Some properties of subunits of DNA gyrase from Pseudomonas aeruginosa PAO1 and its nalidixic acid-resistant mutant

Abstract

Subunits A and B of DNA gyrase were purified from Pseudomonas aeruginosa PAO1 and its mutant, which was resistant to nalidixic acid. Inhibition tests of DNA gyrases reconstituted with a combination of subunits from the two strains showed that an alteration of subunit A but not subunit B caused bacteria to resist fluoroquinolones.New fluoroquinolones, such as norfloxacin (NFX), ofloxacin (OFX), enoxacin, and ciprofloxacin (CPX) show activity against a broad spectrum of gram-negative and gram-positive bacteria, including nalidixic acid (NA)-resistant strains (11,20,25). From studies of in vitro DNA replication systems, these quinolones appear to inhibit DNA syhthesis by inhibiting the activity of DNA gyrase (10, 24). DNA gyrase, which catalyzes the supercoiling of relaxed closed circular DNA by coupling with the hydrolysis of ATP, was found in Escherichia coli (5) and was considered to be an essential enzyme for the DNA replication of chromosomes, plasmids, and bacteriophage (12,13,23). DNA gyrase from E. coli consists of two subunits, A and B (3, 7). Subunit A is a dimer of a 105,000-dalton protein encoded by the gyrA gene, which determines resistance to NA, and subunit B consists of a 95,000-dalton protein encoded by the gyrB gene, which determines resistance to novobiocin and coumermycin Al (4,7,9,19,29).Strains of Pseudomonas aeruginosa are the most important pathogens, because they are often isolated from clinical specimens and are resistant to various antibacterial agents (2). Benbrook and Miller (1) reported that NFX inhibits both the replication of DNA in growing cells of P. aeruginosa and the supercoiling activity of DNA gyrase in vitro. Further, we reported that OFX and CPX, in addition to NFX, strongly inhibit the supercoiling activity of DNA gyrase from P. aeruginosa PAO1 (24).It was reported that the NA resistance mechanisms of P. aeruginosa were chromosomal mutations of nalA or nalB genes (22). The nalA gene is located at approximately 51 min, and the nalB gene is located at approximately 31 min on the genetic map of P. aeruginosa PAO (22), although enzymatic studies have not been done until recently.In this study, we describe the purification procedure and show some properties of subunit A and subunit B proteins of DNA gyrase from a mutant (Nalr), compared with those of the parent strain P. aeruginosa PAO1, to clarify resistance mechanisms of P. aeruginosa to quinolones. P. aeruginosa PAO1 (8) We obtained the following compounds as gifts from the manufacturers: NFX, OFX, enoxacin, CPX, pipemidic acid (PPA), and NA. To determine the MICs of drugs, the agar dilution method was employed. One loopful (ca. 3 x 104 CFU) of a diluted culture was inoculated onto modified Mueller-Hinton agar (Nissui Seiyaku Co., Ltd.) containing serial twofold dilutions of a drug. The MICs were determined after incubation at 37°C for 18 h.P. aeruginosa was grown in 2 liters of medium B (18) and harvested at late log phase. Cells were washed once with 300 ml of saline solution and suspended in an equal volume of 10 mM Tris ...

Resistance to pefloxacin in Pseudomonas aeruginosa

“…In P. aeruginosa, alteration of DNA gyrase subunit A has been recognized as a mechanism of resistance to fluoroquinolones (19,38), and genes associated with this resistance have been identified as nfxA for norfloxacin (18) and cfxA for ciprofloxacin (38); these are alleles of nalA (36). These genes have been mapped on the P. aeruginosa PAO chromosome between hex-9001 and leu-9005 (18).…”

“…These findings suggested that resistance to pefloxacin is associated with altered DNA gyrase in strain PT2-r, with altered permeability in PT1, and with both mechanisms in PT2. The decreased expression of protein D2 and the higher calcium and lipopolysaccharide contents of the outer membrane could be responsible for the permeability deficiency in P. aeruginosa.The activities of fluoroquinolones against Pseudomonas aeruginosa have been fully documented (45), but bacteria can develop resistance to these agents (12,18,19,36,38). This resistance results from reduced permeability of the outer membrane (OM), altered DNA gyrase, or a combination of the two processes (7,8,18,36,38).…”

A pleiotropic, posttherapy, enoxacin-resistant mutant of Pseudomonas aeruginosa

“…As in other bacteria, the primary target for quinolones in Pseudomonas aeruginosa is DNA gyrase. nalA, nfxA, norA, and cipA are alleles ofgyrA and encode A subunits that are less susceptible to inhibition by quinolones (14,16,33,34). Other mutations affecting quinolone activity, but not DNA gyrase, in P. aeruginosa have also been described.…”

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones

“…Some quinolones have been used for the treatment of methicillin-resistant S. aureus infections, but the emergence of quinolone resistance has been reported elsewhere (32). Unlike the mechanism underlying the quinolone resistance of gram-negative bacteria such as Escherichia coli (2,7,9,11,12,15,27,31,(36)(37)(38)(39) and Pseudomonas aeruginosa (4,13,16,29,30,36,40) …”