grlA and gyrA mutations and antimicrobial susceptibility in clinical isolates of ciprofloxacin- methicillin-resistant Staphylococcus aureus.

OBJECTIVE: To determine grlA and gyrA mutations in ciprofloxacin- methicillin-resistant Staphylococcus aureus isolates and their susceptibility to current antimicrobials, including a newer fluoroquinolone gatifloxacin, glycopeptides vancomycin, teicoplanin and oxazolidinone linezolid. METHODS: A total of 56 methicillin-resistant S. aureus (MRSA) isolates were collected during 2003-2006 from inpatients of Süleyman Demirel University Hospital. The isolates were confirmed to be MRSA by the production of coagulase, showing resistance against cefoxitin and having the mecA gene and tested by disk diffusion for susceptibility to vancomycin, teicoplanin and linezolid. The minimum inhibitory concentrations (MICs) of ciprofloxacin and gatifloxacin were measured using the E-test. The quinolone resistance determining regions (QRDRs) of isolates were amplified by PCR and mutations in grlA and gyrA genes were identified by direct sequencing. RESULTS: Sequencing data revealed that 96% of our isolates had mutations in both grlA and gyrA genes. Among these, the grlA mutation of Ser-80-Phe or Tyr and the gyrA mutation of Ser-84-Leu were the most dominant ones being detected in 50 (89%) and 40 (71%) isolates, respectively. Although 96% of isolates were highly resistant to ciprofloxacin (MIC, >or=32 mg/l), only 54% of ciprofloxacin-resistant MRSA isolates were resistant to gatifloxacin and exhibited lower-level resistance (MIC,

Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.

Arrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences. Binding of Tus to a Ter site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion. In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication. In vitro competition assays demonstrated that base pair substitutions at positions 8-19 had significant effects on the free energy of Tus binding (DeltaDeltaG0 of 1.5 to >4. 0 kcal/mol). Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo. Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Ter interactions, suggesting that these base pairs contribute to, but are not absolutely critical for, Tus binding. Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks. Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E. coli genome for sequence similarity to a consensus Ter site sequence.

Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli.

Tus, a DNA-binding protein, mediates arrest of DNA replication in Escherichia coli. Tus binds to DNA sequences called Ter sites, located in the terminus region of the chromosome, and forms replication-arrest complexes that block movement of DNA replication forks in a polar fashion. We have analyzed Tus to determine some of its physical parameters and biochemical characteristics. Native Tus had an 8(20,w) of 3.2, a Stokes' radius of 23 A, an axial ratio of 2, and a molar absorption coefficient of 39,700 M-1 cm-1. The data also indicated that Tus existed as a monomeric protein in solution and when complexed with its cognate DNA binding site. Secondary structure estimated from the circular dichroism spectrum suggested that Tus consisted of 40% alpha-helix, 0% beta-sheet, 15% turn, and 45% aperiodic structure. The isoelectric point of native Tus (pH 7.5) was significantly different than that calculated from its amino acid sequence (pH 10.1), possibly because the tertiary structure of Tus perturbs the ionization of several residues. In addition, partial proteolytic digests of free Tus protein did not produce a subfragment of Tus that retained DNA binding activity, but did demonstrate that Tus was resistant to proteolysis when complexed with a Ter site.