Characterization of the Staphylococcus aureus rRNA methyltransferase encoded by orfX, the gene containing the staphylococcal chromosome Cassette mec (SCCmec) insertion site

Abstract

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

FIGURE 1.

Schematic of the general arrangement of SCCmec. The insertion of SCCmec into the C terminus of orfX at the attachment site attB changes it to attR1. The terminal five amino acids and stop codon are unchanged even though the DNA sequence is altered. Also shown are the regions that define a SCC_mec: the mec operon containing mecA, the gene responsible for β-lactam resistance; the cassette chromosome recombinase (ccr) operon that facilitates the insertion and excision of SCCmec; and three hypervariable regions between them. The sequences of the mec and ccr operons define the SCCmec type. A more detailed description of the different SCC_mec types can be found at the International Working Group on the Staphylococcal Cassette Chromosome Elements. This figure is not to scale and does not show all of the genes in the region.

FIGURE 2.

A, OrfX expression monitored throughout the growth cycle of S. aureus. Shown is a Western blot of samples taken at each of the time points indicted and by diamonds in the growth curve below. B, growth curves comparing the growth of the parent (RN450) and orfX mutant (AW2) at either 18 °C (upper panel) or 37 °C (lower panel) in enriched BHI broth.

FIGURE 3.

Methylation of ribosomes from either the orfX mutant (AW2) or the parent (RN450) with a fully functional orfX (see Fig. 1). The bars are radioactive counts (vertical axis) from ribosomes exposed to OrfX incubated with substrate (AdoMet) containing a ³H-labeled methyl group. Error bars indicate the S.D. of the results from three repeated experiments. The difference between the results obtained with the two strains was statistically significant (p < 0.005; Students t test). Also shown are the controls with 70 S ribosome, with radiolabeled AdoMet and no OrfX, and with OrfX with radiolabeled AdoMet and no 70 S ribosomes.

FIGURE 4.

A, initial difference electron density map (with coefficients F_o − F_c shown at the 2. 8σ level) of the OrfX structure before the bound AdoMet molecule was added to the model. B, final electron density map (with coefficients 2F_o − F_c shown at the 1.0 σ level) of the OrfX structure. All maps are superimposed with the final refined models. C, least-squares superposition of the OrfX structure (blue) with that of the Protein Data Bank 1VH0 structure (magenta). D, least-squares superposition of the active site structures of OrfX (blue) and 1VH0 (magenta).

FIGURE 5.

Overall structure of OrfX (Protein Data Bank code 4FAK). A, ribbon diagram of the dimeric OrfX structure, also with bound AdoMet (yellow stick), a phosphate molecule (brown stick), and PEG-550MME (yellow stick). Monomers A and B are colored green and magenta, respectively. B, least-squares superposition of the OrfX structure (blue) with the YbeA structure (gray).

FIGURE 6.

Active site and AdoMet cofactor-binding mode of OrfX. A, stereo view of the AdoMet-binding site and the bound AdoMet. Monomers A and B of the protein are as yellow and green sticks, respectively. AdoMet is shown as a yellow stick, and the water molecules are red spheres. B, two-dimensional schematic diagram showing interactions between AdoMet and the protein residues or water molecules. Dotted lines indicate hydrogen bond interactions, and dashed lines indicate hydrophobic contacts. C, surface representations of the OrfX-AdoMet complex. The two active sites are shown, with one of them showing the bound AdoMet and phosphate molecules as yellow and cyan spheres, respectively. The protein structure is colored according to the distribution of electrostatic potential from red (−8 kiloteslas) to blue (+8 kiloteslas.

FIGURE 7.

Isothermal titration calorimetry results from the binding of purified dimeric 0.0726 mm OrfX with 1.182 mm AdoMet buffered with 50 mm sodium phosphate (pH 7.5). A shows the exothermic reaction of AdoMet being injected into the cell and binding OrfX. Analysis using Origin 7 produced B. The most favorable χ²/degrees of freedom (726) was in a model predicting sequential binding with the first active site of the dimer being filled, with K_d1 = 52.1 μm, ΔH₁ = −5,135 J/mol, and ΔS₁ = −83.5 J/mol/degree. The second active site of the dimer was then filled, with K_d2 = 606 μm, ΔH₂ = −2.41 × 10⁴ J/mol, and ΔS₂ = −15.9 J/mol/degree. This demonstrates that both active sites in the OrfX dimer are filled with an AdoMet molecule, but that first one active site is filled and then the second one.