Transposon-mediated insertional mutagenesis of the D-alanyl-lipoteichoic acid (dlt) operon raises methicillin resistance in Staphylococcus aureus

Two independent mutants of methicillin-resistant Staphylococcus aureus (MRSA), KAN96H1 and KAN96H2, were isolated by insertional mutagenesis with conjugative transposon Tn918. In both, the minimal inhibitory concentration (MIC) of methicillin was increased to 128 compared to 16 mg/L for the parental strain KAN96. By transduction experiments, we verified that the insertion of Tn918 conferred higher methicillin resistance on KAN96H1, but not on KAN96H2. In KAN96H1, the integration site of Tn918 was located in the 6.1-kb HindIII fragment of the KAN96 chromosomal DNA. We identified a novel D-alanyllipoteichoic acid (dlt) operon of S. aureus in this fragment. The amino acid sequences of four open reading frames of this operon were highly homologous to those of the dlt operon genes of Bacillus subtilis. The nucleotide sequence of the staphylococcal dlt operon is under the accession number D86240 in the DDBJ/GenBank/EMBL databases. In KAN96H1, Tn918 was inserted in the 5′-terminal region of the putative dltB gene which encoded a hypothetical membrane transporter. dlt transcripts of 4.7 kb were detected in KAN96, but were truncated to 2.3 kb in KAN96H1. No corresponding transcripts were observed in KAN96H2. Our results clearly demonstrated that defects in functions of the putative dlt operon resulted in increased methicillin resistance in MRSA.