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J. Bacteriol. doi:10.1128/JB.00912-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In vivo mutations of thymidylate synthase (thyA) are responsible for thymidine-dependency in clinical small colony variants (TD-SCVs) of Staphylococcus aureus

Institute of Medical Microbiology and Hygiene, Institutes of Infectious Disease Medicine, University of Saarland, Homburg/Saar, Germany; Institute of Medical Microbiology, University Hospital Muenster, Muenster, Germany; Department of Medicine and Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin, USA

^* To whom correspondence should be addressed. Email: kahl{at}uni-muenster.de.

	Abstract

Trimethoprim-Sulfamethoxazole (SXT)-resistant thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis (CF) patients often in combination with isogenic normal strains if patients were treated by SXT for extended periods. As SXT inhibits the synthesis of tetrahydrofolic acid, which acts as a co-factor for thymidylate synthase (thyA), the survival of TD-SCVs depends exclusively on the availability of external thymidine. Since the underlying mechanism for thymidine-dependency is unknown, we investigated if alterations in the thyA nucleotide sequences were responsible for this phenomenon. Sequence analysis of several clinical TD-SCVs and their isogenic normal strains with reference to the published S. aureus thyA nucleotide sequences was performed. Three clinical TD-SCVs were complemented by transforming TD-SCVs with the vector pCX19 expressing ThyA from S. aureus 8325-4. Transcriptional analysis of metabolic and virulence genes and regulators (agr, hla, spa, citB, thyA, nupC) was determined by quantitative reverse transcription PCR. The published sequences of thyA and of 2 normal clinical strains were highly conserved, while thyA of 4 normal strains and of 4 SCVs displayed non-synonymous point-mutations. In 8/10 SCVs, deletions occurred implementing stop-codons located in 4/10 SCVs close to or within the active site of the protein (dUMP binding). Complementation of TD-SCVs with thyA almost fully reversed the phenotype, growth characteristics and transcription patterns. In conclusion, we could demonstrate that mutations of thyA were responsible for the phenotype of TD-SCVs. Complementation of TD-SCVs with thyA revealed that a functional ThyA is necessary and sufficient to revert the SCV phenotype and behaviour back to normal.

This article has been cited by other articles:

• Besier, S., Zander, J., Kahl, B. C., Kraiczy, P., Brade, V., Wichelhaus, T. A. (2008). The Thymidine-Dependent Small-Colony-Variant Phenotype Is Associated with Hypermutability and Antibiotic Resistance in Clinical Staphylococcus aureus Isolates. Antimicrob. Agents Chemother. 52: 2183-2189 [Abstract] [Full Text]