrate, 250 mM NADH, 50 mM FMN, 250 mM ferrozine, 1 mM MgSO4, and 0.1 mg/ml bovine serum albumin (BSA).…”

“…Cell fractions were prepared by the method of Mazoy and Lemos 15) with slight modification. Briefly, bacterial cells were grown at 22°C for 30 h in 100 ml of 0.2% casamino acids containing M9 minimal medium 16) (denoted as CM9 medium) or in CM9 medium supplemented with 5 mM iron ion (added as ferric citrate).…”

“…Non-denaturing discontinuous polyacrylamide gel electrophoresis (native PAGE: without SDS and DTT) was performed according to the method described by Mazoy and Lemos 15) with slight modification, by using a 12% (w/v) separating gel and 4% (w/v) stacking gel. Samples were concentrated to 20 ml using the Microcon centrifugal filter device YM-10 (Amicon), mixed with an equal volume of cold sample buffer containing 20 mM Tris-HCl (pH 7.0), 40% glycerol (v/v), and 0.005% (w/v) bromophenol blue as tracking dye, and applied to gels.…”

“…This bacterium can utilize heme or transferrin as its iron source 13) and possesses a heme-binding protein in its cell membranes 14) . Later, Mazoy and Lemos reported the presence of ferric reductase in all cell fractions of V. anguillarum H775-3 and suggested that the activity could be constitutive 15) . They found some stimulation of activity in the cytoplasmic fraction when the bacterium was grown in CM9 medium without a supplement of iron, and speculated on some regulation in the inductive expression of cytoplasmic ferric reductase.…”