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 H₂O₂ resistance, suggesting roles for mco in copper homeostasis and oxidative stress response. Northern blot analysis showed copper-induced mco transcription.

Multicopper oxidases (MCO) are enzymes that are critically involved in copper homeostasis. These enzymes show enhanced oxidase activity for a wide range of substrates (1, 2, 4, 9, 22, 24) and play a role in iron transport in eukaryotes and prokaryotes (9, 23). mco genes have been cloned and identified from animals, plants, insects, fungi, and bacteria (3, 4, 8, 12, 16). However, there is no report of mco in Staphylococcus aureus.

We have been investigating the regulation of metal transport and homeostasis in S. aureus (25, 26). To identify new loci involved in metal homeostasis in S. aureus, we selected streptonigrin-resistant clones from a transposon Tn917-induced mutant library of S. aureus strain ATCC 12600 and characterized mutants by cloning and sequencing. In this study, we identified one of the mutations in the gene encoding multicopper oxidase.

Staphylococcus aureus strains were grown in tryptic soy broth (TSB) or defined medium. Escherichia coli strains were grown in Luria-Bertani broth with the appropriate antibiotic. DNA manipulation, sequencing, cloning, transformation, and overexpression were performed as described earlier (7, 21, 26). DNA sequences were determined with an ABI Prism 310 automated sequencer (Perkin Elmer, Foster City, CA).

The Tn917-flanking sequence of the mutant was cloned in E. coli as described earlier (21). Nucleotide analysis of the flanking region of the Tn917 insertion of one of the streptonigrin-resistant clones showed sequence homology to putative multicopper oxidases. The complete nucleotide sequence of the parental gene copy of S. aureus showed an open reading frame (ORF) comprising 1,389 bp encoding a hypothetical polypeptide of 462 amino acids with a predicted molecular mass of 52 kDa and pI of 9.68. Nucleotide comparison showed a 99% sequence identity with a gene in the S. aureus EMRSA-16 database. Amino acid comparisons of the translated ORF showed 84% identity to putative MCO from Staphylococcus epidermidis and 26 to 41% identities to laccase, CueO, and ascorbate oxidase, which are members of the MCO family. Therefore, we designated this ORF mco. Seventy nucleotides upstream of mco, an ORF designated copA, a copper ATPase, was also identified (Fig. 1).

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.

The multicopper oxidase gene from S. aureus was cloned and overexpressed in E. coli BL21(DE3). MCO activity was determined by using 3,3′-dimethoxybenzidine as described earlier (5, 15, 18). This enzymatic activity was copper dependent, and the presence of 0.5 mM CuSO₄ is optimum for enzymatic activity. The purified MCO showed a specific activity of 9.7 U/mg, compared to 1.6 U/mg in the crude extract (Table 1). The purified MCO also exhibit low levels of ferroxidase (1.58 U/mg) and phenoloxidase (2.3 U/mg) activities compared to those reported for other organisms (11, 13).

TABLE 1.

Overexpression and purification of MCO from E. coli BL21(DE3)(pLysS)^a

Step	Protein (mg)	Total activity (U)b	Sp actc (U/mg)
Crude extract	21	19.9 ± 1.3	1.6 ± 0.2
Ni column elute	0.6	42.0 ± 2.0	9.7 ± 0.3

^aEnzyme activity was measured using 3,3′-dimethoxybenzidine as a substrate. The reaction was initiated by the addition of 0.472 μmol of 3,3′-dimethoxybenzidine (o-dianisidine) and terminated after 5 and 120 min.

^bOne unit of activity catalyzed the oxidation of 1 μmol of substrate per min.

^cSpecific activity is defined as the μmol of substrate oxidized per min per mg of total protein.

Although multicopper oxidase is an important enzyme in many organisms, analysis of the S. aureus database indicated the presence of MCO homologues only in an S. aureus EMRSA-16 strain. We used Southern blot analysis to search for homologous sequences in various S. aureus strains whose genomes have not been sequenced. The mco gene hybridized with a 2.5-kb HindIII DNA fragment of three strains (ATCC 12600, H, and Wood) out of seven laboratory strains. However, the most commonly used S. aureus strains, RN450 and COL, did not show any sequence homology with mco, suggesting that it plays a dispensable role in staphylococci.

The insertion of Tn917 in the multicopper oxidase gene resulted in streptonigrin tolerance and copper sensitivity. As shown in Fig. 2A and B, the mutant strain grows slowly in medium containing more than 1.5 mM CuSO₄. We also checked the mutant's sensitivities to iron, nickel, cobalt, and other metallic ions. So far, we found the mco mutant sensitive only to copper and cobalt (data not shown).

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.

A role of mco in the oxidative stress response has been proposed previously (10, 16). To test whether a mutation in mco has any impact on the oxidative tolerance of S. aureus, the mco mutant and the parent cells were grown in TSB containing various concentrations of H₂O₂ and methyl viologen (paraquat). The mco mutant cells were able to grow in the medium containing 5 mM H₂O₂, whereas the parent cells were unable to grow (Fig. 2C). However, the tolerance levels of the mco mutant and the parent strains to paraquat, another oxidative agent, were similar. Additional experiments using catalase assay activity gels (7) showed that the higher hydrogen peroxide tolerance of the mco mutant was not due to the induced expression of the kat gene, which encodes the catalase (data not shown).

In the presence of heavy metals, MCO catalyzes the formation of H₂O₂. It has been shown that cupric ions and ceruloplasmin, a multicopper oxidase family protein in human serum, have the capacity to oxidize the substrate with the production of superoxide anions and H₂O₂ (17). Moreover, the production of H₂O₂ in the oxidation of heavy metals by laccase, an MCO in Stropharia rugosoannulata, has been reported previously (20). However, the precise role of multicopper oxidase in the hydrogen peroxide tolerance phenotype of the mco mutant is unknown.

Complementation studies were performed to provide genetic evidence that copper sensitivity and H₂O₂ tolerance are due to the transposon insertion within the mco. A 1.8-kb BamHI-HindIII fragment containing mco was cloned in the shuttle vector pLI50 (14) and transformed into an mco mutant strain. As shown in Fig. 2C and D, the complemented strain became H₂O₂ sensitive as predicted; however, it unexpectedly remained copper sensitive (Fig. 2A and B). To explain this result, we hypothesize that the mutation within the mco gene had an additional unanticipated negative effect upon copA expression, resulting in higher intracellular copper levels, which would cause copper sensitivity. In order to test this hypothesis, we measured the intracellular copper contents of the mutant, wild-type, and complemented strains by using an atomic absorption spectrophotometer as described previously (19). The intracellular copper contents in the mutant and complemented strains were 5 pmol and 10 pmol/10⁸ cells, respectively, while the copper content of the wild type was less than 1 pmol/10⁸ cells. These results suggest that the failure to complement the copper-sensitive phenotype may be due to an inability of the cell to efflux copper out of the cell.

Northern blot analysis was performed to examine the effect of copper on mco transcription. As shown in Fig. 3 (lane 2), three transcripts (1.3, 2.0, and 3.5 kb) were induced by elevated concentrations of copper ions (200 μM) in the parent strain, but no induction occurred in the mutant strain (Fig. 3, lane 4). The three different transcripts might represent the products from different genes or of specific degradation. The 3.5-kb transcript likely corresponds to a transcript containing both mco and copA. The nucleotide sequence analysis indicated that mco and copA genes in S. aureus are separated by 70 bp and have no significant sequence identity. Therefore, hybridization of all three transcripts with mco genes is puzzling and possibly indicative of a complex transcriptional regulation that needs further investigation.

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.

In conclusion, we have identified an mco gene which encodes an enzyme that shows oxidase activity in S. aureus. The expression of this gene can be induced by copper. A transposon-induced mutant showed copper and cobalt sensitivity and H₂O₂ tolerance. Since mco was not found in all strains of S. aureus, this suggests that it plays a dispensable role in staphylococci. It has been suggested that the mco role in copper homeostasis may be compensated for by other gene systems (6). Further studies are required to determine the role of mco in heavy metal homeostasis and oxidative response.

Nucleotide sequence accession number.

The complete nucleotide sequence of the parental gene copy of S. aureus has been deposited to the GenBank database under accession no. AY259130 gi 33391259.