doi:10.1016/j.femsle.2005.06.038 
Copyright © 2005 Federation of European Microbiological Societies Published by Elsevier B.V.
Staphylococcal Drp35 is the functional counterpart of the eukaryotic PONs
Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoh-dai, Tsukuba 305-8575, Japan
Received 2 April 2005;
revised 7 June 2005;
accepted 9 June 2005.
Available online 5 July 2005.
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Abstract
Drp35 has been identified as a protein that is induced in Staphylococcus aureus in response to exposure to certain antibiotics. Here we demonstrate that Drp35 is a lactonase that does not contribute directly to the resistance to the inducer antibiotics except for bacitracin. The detailed analysis on the expression of Drp35 revealed that in addition to a broad range of antibiotics, agents such as detergents that perturb the membrane integrity could induce its expression. The significance of this characteristic expression is discussed in relation to its activity similarity to the eukaryotic counterparts, paraoxonase family proteins.
Keywords: Staphylococcus aureus; Drp35; Paraoxionase; Membrane integrity
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Bacterial strains
- 2.2. Construction of drp35 disruptant, NM6
- 2.3. Construction of drp35 overexpressing strain, NMDR
- 2.4. Western blot analysis
- 2.5. Antibiotic susceptibility tests
- 2.6. Preparation of the recombinant Drp35 protein
- 2.7. Enzyme assay
- 3. Results
- 3.1. Is Drp35 required for the antibiotic resistance?
- 3.2. Drp35 is functional protein with calcium-dependent lactonase activity
- 3.3. Expression profile of Drp35
- 4. Discussion
- 4.1. Drp35 contributes to the bacitracin resistance
- 4.2. Drp35 acts in cells with perturbed membrane integrity
- 4.3. Drp35 is the bacterial counterpart of eukaryotic paraoxonase family proteins
- Acknowledgements
- References
Fig. 1. The sequence alignment of Drp35 and paraoxonase family proteins. Residues identical to S. aureus Drp35 are shaded in yellow. The PON1 structure (β-propeller) and important residues [11] are indicated below the alignment. The sequences were retrieved from the KEGG database and aligned by DNAstar software. S. aureus Drp35 (Entry No. sau:SA2480), Staphylococcus epidermidis Drp35 (sep:SE0263), Mus musculus PON1 (mmu:18979), PON2 (mmu:330260), PON3 (mmu:269823), Homo sapiens PON1 (hsa:5444), PON2 (hsa:5445), PON3(hsa:5446).
Fig. 2. Drp35 can hydrolyze dihydrocoumarin (a) and 2-coumaranone (b) 0.6 μg ml−1 of the purified Drp35 protein (♦) or 0.6 μg ml−1 of a negative control protein, quinone oxidoreductase [19] (
) was used for the assay. The y-axis represents the degraded amount of the substrate at μmol/mg protein.
Fig. 3. The induction and subcellular location of Drp35. The cells of N315 at the early logarithmic phase were exposed to various cell wall-affecting antibiotics and detergents at 37 °C for 30 min. (a) no antibiotic (lane 1), oxacillin 64 μg ml−1 (lane 2), vancomycin 1 μg ml−1 (lane 3), fosfomycin 4 μg ml−1 (lane 4), bacitracin 80 μg ml−1 (lane5). (b) no detergent (lane 1), 0.5% each of Nonidet P40 (lane 2), TritonX-100 (lane 3), SDS (lane 4), CHAPS (lane 5). The 25 μg of whole protein extracts were submitted to Western blot analysis with anti-Drp35 antibody. (c) N315 cells were incubated with (lanes 2, 4, 6) or without (lanes 1, 3, 5) 80 μg ml−1 of bacitracin. Soluble proteins (lanes 3, 4) and membrane proteins (lanes 5, 6) were submitted to Western blot analysis. Lanes 1 and 2 contains whole cell proteins. Drp35 was the soluble protein and was never detected in the membrane fraction.
Table 1.
MIC values for S. aureus strains: N315, NM6, NMDR, and NM6v
Note that the MIC values of bacitracin was precisely determined by using a series of concentrations at 10 μg/ml intervals.
Abstract
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