The 3= end of rsbU, encoding the positive regulator of the stress factor sigma B, was identified as a hot spot for spontaneous IS256 insertion in Staphylococcus aureus SA137/93G. Interestingly, subinhibitory concentrations of chloramphenicol in combination with heat stress, as well as linezolid and spectinomycin at physiological temperatures, selected for such rsbU:: IS256 insertion mutants. In consequence of the inactivation of rsbU, the IS256 transposition frequency was increased 4-fold in S. aureus HG001.
Staphylococcus aureus is a major human pathogen and has been able to acquire resistance to nearly all clinically used antibiotics. Insertion sequences (IS elements) have been instrumental in the development of resistance, since they represent the active component of transposons and, thus, mobilize resistance genes for horizontal gene transfer (1) or modulate the expression of resistance-conferring genes (2). Therefore, investigation of the transposition process of IS elements, especially in the presence of subinhibitory concentrations of antibiotics, will help us to understand and ultimately, perhaps, interfere with resistance development.IS256 is a highly active insertion sequence that, as a component of the composite transposon Tn4001, has played a pivotal role in the development of aminoglycoside resistance in S. aureus (3, 4). IS256 has been detected in the genome of several clinical isolates of enterococci and staphylococci (5, 6), e.g., in the clinical methicillin-resistant S. aureus (MRSA) isolates of sequence type 228 (ST228) (1 copy), ST239 (8 to 21 copies), and ST247 (at least 14 copies) (7-11). The accumulation of multiple IS256 copies in the genomes is due to its transposition process, which is mediated by a copy-and-paste mechanism (12). The effect of multiple IS256 elements on genomic flexibility and resistance development is exemplified by S. aureus SA137/93G (more than 14 copies), an ST247 strain that displays homogeneous vancomycin-intermediate resistance (vancomycin-intermediate S. aureus [VISA] phenotype) (13), which is mainly caused by the insertion of IS256 into the tcaA gene (11,14). A variety of intrinsic and extrinsic mechanisms regulate the transposition activity of mobile elements in bacteria (15). Whereas subinhibitory concentrations of ciprofloxacin and vancomycin lead to the activation of IS256 transposition (16), the alternative sigma factor B, which controls the expression of genes involved in stress response (17), is a negative regulator of IS256 transposition in S. aureus (18). In the present study, we demonstrate that frequent insertion of IS256 into the rsbU gene, which encodes a positive regulator of SigB (19), resulted in the autoactivation of IS256 transposition in S. aureus. rsbU is a hot spot for spontaneous IS256 insertions. All strains, plasmids, and oligonucleotides are shown in Tables 1 and 2. Spontaneous insertion of IS256 into rsbU was first detected during attempts to integrate temperature-sensitive plasmids harboring a chloramphenicol resistance gene into the...