Mutation at the “Exit Gate” of the Salmonella Gyrase A Subunit Suppresses a Defect in the Gyrase B Subunit

Abstract

In Salmonella enterica serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele gyrB651) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the gyrA gene (allele gyrA659), a T467S substitution, which partially suppresses the supercoiling defect of gyrB651. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a gyrA mutation that compensates for a gyrB defect.DNA gyrase is a member of the family of type II DNA topoisomerases, a group of enzymes that catalyze the interconversion of different topological forms of DNA. DNA gyrase introduces supercoils into DNA through the breakage of a DNA duplex, the passage of another DNA segment through the break, and the resealing of the break (8). This activity involves the opening and closing of a series of molecular gates, coupled to ATP hydrolysis. DNA gyrase is a heterotetramer of two A subunits (97 kDa) and two B subunits (90 kDa), encoded by the gyrA and gyrB genes, respectively (8). The 64-kDa Nterminal domain of GyrA is thought to intervene in the breakage-reunion reaction, whereas the 33-kDa C-terminal domain is involved in the stabilization of the gyrase-DNA complex. The GyrB protein contains a 43-kDa N-terminal ATP binding domain and a 47-kDa C-terminal domain that interacts with GyrA and DNA. Structural analysis indicates that the GyrA breakage-reunion domain resembles a clamp with two sets of jaws at opposite ends (18). Experimental studies, using yeast topoisomerase II or DNA gyrase, indicate that DNA is transported by a "two-gate" mechanism, entering the interior of the enzyme through an upper N gate at the GyrA head dimer interface and leaving the enzyme through an "exit gate" at the GyrA bottom dimer interface closer to the C termini (19,20,26).DNA gyrase is the target of two classes of inhibitors, the quinolones, such as nalidixic acid (Nal), which trap DNA gyrase covalently bound to its cleaved substrate, and the coumarins, such as novobiocin (Nov), which inhibit the ATPase activity (15). Mutations conferring Nal resistance are for the most part located in a region of the gyrA gene specifying the N-terminal domain portion between amino acids 51 and 106, the so-called quinolone resistance-determining region (22). Resistance to quinolones (albeit to a lower level) can also result from mutations in the gyrB gene, and the affected sites identified are amino acids D426, K447, and S464 in the GyrB C-terminal domain (12,27). In addition to their effect on quinolone sensitivity, mutations at sites D426 and K447 also inhibit the gyrase catalytic activity (13). It has been proposed that the quinolone-binding site is a pocket surrounded by surfaces involving the quinolone resistance-determining regions of both the GyrA and GyrB proteins (13,27).Isolation of the gyrB651 allele and its phenotypic suppressor, gyrA659. Mutants of Salmonella enterica ...

Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile

“…In a previous study investigating fluoroquinolone resistance in Salmonella enterica, Blanc-Potard et al characterised a pleiotropic mutation in gyrB causing ciprofloxacin resistance and concomitant novobiocin sensitivity. They subsequently identified an epistatic and compensatory mutation in gyrA that restored novobiocin sensitivity to wild-type levels while further augmenting fluoroquinolone resistance [14]. Notably, the epistatic mutation (Thr467Ser) was located at the dimer interface region, thus confirming the ability of mutation at this region in GyrA to influence both ciprofloxacin and novobiocin sensitivity, a finding consistent with the current observations in C. difficile.…”

“…Notably, the epistatic mutation (Thr467Ser) was located at the dimer interface region, thus confirming the ability of mutation at this region in GyrA to influence both ciprofloxacin and novobiocin sensitivity, a finding consistent with the current observations in C. difficile. We speculate that the Ala384Asp mutation in C. difficile may be functionally analogous to the Thr467Ser mutation in Salmonella GyrA that results in a predicted destabilisation of the primary interface favouring a quinolone-resistant DNA gyrase conformation [14].…”

Type II topoisomerase mutations in clinical isolates of Enterobacter cloacae and other enterobacterial species harbouring the qnrA gene

“…In Gram-negative bacilli, gyrB mutations are rare in clinical strains and they have been described at positions 426, 431, 447, 464 and 466 [44][45][46][47]. The Ser464Phe mutation has been linked to quinolone resistance in S. enterica [45], P. aeruginosa [47] and P. mirabilis [48].…”

Systematic genome engineering approaches to investigate mutational effects and evolutionary processes

“…Most notably, the analysis revealed a novel 46-aminoacid-long region of GyrB, which was found to be mutated in more than 22% of the analyzed mutants. To understand the role of this region on GyrB, we subsequently performed protein structure analyses which demonstrated that this region is in the close proximity of GyrA, and forms the binding site of fluoroquinolone antibiotics [178,179] (Figure 24). Also, it should be noted that the lack of resistance-conferring mutations at ParC and ParE is in-line with previous observations [175].…”