Characterization of a multicopper oxidase gene from Staphylococcus aureus

Abstract

A multicopper oxidase gene from Staphylococcus aureus was cloned and overexpressed. Purified recombinant multicopper oxidase oxidized the substrate 3,3'-dimethoxybenzidine in the presence of copper. Disruption of mco showed copper sensitivity and H(2)O(2) resistance, suggesting roles for mco in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced mco transcription.

FIG. 1.

Schematic representation of the multicopper oxidase and copper ATPase genes. The arrow indicates predicted gene and orientation. The transposon insertion site within the gene is indicated by an inverted triangle. The solid bar indicates the DNA fragment which was used as a probe for the Northern analysis.

FIG. 2.

Effects of copper and hydrogen peroxide on growth. Overnight cultures were diluted 1:500 in TSB with different concentrations of CuSO₄ (A) or H₂O₂ (C) and incubated at 37°C with shaking. Cell growth was monitored by measuring optical density at 600 nm for 18 h. Growth curves with 2.5 mM CuSO₄ (B) and 1.5 mM H₂O₂ (D) are shown. Overnight cultures were diluted 1:500 in TSB with 1.5 mM H₂O₂ or 2.5 mM CuSO_4. Cell growth was monitored by measuring absorbance at 600 nm at various intervals of incubation at 37°C with shaking. Symbols: ⧫, wild type; ▪, mco mutant; ▴, mco complemented strain. Each point represents the mean value ± standard deviation (represented by bar) of three experiments.

FIG. 3.

Northern blot analysis. (A) Ten micrograms of total RNA was electrophoresed on formaldehyde agarose gels (1.2%) and transferred to a nitrocellulose membrane. The blot was probed with radiolabeled mco. Lane 1, RNA extract from the wild type grown in defined medium (DM); lane 2, RNA extract from the wild type grown in DM with 200 μM CuSO₄; lane 3, RNA extract from mutant grown in DM; and lane 4, RNA extract from the mutant grown in DM with 200 μM CuSO₄. Arrows indicate the multiple transcripts of approximately 1.3 (a), 2.0 (b), and 3.5 (c) kb. Molecular size markers (in kilobases) are indicated on the left. (B) Same blot, probed with radiolabeled 16S rRNA gene.