Pentapeptide repeat proteins QnrB1 and AlbG require ATP hydrolysis to rejuvenate poisoned gyrase complexes

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of “poisons”, namely natural product toxins (e.g. albicidin. microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We compared activities of two such proteins, QnrB1 and AlbG in vitro. Each of them provided specific protection against its cognate toxin (fluoroquinolone or albicidin), which strictly required ATP hydrolysis by gyrase. Through a combination of fluorescence anisotropy, pull-downs and photocrosslinking we show that QnrB1 binds to the GyrB protein. We further probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped strong and specific crosslinks to the N-terminal ATPase/transducer domain. We propose a model in which protective PRPs bind to the enzyme as T-segment DNA mimics to promote dissociation of the bound poison molecule.

have been proposed to explain the protective effects of QnrB1 and related PRPs. The righthelical shape and stretches of negative charge along the length of MfpA and other PRPs are similar to that of double-stranded DNA, leading to the idea that PRPs can act as G-segment mimics (20). In this concept, as a result of competition between the PRP and G-segments for gyrase binding, there would be fewer complexes with DNA present, hence fewer lethal double stranded DNA breaks. In agreement with this hypothesis, MfpA was shown to inhibit gyrase in vitro (20,25) and Qnr protein was shown to reduce DNA binding to gyrase in a filter assay (26). However, later it was shown that QnrB1 does not inhibit gyrase supercoiling activity, at least at the concentrations required for rescue (21,22,25). This observation is incompatible with a model in which PRPs compete with G-segment binding, as clearly supercoiling cannot proceed without G-segment present. This led to the idea of a direct recognition of the gyrasedrug complex by the PRP, which results in loss of the drug from gyrase (21). These observations and apparent DNA-mimicry can be reconciled by the proposed "T-segment mimicry" model, where the transport of the PRP through the enzyme destabilises the enzymedrug complex and allows for dissociation of the drug from the enzyme (27).
In order to clarify the mechanisms whereby PRPs protect gyrase from poisons, we have
Plasmids pET28-albG (encoding AlbG with a thrombin-cleavable His-tag, (13), pBAD-mcbABCDEFG (wt MccB17 operon) and pET28-qnrB1 (encoding QnrB1 with a C-terminal 6xHis tag) are gifts from Dr. Mikhail Metelev (Uppsala University). Plasmids pET21-GyrA and pET21-GyrB coding for full-length untagged GyrA and GyrB subunits amplified from E. coli MG1655 by PCR and cloned between NdeI and BamHI sites were previously produced in the laboratory. Plasmids pET21-3xFLAG-GyrB and pET21-GyrA-FLAG were constructed by amplifying GyrA and GyrB genes (as above) with N-terminal (GyrB) 3xFLAG tag and Cterminal (GyrA) FLAG tags and cloning using Nde I and Xho I sites into pET21. pET28-GyrB47 was constructed by amplifying coding sequence for gyrase TOPRIM domain (393-804) from E. coli MG1655 and cloning it into pET28 using Nde I and Xho I restriction sites behind the N-terminal hexahistidine tag. pAJR10.18 (GyrA59) and pAJ1 (GyrB43) were gifts of A. Maxwell, John Innes Centre. For pBAD-albG, pBAD-mcbG and pBAD-qnrB1, corresponding genes were amplified from the corresponding template plasmids and cloned into pBAD vectors using Nco I and Xho I restriction sites.

Purification of gyrase subunits and domains
E. coli full-length GyrA and GyrB subunits were purified similarly to (29). Plasmids pET21b containing corresponding genes were transformed into BL21 (DE3) Gold (Agilent). For GyrA, 2 L of TB culture supplemented with 100 µg/ml ampicillin was incubated at 37°C with shaking to OD600 = 0.75, induced with 500 µM isopropyl-β-d-thiogalactopyranoside (IPTG) and incubated for a further 4 hours at 37°C. Cells were harvested by centrifugation at 6000 g for 15 minutes at 4°C. Pellets were resuspended in buffer A (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM DTT) and supplemented with protease inhibitor cocktail (Pierce). Cells were lysed using a French press and cell debris removed by centrifugation at 40000 g for 20 minutes at 4°C. Clarified lysate was loaded onto 10 ml Q XL column (GE Healthcare) in buffer A, washed with 10 CV of buffer A, 10 CV of buffer A supplemented with 0.1 M NaCl and eluted with gradient (0.1-1 M) of NaCl in buffer A over 5 CVs. Collected fractions were dialysed into buffer A overnight at 4°C and further purified by MonoQ HR 16/10 (20 ml) using the same method as described for Q XL. Peak fractions were pooled together, concentrated using Amicon concentrators (Millipore) and loaded onto a Superdex 200 Increase 10/300 (Cytiva) column equilibrated in buffer A. Fractions containing GyrA were aliquoted, frozen in liquid nitrogen and stored at -80°C in buffer A. For GyrB, 2 L of TB culture was grown at 37°C with shaking to OD600 = 0.8, induced with 500 µM IPTG and incubated for a further 3 hours at 28°C. Cells were lysed and processed in buffer A as described for GyrA and protein was purified using heparin affinity (Heparin FF 16/10 column) and anion exchange (MonoQ HR 16/10 column) chromatography (Cytiva), eluting with a 0-1 M gradient of NaCl.
Peak fractions from MonoQ were pooled, dialysed into buffer A, aliquoted and frozen at -80°C.
FLAG-tagged versions of both GyrA and GyrB were purified similarly.
GyrB 43 kDa domain (GyrB43) was purified similarly to (30). The plasmid pAJ1 (31) was transformed into BL21 (DE3) Gold cells. 1 L culture of TB inoculated with transformed cells was incubated at 37°C with shaking to OD600 = 0.8 after which it was induced with 500 µM IPTG. The temperature was reduced to 25°C and the culture was incubated with shaking overnight. Cells were harvested and processed similarly to GyrA and GyrB proteins. GyrB43 was first purified on Q XL similarly to GyrA and B proteins. Fractions containing protein of interest were combined and ammonium sulfate was added to 1.5M. Salt-adjusted protein was loaded to 10 ml Phenyl Sepharose HS FF (Cytiva) column equilibrated in 1.5M (NH4)2SO4 and protein was eluted by 20 CV gradient from 1.5 M to 0 (NH4)2SO4. Collected fractions were pooled together, dialysed overnight in buffer A and loaded onto a 20 ml MonoQ column as described for GyrA and GyrB proteins. Peak fractions from MonoQ were pooled, dialysed overnight into buffer A, concentrated to 10 mg/ml, aliquoted, frozen in liquid N2 and stored at -80°C.
GyrB47 purification was described in (32). 2 L culture of BL21 (DE3) Gold pET28-GyrB47 was grown in TB at 37°C. At OD600 = 0.75 IPTG was added to 0.5 mM and cells were grown for further 4 hours at 37°C. After harvesting, cells were resuspended in a lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 20 mM imidazole, 10% glycerol) and lysed using a French press. Cleared lysate was loaded on a pre-equilibrated 5 ml HisTrap FF column (Cytiva), washed with the lysis buffer and eluted with the lysis buffer containing 250 mM imidazole.
Peak fractions were dialysed overnight into buffer A and further purified on a 5 ml Q HP column (Cytiva). A step gradient of NaCl was used and protein eluted at 40% NaCl.
GyrA59 was purified similarly to (33). Plasmid pAJR10.18 was transformed into BL21(DE3) Gold cells. 2 L of culture was grown in LB at 37°C to OD600 = 0.85, induced with 0.5 mM IPTG, and grown for further 4 hours at 37°C. Cells were lysed by sonication in buffer A. Lysate was cleared by centrifugation at 87 000 g for 30 minutes at 4°C and GyrA59 was purified first on 16/10 Heparin FF (Cytiva) column using 0.1-1 M gradient of NaCl in buffer A. After overnight dialysis to buffer A, GyrA59 was further purified on MonoQ (16/10, Cytiva) using a gradient 0-0.7M NaCl over 6 CV. Fractions containing GyrA59 were pooled, concentrated and loaded onto Superdex 200 16/600 gel filtration column. Pure protein, concentrated to ~5 mg/ml was aliquoted, flash-frozen in liquid N2 and stored at -80°C.

AlbG purification
Plasmid pET28-AlbG containing the albG open reading frame with a thrombin-cleavable Nterminal hexahistidine tag was transformed into BL21 (DE3) Gold cells. 3 L of 2xYT liquid media supplemented with 30 µg/ml kanamycin were inoculated using 1:100 ratio of starter overnight culture and incubated at 37°C with shaking to OD600 = 0.8. After induction with 500 µM IPTG the temperature was reduced to 24°C and the cultures were incubated overnight with shaking. Cells were harvested by centrifugation at 7000 g for 30 minutes at 4°C. Pellets were resuspended in AlbG lysis buffer (50 mM Tris-Cl pH 8, 20 mM imidazole, 300 mM NaCl, 5% glycerol) supplemented with protease inhibitors (Pierce). Resuspended cells were incubated for 30 minutes on ice with 2 mg/ml lysozyme and 5 μg/ml DNase I. Cells were lysed using a French press and lysate was cleared by centrifugation at 87 000 g for 30 minutes at 4°C. AlbG was purified by Ni-affinity chromatography (5ml HisTrap HP, Cytiva), dialysed overnight into buffer A and further purified by anion exchange (Q XL 5 ml, Cytiva) using step elution by increasing concentrations of NaCl. Peak fractions eluted between 0.2-0.3 M NaCl were dialysed into buffer A, concentrated and stored at -80 C.

QnrB1 purification
Plasmid pET28-QnrB1 containing the qnrB1 open reading frame with C-terminal hexahistidine tag was transformed into BL21 (DE3) Gold cells 3 L of TB liquid media supplemented with 30 µg/ml kanamycin were inoculated with1/100 ratio of starter overnight culture and incubated at 37°C with shaking to OD 600 = 0.8. After induction with 500 µM IPTG the temperature was reduced to 24°C and the cultures were incubated overnight with shaking. Cells were harvested by centrifugation at 7000 g for 30 minutes at 4°C. Pellets were resuspended in QnrB1 lysis buffer (50 mM Tris-Cl pH 8.0, 200 mM (NH4)2SO4, 10% glycerol, 20 mM imidazole) supplemented with protease inhibitors (Pierce). Resuspended cells were incubated for 30 minutes on ice with 1 mg/ml lysozyme with periodic mixing. Cells were lysed using a French press and lysate was cleared by centrifugation at 87 000 g for 30 minutes at 4°C. QnrB1 was purified by Ni-affinity chromatography (HisTrap HP 5 ml, Cytiva). The column was Peak fractions eluted by 0-1 M NaCl gradient were pooled, concentrated, and loaded onto a 10/300 Superdex S75 Increase SEC column (Cytiva) previously equilibrated with QnrB1 storage buffer (20 mM Tris pH 7.5, 50 mM NaCl, 5% glycerol, 50 mM arginine hydrochloride, 2 mM DTT). Peak fractions were concentrated, snap-frozen in liquid N2 and stored at -80°C.

Purification of QnrB1 para-benzoyl-phenylalanine (pBpa) mutants
Plasmids pBAD-qnrB1[x]pBpa, carrying qnrB1 amber mutants with N-terminal 6xHis, were transformed into One Shot BL21 Star (DE3) cells (Thermo) together with the pEVOL-pBpa plasmid (34). 100 ml of LB liquid media supplemented with 30 µg/ml kanamycin and 100 µg/ml ampicillin was inoculated using a 1:100 ratio of starter overnight culture and incubated at 37°C with shaking to OD600 = 0.3. At this point para-benzoyl-phenylalanine (pBpa) was added to a final concentration of 1 mM. The incubation was continued until the culture reached OD600 = 0.6. The expression of orthogonal aaRS was induced by the addition of arabinose (10 mM). After 6 h of expression at 37°C with shaking, the cells were collected by centrifugation at 7000 g for 30 minutes at 4°C. Pellets were resuspended in QnrB1 lysis buffer (50mM Tris-Cl pH 8.0, 200 mM (NH4)2SO4, 10% glycerol, 20 mM imidazole) supplemented with protease inhibitors (Pierce). Resuspended cells were incubated with mixing for 30 minutes on ice with 1 mg/ml lysozyme. Cells were lysed by sonication and lysate was cleared by centrifugation at 87 000 g for 30 minutes at 4°C. Lysate was loaded onto a Ni 2+ -affinity chromatography column

Purification of gyrase-targeting toxins
Microcin B17 was obtained from Escherichia coli BW25113 transformed with pBAD-mcbABCDEFG plasmid using a method described previously (35). Briefly, transformants were cultured in 2 L of 2xYT media until OD600 = 0.7, induced with 10 mM arabinose and left overnight at 37°C. Cells were collected by centrifugation at 4000 g, resuspended in 100 mM acetic acid/1 mM EDTA solution and lysed by boiling for 10 min on a water bath. Lysate was clarified by centrifugation (12 000g) and loaded onto Agilent BondElute 10 g C18 cartridge, equilibrated with 0.1% TFA. Cartridge was extensively washed first with 20 CV 0.1% TFA, then with 20 CV of 10% MeCN in 0.1% TFA before elution with 30% MeCN/0.1% TFA.

MIC measurements
Minimal inhibitory concentrations of compounds were measured by broth microdilution in 96well plates as described by the Clinical & Laboratory Standards Institute (37). All measurements were performed as triplicates. The error is expressed by standard deviation of the mean.

Gyrase activity assays
For supercoiling assays, 1 unit of E.coli gyrase (1 unit defined as the amount of gyrase required for full conversion of 500 ng relaxed pBR322 DNA into the completely supercoiled form in 30 min at 37°C in a 30 µl reaction, corresponded to 4 nM concentration) was incubated in a total volume of 30 μl in assay buffer (35 mM Tris-Cl pH 7.5, 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5 % (w/v) glycerol, 0.1 mg/ml albumin) and 500 ng relaxed pBR322 DNA (Inspiralis Ltd.). The compounds and/or proteins were replaced by corresponding buffers in control reactions. Reactions were stopped by the addition of chloroform:isoamyl alcohol (24:1) and STEB (20% sucrose, 50 mM Tris-Cl pH 8, 5 mM EDTA, 0.25 mg/ml bromophenol blue). The aqueous layers from the assays were run on 1% agarose TAE gels at 80 V for 2.5 hours in 1 x TAE buffer. Once complete, the gels were stained with 10 μg/ml ethidium bromide solution (Sigma) for 15 minutes and de-stained with 1 x TAE buffer for 15 minutes and visualised using a gel documentation system (UVP). Quantification of the amount of supercoiled DNA was performed using Fiji software (38). Percentage of supercoiled DNA was plotted against QnrB1 concentration and the data was fitted to the equation: % = [ 1] , where a and b are function parameters found after fitting the function using Origin (Pro), Version 2020 b . IC50 (concentration of QnrB1 required for 50% supercoiling inhibition). Gyrase relaxation assays were carried out in a similar manner, but ATP and spermidine were omitted and ~5 U of gyrase were used. For gyrase cleavage assays ~5 U of gyrase were used and reactions were terminated by the addition of 0.2% sodium dodecyl sulphate (SDS) and 0.2 mg/ml proteinase K, followed by incubation for 30 minutes at was replaced with CaCl2. The buffer was supplemented with 1 mM ATP or 0.5 mM ADPNP as required. When tested compounds and/or proteins were absent in control reactions, they were replaced by DMSO or corresponding buffers. At selected time points, 20 µl aliquots were withdrawn and stopped by addition of 2 µl 5% SDS and 2 µl 250 mM EDTA. After the time course, collected samples were treated with proteinase K (0.2 mg/ml) for 30 minutes at 37°C and extracted by chloroform:isoamyl alcohol (24:1). The aqueous layers from the assays were mixed with STEB and run on 1% agarose TAE gels at 80 V for 2.5 hours in 1 x TAE buffer.
Once complete, the gels were stained with 10 μg/ml ethidium bromide solution (Sigma) for 15 minutes and de-stained with 1xTAE buffer for 15 minutes and visualised using a gel documentation system (UVP).
Cleavage complex stability assays were performed as follows: an initial 60 µl reaction was set up using 80 units of gyrase in the assay buffer, 50 nM of relaxed DNA and 20 µM CFX. which was incubated at 37°C for 10 minutes. Then 20 ul aliquots were withdrawn and diluted 20-fold with the buffer supplemented with 5 µM QnrB1/ 5 µM AlbG and/or 0.5 mM ATP as required.
For AlbG containing assays 1 µM of albicidin was used to form a starting complex. At chosen time points 20 µl from each reaction was pipetted to a separate tube and stopped by adding 2 µl of 5% SDS and 2 µl 250 mM EDTA. After the time course, samples were treated with proteinase K (0.2 mg/ml) for 30 minutes at 37°C, then a chloroform-isoamyl alcohol mixture (24:1) and STEB (20% sucrose, 50 mM Tris-Cl pH 8, 5 mM EDTA, 0.25 mg/ml bromophenol blue) were added. The aqueous layers from the assays were run on 1% agarose TAE gels at 80 V for 2.5 hours in 1 x TAE buffer. Once complete, the gels were stained with 10 μg/ml ethidium bromide solution (Sigma) for 15 minutes and de-stained with 1 x TAE buffer for 15 minutes and visualised using a gel documentation system (UVP).

Electrophoretic mobility shift assays (EMSA)
147 bp pBR322 dsDNA fragment with known strong gyrase binding site (39) was produced by PCR and purified with Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro kit. 20 nM of the fragment was mixed with 0.2 µM of reconstituted gyrase in EMSA buffer (30 mM Tris-Cl pH 7.5, 75 mM KCl, 6% glycerol, 2 mM MgCl2, 1 mM DTT). QnrB1 or AlbG were added as indicated. In control reactions, QnrB1/AlbG were supplemented by their storage buffers added. Reactions were incubated for 30 min at 25°C and run on 6% polyacrylamide gels in TBM buffer (90 mM Tris-borate, pH 7.5, 4 mM MgCl2) at 150V at room temperature.
After the run gels were stained with SYBR Gold (Thermo) for 20 mins and visualized under UV light.

ATPase assays
Gyrase and GyrB 43 ATPase assays were carried out using Inspiralis kits according to the protocol provided by Inspiralis Ltd based on Tamura & Gellert (40). Each reaction contained 50 mM Tris.HCl (pH 7.5), 1 mM EDTA, 5 mM magnesium chloride, 5 mM DTT, 10 % (w/v) glycerol, 0.8 mM PEP, 0.4 mM NADH and ~1U of PK/LDH mix (Sigma). For GyrB43 assays, the concentration of GyrB43 was 4 µM. For gyrase assays, 50 nM gyrase tetramer (A2B2) was used. Linear pBR322DNA (Inspiralis) was used at 10.5 nM where indicated. Assays were performed in microtitre plates with a reaction volume of 100 µl. The absorbance at 340 nm was measured continuously in a plate reader(SpectraMAX190, Molecular Devices) and used to evaluate the oxidation of NADH (using an extinction coefficient of 6.22 mM -1 cm -1 ), which relates stoichiometrically to the production of ADP.The results were fitted to a Michaelis -

Menten plot according to equation
using Origin (Pro), Version 2020 b, (OriginLab Corporation). Novobiocin (50 µM) was used as a control for gyrase-independent ATPase activity, which was found negligible.

Fluorescence anisotropy measurements
QnrB1 was N-terminally labelled with AlexaFluor 488-carboxylic acid-2,3,5,6tetrafluorophenyl ester-5 isomer (5-TFP, Thermo), according to a procedure described for YacG protein (32). Briefly, purified QnrB1-His was exchanged to amino labelling buffer ( GyrB dimerisation and the reaction was pre-incubated at RT together with a control reaction. An equal amount of equilibrated FLAG M2 agarose (Sigma) was added to each reaction. After addition of QnrB1, reactions were incubated for a further 60 min at RT with gentle shaking.

In vivo benzoyl-phenylalanine (pBpa) -driven UV crosslinking
E. coli GyrA-SPA and GyrB-SPA were co-transformed with pEVOL-pBpa plasmid and pBAD-QnrB1[x]pBpa plasmid containing appropriate amber codon substitutions. Cells were grown at 37°C with shaking in LB medium supplemented with antibiotics and 1 mM pBpa.
Protein expression was induced by addition of 10 mM arabinose at OD600 = 0.6 and culture was allowed to grow for a further 3 hours. After that time cells were centrifuged (5000 g, 10 min), the medium was removed and cells were resuspended in phosphate buffered saline (PBS,

Pentapeptide repeat proteins offer specific protection against their cognate toxins in vivo
To compare protective activities of different PRPs in identical conditions, we created a set of To investigate the biochemical basis of protection offered by PRPs, we purified hexahistidinetagged QnrB1 and AlbG (we were unable to purify functional McbG in sufficient quantity and purity) and first tested their activities in gyrase supercoiling assays. Similar to previous reports (26), QnrB1 provided limited protection (supercoiling was not completely restored) against 5 μM CFX ( Figure 1C). Calculated EC50QnrB1 (concentration of QnrB1 required to observe half of maximum protective effect) was 0.2 μM. Complete rescue was observed when CFX concentration was lowered to 1 µM. As can be seen from the same figure, high concentrations of QnrB1 (>10 µM) were found to inhibit gyrase activity, and this high concentration inhibitory effect was also observed without CFX present (Supplementary Figure S1A). Calculated IC50 QnrB1 was 11 µM, >50 times higher than EC50. The abovementioned Δ107-109 mutant of QnrB1 (QnrB1 ΔTTR) showed the same level of supercoiling inhibition as WT QnrB1 but was unable to rescue gyrase supercoiling inhibited by CFX. (Supplementary Figure S1BC).
When tested against albicidin, AlbG provided a protective effect (supercoiling was restored) over a wide range of concentrations ( Figure 1D) with EC 50 AlbG=1.2 μM. In contrast to QnrB1, AlbG did not inhibit E. coli gyrase at any concentration tested (up to 50 µM) ( Figure 1E). Akin to the in vivo results, we saw no protection when we swapped the inhibitors and tested protection by QnrB1 against ALB or protection by AlbG against CFX (Figure 1EF). Due to the lack of inhibition by AlbG, we wondered whether the mechanism of gyrase protection might be different (e.g. not involving gyrase, such as sequestering of albicidin by AlbG).
However, incubation of albicidin with purified AlbG did not show any signs of inactivation of the toxin (Supplementary Figure S2).
It was previously shown that QnrB1 reduced the amount of DNA gyrase complexes with cleaved DNA (DNA cleavage) stabilised by CFX (26). We have confirmed this result, showing that in the cleavage assay, the amount of linear DNA in the presence of 5 µM QnrB1 is decreased ~50% across concentrations of ciprofloxacin tested (up to 20 µM) (Figure 2A, C).
Inhibition of FQ-induced cleavage was also observed when negatively supercoiled DNA was used as a substrate, but the effect was weaker (Supplementary Figure S3AB).
AlbG similarly reduced the amount of linear DNA formed in the presence of albicidin ( Figure   2DE. However, in contrast with QnrB1, here the magnitude of the effect faded with increasing concentration of albicidin, vanishing at [ALB] >15 µM ( Figure 2EF). As for supercoiling assays, QnrB1 and AlbG showed no cross-protection towards ALB and CFX (not shown).
As GyrB ATPase domains and GyrA CTDs are not required for FQ-stimulated cleavage, we have produced different previously reported truncated gyrase complexes: GyrA2592/B2 andGyrA2/B472 and tested if DNA cleavage by these complexes(using negatively supercoiled DNA as a substrate) can be inhibited by QnrB1. GyrA592/B2 complex lacks DNA-wrapping domains (GyrA CTDs) but can relax negatively supercoiled DNA in the presence of ATP, similarly to Topo IV (42). In contrast, GyrA2/B472 lacks the GyrB43 ATPase-transducer domain but is still capable of ATP-independent relaxation (43). No protective effect of QnrB1 was seen in cleavage assays with any of these enzymes apart from A592/B2 (Supplementary Figure 3CD), where the amount of linear DNA trapped by CFX was observed to decrease in the presence of 5 µM QnrB1. Therefore, we assumed that DNA wrapping is not required for the QnrB1 to act, but the ATPase domain is indispensable.
To further investigate the potential role of DNA wrapping, we tested short linear DNA fragments (76, 100, 133, 147, 220 or 300 bp) as a substrate for full-length gyrase cleavage (Supplementary Figure S4); again, reduction of cleavage was clearly visible even with the shortest fragment tested, meaning that QnrB1 indeed does not require a DNA node to act.

QnrB, but not AlbG decreases DNA binding to gyrase
The observed specific protective action of both AlbG and QnrB1 seems inconsistent with the  we performed cleavage assays without a nucleotide, we found that the CFX-induced cleavage was not reversed by QnrB1 (Supplementary Figure S6A). Similar experiments have been done with albicidin, where we tested a range of poison concentrations; again, no protection was observed (Supplementary Figure S6B). Therefore, in order for PRPs to act, they require either the dimerization of the ATPase-gate following ATP binding, or ATP hydrolysis and subsequent "resetting" (44) of the enzyme. To choose between these scenarios for QnrB1, we performed time-course experiments to closely monitor CFX-dependent cleavage complex formation in three different conditions: in the presence of ATP, its non-hydrolysable analogue ADPNP or in the absence of nucleotide ( Figure 3AB). To give QnrB1 an opportunity to bind to the gyrase before N-gate dimerization, nucleotides were added last to the mixture, following preincubation of gyrase and QnrB1. Addition of QnrB1 had no effect on cleavage in case of ADPNP or when nucleotide was omitted; however, in the presence of ATP, a decrease in linear DNA was readily observed, allowing more complete supercoiling in the presence of QnrB1.
The negative result with ADPNP suggests that N-gate dimerization by itself is not sufficient for QnrB1 to inhibit cleavage.
If PRPs merely prevent cleavage complex formation (based on G-segment mimicry or similar mechanism), they must interact with the gyrase before the G-segment is cleaved and covalently bound to the enzyme. Conversely, if PRPs actively promote dissociation of the drugs and DNA re-ligation, they should be able to revert already existing cleavage complexes. We tested if QnrB1 can destabilise pre-formed cleavage complexes, consisting of gyrase, DNA and CFX. Figure 3C shows that in the presence of ATP and QnrB1, the pre-formed cleavage complex was quickly dissociated whereas without QnrB1, the complex was stable for at least 2 hours.
No dissociation was observed without the nucleotide or with ADPNP (not shown). Thus, QnrB1 is able to interact with gyrase after cleavage complex formation to rescue the stalled enzyme at the stage after DNA binding, and this process requires ATP hydrolysis. We have repeated the experiments with the AlbG/albicidin pair and observed the similar effect, but albicidin cleavage complexes appeared more stable in agreement with higher potency of ALB ( Figure 3D).

Protective effects of QnrB1 and AlbG do not depend on strand passage
We showed that the inhibition of cleavage complex formation by QnrB1 and AlbG depends on ATP hydrolysis by gyrase. Two possible mechanisms can explain such behaviour: either PRPs require the energy of ATP hydrolysis and associated large-scale conformational changes to achieve removal of bound drug, or ATP-driven strand passage provides PRPs with access to a temporarily exposed drug binding site within the enzyme. The strand passage requirement was suggested previously for MccB17 and gyrase-binding toxin CcdB (40,41). In these two cases, strand passage, occurring during ATP-independent relaxation of negatively supercoiled DNA, allowed for binding of toxins. We have investigated if ATP-independent relaxation of negatively supercoiled DNA, inhibited by CFX or albicidin, can be rescued by QnrB1 or AlbG.
We saw no protection in both cases (Supplementary Figure S7AB) but high-concentration inhibitory effects of QnrB1 were still observed, suggesting that QnrB1 is still able to interact with gyrase. Interestingly, AlbG was also found to inhibit gyrase under these conditions.

However, both QnrB1 and AlbG on its own could not inhibit ATP-independent relaxation
(Supplementary Figure S7CD); instead, QnrB1 slightly promoted it (Supplementary Figure   S7E). To further test the potential requirement for strand passage, we analysed relaxation activities of A592/B2 and A2/B472 gyrase complexes mentioned above. Rescue of CFXpoisoned ATP-dependent (A592/B2) or ATP-independent (A2/B472) relaxation respectively was not observed and the enzyme was strongly inhibited (Supplementary Figure S8AB).
However, the effects of QnrB1 on its own on ATP-dependent and ATP-independent relaxation were different: while A59/B relaxation was clearly inhibited, relaxation by A/B472 was stimulated akin to the results with the full-length enzyme (Supplementary Figure S8CD).
In summary, these observations led us to think that strand passage on its own is not important for QnrB1 activity. Therefore, we hypothesized that QnrB1 interacts with ATPase domains of GyrB, which allosterically leads to the removal of the drug.

QnrB1 stimulates gyrase ATPase activity
We proceeded to test if QnrB1 and AlbG can stimulate DNA-independent and DNA-stimulated ATPase activity of gyrase. Figure 4A shows that QnrB1 indeed increased the ATP hydrolysis rate about 3-fold in the absence of DNA while the DNA-stimulated rate was not affected (Km -QnrB1 = 0.46 ± 0.15 µM, Km + QnrB1 = 0.96 ± 0.17 µM). Strikingly, when tested with isolated GyrB43 subunit, stimulation became much stronger ( Figure 4BCD). The ATPase reaction rate of GyrB43 with 5 µM QnrB1 was ~17 times higher than the rate with no QnrB1 present (Vmax Lastly, we could not observe any stimulation effect for AlbG ( Figure 4B)

QnrB1 binds to the GyrB subunit in vitro
We sought to determine whether GyrB43 ATPase domain is indeed involved in QnrB1 binding.
We measured interactions between gyrase and N-terminally labelled QnrB1 using a fluorescent anisotropy (FA)-based assay, similarly to the work done previously for gyrase regulator YacG (32). As can be seen from Figure 5A, Figure S10), suggesting that QnrB1 cannot interact with the "restrained" GyrB conformation, stabilised by the nucleotide analog (45).
We used an orthogonal technique (pull-down) to confirm these findings. N-terminally FLAGtagged QnrB1 was able to bind purified GyrB, both independently and in the context of GyrB2A2 complex (Figure 5B). GyrA on its own didn't interact with FLAG-QnrB1, but it was retained on the resin in the presence of GyrB, suggesting that gyrase complex is not disrupted by QnrB1 binding. When reactions were pre-incubated with ADPNP, no binding was observed.
Once again, preincubation with ADPNP completely abolished binding. We have tried to detect interactions of QnrB1 with smaller parts of GyrB (i.e. ATPase-transducer GyrB43, ATPase GyrB24 and TOPRIM GyrB47) but only full-length GyrB could pull-down QnrB1 (Supplementary Figure S12).

In vivo and in vitro crosslinking further support QnrB1-GyrB interaction
Attempts to purify QnrB1-gyrase complex by size-exclusion chromatography were unsuccessful, suggesting the transient nature of the complex. We used an orthogonal photo-crosslinkable amino acid benzoyl-phenylalanine (pBpa) to stabilise the native QnrB1-gyrase complex in E. coli cells (34). To pinpoint which residues of QnrB1 are likely to be involved, we selected by visual inspection of published crystal structure (PDB:2XTW) 29 surfaceexposed QnrB1 residues, which were not part of pentapeptide repeats and did not have obvious structural role, and replaced them with the pBpa (Supplementary Table S1 and Figure 5C).
E. coli DY330 derivatives, in which chromosomal GyrB or GyrA genes are fused with SPA purification tags (GyrA-SPA and GyrB-SPA) (47,48) were used as hosts for expression of QnrB1 pBpa variants. Four residues (Q51, R77, Y123, R167) were found to produce crosslinks, all of them with GyrB ( Figure 5D, Supplementary Table S1 and Supplementary figures 18-20). All crosslinked residues were found on one face of QnrB1 (Face 2), suggesting a defined interaction interface ( Figure 5C).

Specificity of PRP interactions with toxins
The G-segment DNA mimicry model originally proposed for MfpA postulated that this protein acts by reducing DNA binding to gyrase, which limits the formation of toxic cleavage complexes. The proposed reduction in DNA binding should reduce susceptibility to all agents that stabilise cleavage complexes involving the G-segment-gyrase interface. However, Qnr does not protect against proteinaceous gyrase poison CcdB (45) nor against natural product simocyclinone D8, which binds to the GyrA subunit in the 'saddle' region and prevents Gsegment DNA binding. Moreover, QnrB1 was found to act synergistically with simocyclinone (47). In our work we show that the activity of three different PRPs, QnrB1, AlbG and McbG show a high level of specificity towards three different gyrase poisons namely CFX, albicidin and MccB17. QnrB1 provided maximum protection against CFX and had almost no effect on the action of albicidin or microcin B17. Similarly, AlbG was extremely specific to albicidin.
These results were recapitulated in vitro with purified gyrase. Given the overall structural similarity between different topoisomerase-interacting PRPs, a universal higher level mechanism of action would be expected, however this should allow for specific interactions depending on the particular gyrase poisons.

Effects of QnrB1 and AlbG on supercoiling and relaxation reactions of DNA gyrase
Topoisomerase-interacting PRPs were proposed to be gyrase regulators with different activities. Mycobacterium tuberculosis MfpA, Klebsiella pneumoniae Qnr and Enterococcus faecalis EfsQnr were all reported to both protect E. coli gyrase from quinolone inhibition and at the same time inhibit gyrase supercoiling activity. In this study we tried to carefully disentangle "inhibition" and "rescue" modes of action of QnrB1 using E. coli gyrase as a model. In supercoiling reactions, QnrB1 required >10000-fold excess over the enzyme for efficient inhibition. The same level of inhibition was observed with a loop mutant protein, devoid of any protective activity. In contrast, AlbG from Xanthomonas albilineans did not produce any visible inhibition of supercoiling even at >10000-fold excess over gyrase.
DNA gyrase is capable of ATP-independent relaxation of negatively supercoiled DNA, where strand passage is believed to proceed in the reverse direction ('bottom to top'). Strikingly, this reaction was not inhibited by QnrB1 at any concentration (in fact, we consistently observed slight stimulation of relaxation activity at the highest concentrations of QnrB1 -see Supplementary Figure S7). Similarly, ATP-independent relaxation of negatively supercoiled DNA by GyrA/GyrB47 complex was not inhibited but rather stimulated by high doses of QnrB1 (Supplementary Figure S8). (see below) In the case of ATP-dependent, active relaxation by the GyrA59/GyrB complex strand passage is supposed to happen 'top to bottom' from N-gate to the C-gate (42). This reaction was strongly inhibited by QnrB1. Therefore, QnrB1 is only able to inhibit gyrase activity which requires normal "top to bottom" strand passage, coupled with ATP hydrolysis.
Lastly, we did not observe any inhibitory activity for AlbG, which comes from a Gramnegative bacterium X. albilineans. Xanthomonas gyrase has a significantly lower homology with the E. coli enzyme compared to the Klebsiella gyrase (59% versus 92% for GyrA and 61% versus 95% for GyrB). We hypothesize that AlbG has much stronger binding to its cognate Xanthomonas enzyme, as required for the host organism protection against a very potent toxin. It is worth noting that Xanthomonas gyrase was reported to have significantly different enzymatic properties and multiple antibiotic resistance (48).

Inhibition of DNA binding by QnrB1/AlbG
The main assumption of the "G-segment DNA mimicry" hypothesis is that PRPs bind to gyrase and reduce the amount of bound DNA. The only direct evidence for this was reduced DNA binding in filter assays with a Qnr variant (26). We have directly tested the ability of QnrB1 Finally, no effect on DNA binding was found for AlbG, despite the latter protein's clear ability to relieve albicidin inhibition. We suggest that in line with the results above showing that AlbG has poor affinity for E. coli gyrase, which is nevertheless sufficient to rescue it from albicidin inhibition.

Rescue of poisoned gyrase complexes by QnrB1 and AlbG
We have shown that a significantly reduced excess of QnrB1 over gyrase (100-fold) is required to effectively relieve FQ inhibition, compared with the >10000-fold excess required to inhibit gyrase. A slightly higher working concentration was required for AlbG (200-fold excess over gyrase), while no inhibition was observed for this protein as discussed above. Being the first to characterise AlbG activity in vitro against its cognate gyrase poison, we have additionally shown that it does not act by sequestering albicidin, analogous to the recently described AlbA (41). We did not see any signs of inactivation of the toxin after incubation with AlbG, confirming that direct interaction with the gyrase is the most likely mode of action for AlbG.
Both QnrB1 and AlbG decreased CFX-or albicidin-induced cleavage complex formation respectively. The concentration of CFX did not affect the ability of QnrB1 to inhibit cleavage complex formation, whilst AlbG failed to efficiently protect gyrase at [ALB] higher than 15 μM, which is not surprising given the differences between X. albilineans and E. coli gyrase discussed above. The cleavage inhibition activity strictly required ATP hydrolysis by gyrase, ruling out any models where PRPs are expected to simply bind to the enzyme and block the gyrase poison binding site. Moreover, we have shown that both QnrB1 and AlbG are able to actively disrupt pre-formed gyrase cleavage complexes in the presence of ATP (but not the non-hydrolysable analog ADPNP). In our view, these findings are hard to reconcile with the G-segment DNA mimicry model. These results are better aligned with the hypotheses suggesting specific recognition of topoisomerase poisons by PRPs (13) or T-segment DNA mimicry theory (see below).
In the absence of ATP, QnrB1 still binds to gyrase-FQ complexes, as manifested by inhibition of DNA relaxation reactions in the presence of FQs. The same is true for AlbG which was found to inhibit ATP-independent relaxation, but only in the presence of albicidin. Therefore, ATP hydrolysis is required for protection, but not for binding of PRPs to the gyrase.

Interactions of QnrB1 and AlbG with DNA gyrase
Both QnrB1 and MfpA were reported to bind E. coli gyrase in native gel retardation (26) and surface plasmon resonance (20) experiments, respectively. Models were proposed for PRPs (20,23) to bind to the positively charged GyrA dimer. In the more recent pull-down experiment (49), GST-tagged QnrB1 protein was shown to bind E. coli GyrB much more strongly than GyrA. Likewise, in two-hybrid system experiments (50) the QnrB1 interaction signal for GyrB was 7-to 11-fold higher than the signal for GyrA. The same work has shown that GyrA, but not GyrB interaction depends on the presence of the QnrB1 loops and is affected by sublethal doses of FQs.
Our study presents overwhelming evidence that the main binding partner of QnrB1 is GyrB.

Stimulation of ATPase activity by QnrB1
The association between QnrB1 and GyrB is additionally supported by the observed stimulation of ATP hydrolysis. This stimulation is particularly strong in the case of GyrB43 (17-fold). In contrast, only modest (3-fold) stimulation was observed for the full-length gyrase, compared with 7-fold stimulation by DNA. The difference in the magnitude of the QnrB1 effect might be attributed to the much higher baseline ATPase activity, exhibited by the full-length GyrB subunit, compared to GyrB43 (we used 4 µM of GyrB43 versus 50 nM of full-length subunits to achieve comparable reaction rates). QnrB1 cannot stimulate ATPase activity of the full-length gyrase to the same extent as DNA; at the same time, it stimulates ATP hydrolysis of GyrB43. Therefore, QnrB1 is unlikely to bind to the GyrB in exactly the same way as DNA.
One possibility is that QnrB1 induces dimerisation or stabilises the GyrB43 dimer and thus promotes ATP hydrolysis.
No stimulation of ATPase activity was observed for AlbG. Again, this is in line with the absence of binding to GyrB, lack of gyrase inhibition, and inability to compete with DNA, suggesting that all these effects are connected to the stronger binding of QnrB1 to the E.coli gyrase.

Role of loops in the activity of QnrB1 and AlbG
A 12-amino acid loop, protruding from the rod-like QnrB1 scaffold, was shown to be the main determinant of protection against FQs (21,24), with a deletion of three amino acids (107-109, TTR) completely abolishing protection (24). A similar structural element has been found in AlbG (13). Interestingly, in two-hybrid system experiments, loop deletions did not perturb the interaction with GyrB. This corresponds well with our findings that QnrB1 ΔTTR was found to bind GyrB equally strongly as WT protein in pull-down and crosslinking experiments and activated ATPase activity to the same extent. Moreover, QnrB1 ΔTTR inhibited gyrase supercoiling activity to the same extent as WT protein, despite having no visible protective activity. In this study we also for the first time analysed the role of the AlbG loop and found that similarly to QnrB1, its deletion abolished protection against albicidin in vivo.
We assume that the loop, as was suggested before (50) is important for the precise positioning of the PRP, required to remove the bound drug, but does not by itself constitute the main binding interface (see below) .
T-segment mimicry model as a possible mechanism of action of topoisomerase acting

PRPs
Given the significant structural similarities between PRPs, we expect that the overall mechanism of topoisomerase protection must be universal but should account for the observed toxin-specificity. Moreover, it should account for the two clearly distinct modalities of PRP action (general interaction with the enzyme and drug-specific protection). This is highlighted by the example of the QnrB1 ΔTTR mutant, which is still able to bind gyrase, stimulate ATP hydrolysis and cause inhibition of supercoiling at high concentration, but is completely devoid of protective activity (21,24). In contrast, AlbG, retaining its loop, does not bind Ec gyrase well, but retains the capacity to protect the enzyme against gyrase poison.
The suggestion that PRPs are DNA mimics is attractive, but the original "G-segment" DNA mimicry model is not supported by our and others' data (21). We have previously hypothesized We propose an alternative model for QnrB1 action which incorporates elements of the" Tsegment DNA mimicry model" (Figure 6) and suggest that the same model applies to all topoisomerase-interacting PRPs. In this model, to bind to the gyrase in physiological conditions (i.e. without gyrase poison present and with DNA wrapped around the enzyme) QnrB1 must outcompete T-segments, which requires very high excess of QnrB1 over gyrase and is unlikely to happen. However, sufficient QnrB1 interferes with normal top to bottom strand passage and inhibits supercoiling and ATP-dependent relaxation, as observed in our experiments with QnrB1 ( Figure 6, "Gyrase inhibition"). The same interference helps to promote ATPindependent relaxation, which is thought to occur in the reverse direction, i.e. DNA entering from the C-gate. Inhibition of top to bottom strand passage thus helps to shift the thermodynamic relaxation equilibrium towards more relaxed DNA species. Binding of QnrB1 also destabilises the wrapped DNA complex, reducing DNA binding observed in EMSAs and FA assays. The binding of QnrB1 to gyrase does not require loops, but likely requires amino acids on Face 2.
When the DNA is not present, the QnrB1 does not have to compete with the T-segment and can bind gyrase. Cleavage complex stabilisation by the gyrase poison should similarly allow QnrB1 to interact with the enzyme, presumably by removing competition with the T-segment.
The protective activity of PRPs strictly requires ATP hydrolysis by gyrase, moreover, QnrB1 binding actively promotes ATP hydrolysis. If we consider that the protective mechanism involves replacement of bound gyrase poison by the QnrB1 molecule then to efficiently replace bound toxin, the QnrB1-stabilised conformation must have lower energy than the toxinstabilised one. Energy input is then required for a subsequent QnrB1 release, otherwise the enzyme would be constantly inhibited by the QnrB1 itself. Therefore, the requirement for the energy input in the form of ATP is logical. We suggest that QnrB1/AlbG act as "spokes in the wheel" which, upon ATP hydrolysis, are "pushed" through the enzyme and physically dislodge bound drugs or toxins (Figure 6, "Gyrase rescue from FQ"). This dislodgement is poisonspecific and requires specific amino acids in loops of QnrB1 or AlbG. Analogous mechanisms of transient interaction with the enzyme are observed for ribosome protective factors such as TetO and TetM which hydrolyse GTP in order to release themselves from the ribosome (53) Further clarification of this model requires knowledge of fine molecular details of interactions between PRPs and their targets. High-resolution structures of QnrB1 "caught in the act" will be difficult to obtain, as they require full complex assembly and likely ATP hydrolysis. No structures of the full enzyme with T-segment captured at the moment of strand passage have been reported to date. However, the recent development of a cryo-EM platform for structural studies of the E. coli gyrase complex (54) has the potential to address such challenges. Such structural information may also allow to design novel gyrase inhibitors based on PRP peptides, novel antibacterials which avoids PRP-driven resistance or small molecules which will block the PRP-gyrase interactions, to keep the potency of existing drugs such as FQs.