Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

Abstract

In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases that modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (33). DNA gyrase, encoded by gyrA and gyrB, forms an A 2 B 2 tetramer that removes the positive supercoils introduced during DNA replication and helps to maintain the steady-state balance of negative supercoiling of the bacterial DNA (30). Topoisomerase IV, encoded by parC and parE, forms a C 2 E 2 tetramer. Its primary role is the decatenation of daughter chromosomes following DNA replication, and other functions are DNA relaxation and double-strand DNA breakage (1,6,14,33). Both enzymes are the target of two antibiotic families: the quinolones, such as ciprofloxacin, sparfloxacin, and grepafloxacin, and the coumarins, such as novobiocin. The quinolones form a ternary complex with topoisomerases in the presence of DNA, resulting in double-stranded breaks (4). The coumarins inhibit ATP-dependent functions of these enzymes by preventing binding and hydrolysis of ATP (27).In Streptococcus pneumoniae, it has been shown that the primary and the secondary targets of quinolones are either the gyrase or the topoisomerase IV, depending upon the molecule (21, 29). Mutants resistant to quinolones most frequently harbor mutations in the quinolone resistance-determining regions (QRDR) of GyrA and ParC and less frequently in that of ParE (7). The level of quinolone resistance increases when a mutation is present in the secondary target in addition to one in the primary target (21, 29). In S. pneumoniae, in vitro-selected resistance to novobiocin is associated with the S127L substitution in the GyrB subunit (17). This position is located upstream from and close to positions 136 and 164 described in the ATP binding domain of GyrB in Escherichia coli and also involved in coumarin resistance (3). The substitution in ParE of E. coli at a position equivalent to that of position 136 in GyrB resulted in a decreased inhibition of topoisomerase IV by novobiocin (6).In previous studies (11, 26), we showed in S. pneumoniae that the H103Y substitution in the N terminus of the ParE subunit of the topoisomerase IV resulted in an original phenotype of resistance only if it was as...