Functions of a GyrBA Fusion Protein and Its Interaction with QnrB and Quinolones

Abstract

In order to study the interactions between Escherichia coli DNA gyrase and the gyrase interacting protein QnrB in vivo, we constructed a gyrB-gyrA fusion and validated its ability to correct the temperature-sensitive growth of gyrA and gyrB mutants. Like wild-type gyrA, the gyrB-gyrA fusion complemented a quinolone-resistant gyrA mutant to increase susceptibility. It functioned as an active type II topoisomerase, catalyzed negative supercoiling of DNA, was inhibited by quinolone, and was protected by QnrB.

TEXT

Bacteria usually express two type II topoisomerases, DNA gyrase and topoisomerase IV (topo IV). DNA gyrase, a GyrA₂GyrB₂ tetramer encoded by the gyrA and gyrB genes, is the only type II topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. Topo IV is a ParC₂ParE₂ tetramer that mediates the topological unlinking of catenated daughter chromosomes (1, 2). Quinolones interact with gyrase and topo IV, but in most cases, target gyrase in Gram-negative bacteria and topo IV in Gram-positive bacteria (3). Quinolone inhibition of DNA gyrase occurs via formation of a ternary cleavage complex among gyrase, DNA, and quinolone that blocks the progression of the DNA replication fork, leading to accumulation and eventual release of double-stranded DNA breaks that are ultimately lethal to the cell (4). Qnr belongs to the pentapeptide repeat protein (PRP) family and can protect Escherichia coli DNA gyrase from ciprofloxacin inhibition as measured by an in vitro supercoiling assay (5, 6). It remains unclear, however, how Qnr proteins are able to protect from quinolone action without impairing gyrase catalytic function.

In order to test the suitability of an Escherichia coli GyrBA fusion protein for whole-cell protein-protein interaction studies of Qnr binding to gyrase using bacterial 2-hybrid assays, we tested the ability of a gyrBA fusion to complement temperature-sensitive growth of gyrA and gyrB mutants, the functions of purified GyrBA, and its interactions with quinolones. Although GyrBA fusion proteins purified from Bacillus subtilis or E. coli have been shown to introduce negative supercoils into DNA in vitro (7, 8), efforts to show the ability of one fusion to substitute for topoisomerase II in yeast cells in vivo failed, and the ability of gyrBA to complement bacterial gyrase mutants and the interaction of GyrBA with quinolones has not been reported (8). Therefore, it was important to validate the whole-cell functionality of our gyrBA fusion. Previously, catalytic activity and in vivo complementation have been shown for an E. coli ParEC fusion (9). Recently, a naturally occurring type II topoisomerase (topoisomerase VIII) was reported, in which the A and B subunits were fused into a single polypeptide (10).

In this study, the gyrB and gyrA genes from E. coli J53 (11) were amplified and fused by overlap extension PCR such that the coding region for GyrB (amino acids 1 to 803) was fused to the coding region for GyrA (amino acids 1 to 874) via a Gly-Ala-Pro (GAP) linker and then cloned into BamHI and XhoI sites of pET28a. Individual gyrA and gyrB subunits were similarly amplified and cloned into pET28a via BamHI and XhoI sites. pET28a-gyrBA was introduced into E. coli BL21(DE3) and expressed after growth to an optical density at 600 nm (OD₆₀₀) of 1.2 with vigorous shaking at 37°C followed by induction with 1 mM IPTG (isopropyl-beta-d-thiogalactopyranoside) (Sigma-Aldrich, USA) and further incubation for 2 h at 30°C. The cell pellets were harvested by centrifugation at 4,000 × g for 30 min, resuspended in buffer A (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl), and disrupted by sonication. The extract was centrifuged at 13,800 × g for 30 min at 4°C, and the supernatant was mixed with HIS-Select nickel affinity gel (Sigma-Aldrich, USA) for affinity purification (12, 13). Fractions were eluted with increasing concentrations of imidazole (Sigma-Aldrich, USA). On analysis by Novex NuPAGE SDS-PAGE gel, a single polypeptide between 160 and 220 kDa was observed, consistent with expression of the fusion protein. Proteins were dialyzed against buffer B (50 mM Tris-HCl, pH 7.5, 50 mM NaCl) and stored at −80°C in buffer B supplemented with 50% glycerol.

Evidence of the catalytic activity of GyrBA was obtained by examining its effects on DNA topology. DNA supercoiling assays were performed using E. coli gyrase assay kits (New England BioLabs, USA) according to the manufacturer's instructions. On DNA supercoiling activity, 25 μM ciprofloxacin (MP Biomedicals, France) was added to evaluate its inhibitory effect. DNA cleavage assays were performed to assess the interaction between GyrBA and quinolone as previously described (12). DNA substrates, relaxed pHOT-1 DNA and negatively supercoiled pHOT-1 DNA, were purchased from TopoGEN (USA).

Like tetrameric wild-type (WT) DNA gyrase, GyrBA catalyzed ATP-dependent negative supercoiling activity, and this activity was inhibited by ciprofloxacin (Fig. 1A). GyrBA also generated linear DNA cleavage complexes when incubated in the presence of ciprofloxacin followed by a denaturing agent (Fig. 1B). Cleavage complex formation occurred in the absence of ATP. The catalytic activity of the GyrBA protein in the supercoiling reaction was 2-fold lower than that of wild-type DNA gyrase, while its cleavage ability was 25-fold higher than that of wild-type DNA gyrase (Fig. 1). Thus, the purified E. coli GyrBA fusion protein has catalytic function as an active topoisomerase, and it retains sensitivity to quinolone inhibition of negatively supercoiling activity and the ability to form quinolone-mediated cleavage complexes.

FIG 1.

GyrBA catalytic activities and interactions with ciprofloxacin. (A) ATP-dependent DNA supercoiling by GyrBA and gyrase and inhibition by ciprofloxacin. Lane 1 shows negatively supercoiled (SC) pHOT-1 at 0.4 μg. Lanes 2 to 12 show relaxed pHOT-1 at 0.4 μg, with GyrBA at 10 ng (lane 3), 20 ng (lane 4), 40 ng (lane 5), 80 ng (lanes 6 to 7), and 25 μM ciprofloxacin (lane 7). Lanes 8 to 11 show gyrase at 10 ng (lane 8), 20 ng (lane 9), 40 ng (lane 10), 80 ng (lane 11 to 12), and 25 μM ciprofloxacin (lane 12). (B) DNA cleavage by GyrBA and gyrase in the presence of ciprofloxacin. Lane 1 shows relaxed pHOT-1 at 0.4 μg. Lanes 2 to 12 show negatively supercoiled (SC) pHOT-1 at 0.4 μg, with 25 μM ciprofloxacin (lanes 3 to 6) and GyrBA at 17.4 ng (lane 3), 43.5 ng (lane 4), 87 ng (lane 5), and 130.5 ng (lane 6) and with GyrBA at 130.5 ng without ciprofloxacin (lane 7). Lanes 8 to 11 show 25 μM ciprofloxacin (lanes 8 to 11) and gyrase at 174 ng (lane 8), 435 ng (lane 9), 870 ng (lane 10), 1,300 ng (lane 11), and gyrase at 1,300 ng without ciprofloxacin. (According to the manufacturer's instruction, 1 unit of DNA gyrase is 174 ng.) Rel, NC, L, and SC indicate relaxed, nicked circular, linear, and supercoiled forms of pHOT-1, respectively. (A) Intensities of supercoiled (SC) bands in lanes 4 and 8 are similar. (B) Intensities of linear (L) bands in lanes 3 and 9 are similar.

Temperature-sensitive gyrase subunit mutants of E. coli, KNK453 [gyrA(Ts)] and N4177 [gyrB(Ts)] were used to test the whole-cell functions of gyrBA. These strains grow at 30°C but fail to grow at 42°C (14, 15). Plasmids pET28a, pET28a-gyrA, pET28a-gyrB, and pET28a-gyrBA were introduced into KNK453 and N4177 by electroporation. The growth of KNK453 or N4177 and their transformants was tested by spotting cultures on LB plates containing 50 μg/ml kanamycin (Sigma-Aldrich, USA) and 0.5 mM IPTG as needed. The plates were incubated overnight at 30°C or 42°C.

The ability of N4177 [gyrB(Ts)] to grow at 42°C was rescued in the presence of pET28a-gyrB and pET28a-gyrBA but not by pET28a or pET28a-gyrA. Similarly, the growth of KNK453 at 42°C was complemented only by pET28a-gyrA or pET28a-gyrBA (Table 1). Notably, gyrA complementation was observed only after induction of gyrA or gyrBA expression with 0.5 mM IPTG. These results indicate that gyrBA encodes a functional gyrase in vivo.

TABLE 1.

E. coli gyrBA fusion complements temperature-sensitive growth of E. coli KNK453 [gyrA(Ts)] and N4177 [gyrB(Ts)]

Temperaturea (°C)	KNK453 [gyrA(Ts)]b,c				N4177 [gyrB(Ts)]c
	pET28a	pET28a-gyrB	pET28a -gyrA	pET28a -gyrBA	pET28a	pET28a-gyrA	pET28a-gyrB	pET28a- gyrBA
30	+	+	+	+	+	+	+	+
42	−	−	+	+	−	−	+	+

^aA temperature of 30°C is the permissive temperature, and 42°C is the restrictive temperature for E. coli strains KNK453 and N4177.

^bFor KNK453, 0.5 mM IPTG was added in LB plates to induced gyrA and gyrBA.

^c+, Robust growth; −, no growth.

Amino acid substitution S83L in GyrA causes decreased susceptibility to fluoroquinolones in E. coli (16). To test the interaction between quinolones and GyrBA in vivo, MICs of J53 (gyrA S83L), wild-type J53, and their transformants were tested by broth microdilution. J53 (gyrA S83L) with plasmids pET-gyrA or pET-gyrBA carrying a susceptible gyrA gene allele reduced quinolone MICs by 2-fold to 16-fold, but plasmid pET28 and pET-gyrB did not affect the MICs. None of the four plasmids affected the MICs of WT E. coli J53 (Table 2). These results show that in J53 (gyrA S83L), a wild-type gyrA gene on plasmids is dominant over the resistant gyrA S83L chromosomal allele as previously reported (17), and plasmid-borne gyrBA behaved similarly. Thus, GyrBA interacts with quinolones similarly to wild-type gyrase in vivo as well as in vitro.

TABLE 2.

E. coli gyrBA fusion reduces quinolone MICs for E. coli J53 (gyrA S83L)

Host cells	Plasmid	MIC (μg/ml) for:
		Ciprofloxacin	Norfloxacin	Nalidixic acid
E. coli J53 (gyrA S83L)	None	0.5	1	4,096
	pET28a	0.5	1	4,096
	pET28-gyrB	0.5	1	4,096
	pET28-gyrA	0.25	0.5	512
	pET28-gyrBA	0.125	0.25	256
Wild-type E. coli J53	None	0.016	0.06	8
	pET28a	0.016	0.06	8
	pET28-gyrB	0.016	0.06	8
	pET28-gyrA	0.016	0.06	8
	pET28-gyrBA	0.016	0.06	8

QnrB was previously shown to protect E. coli DNA gyrase from inhibition by quinolones in vitro (5). To test the ability of QnrB to counteract quinolone inhibition of GyrBA, pTrcHisA-qnrB1 or pTrcHisA was introduced into E. coli J53 gyrA S83L containing pET28a, pET28a-gyrB, pET28a-gyrA, or pET28a-gyrBA, and quinolone MICs of these strains were determined. In the absence of QnrB, quinolone susceptibility increased when a sensitive GyrA was present, since quinolone sensitivity dominates resistance (17). The presence of qnrB caused an increase in MICs of ciprofloxacin and norfloxacin (Sigma-Aldrich, USA) in E. coli J53 gyrA S83L containing pET28a, pET28a-gyrBA, pET28a-gyrA, and pET28a-gyrB, with higher increments in the presence of pET28a-gyrBA or pET28a-gyrA, which themselves generated greater susceptibility over that of the resistant chromosomal gyrA allele (Table 3). Thus, qnrB can cause increased resistance in the presence of dominant gyrBA or wild-type gyrA.

TABLE 3.

qnrB protects gyrBA from ciprofloxacin and norfloxacin inhibition in E. coli J53 (gyrA S83L)

Host cells	Plasmid	MIC (μg/ml) for:
		Ciprofloxacin		Norfloxacin
		pTrcHisA	pTrcHisA-qnrB1	pTrcHisA	pTrcHisA-qnrB1
E. coli J53 (gyrA S83L)	None	0.5	1	1	2
	pET28a	0.5	1	1	2
	pET28a-gyrB	0.5	1	1	2
	pET28a-gyrA	0.25	1	0.5	2
	pET28a-gyrBA	0.125	1	0.25	2

To demonstrate further that QnrB protects from quinolone inhibition by targeting plasmid-expressed GyrBA, we tested ciprofloxacin MICs of E. coli temperature-sensitive strains N4177 and KNK453 containing pET28a, pET28a-gyrB, pET28a-gyrA, or pET28a-gyrBA at 30°C and 42°C. pET28a-gyrB and pET28a-gyrBA complemented the growth of N4177 at 42°C, and the MICs of ciprofloxacin in N4177 containing pET28a-gyrB and pET28a-gyrBA were increased by about 16-fold in the presence of QnrB (Table 4). Similar results were observed in KNK453 (Table 4). These results are consistent with the interaction of QnrB with GyrBA as occurs with tetrameric gyrase.

TABLE 4.

qnrB protects gyrBA from ciprofloxacin inhibition in N4177 and KNK453

Host cells	Plasmid	MIC (μg/ml) for:
		30°C		42°C
		pTrcHisA	pTrcHisA-qnrB1	pTrcHisA	pTrcHisA-qnrB1
E. coli N4177	None	0.008	0.25
	pET28a	0.008	0.25
	pET28a-gyrA	0.008	0.25
	pET28a-gyrB	0.008	0.25	0.016	0.25
	pET28a-gyrBA	0.008	0.25	0.016	0.25
E. coli KNK453a	None	0.06	1
	pET28a	0.06	1
	pET28a-gyrB	0.06	1
	pET28a-gyrA	0.06	1	0.06	1
	pET28a-gyrBA	0.06	1	0.06	1

^aIPTG 0.5 mM was added to induce GyrA and GyrBA expression in E. coli KNK453.

Our studies demonstrate that the GyrBA fusion protein functions as an active topoisomerase in vivo and in vitro. In addition, the fusion protein interacts with quinolones and QnrB similarly to wild-type gyrase. Thus, this functional construct allows studies of the physical interactions of gyrase-binding proteins, such as Qnr, with gyrase holoenzyme in addition to studies with individual but catalytically inactive GyrA and GyrB subunits.