Vibrio cholerae ParE2 poisons DNA gyrase via a mechanism distinct from other gyrase inhibitors

Abstract

DNA gyrase is an essential bacterial enzyme required for the maintenance of chromosomal DNA topology. This enzyme is the target of several protein toxins encoded in toxin-antitoxin (TA) loci as well as of man-made antibiotics such as quinolones. The genome of Vibrio cholerae, the cause of cholera, contains three putative TA loci that exhibit modest similarity to the RK2 plasmid-borne parDE TA locus, which is thought to target gyrase although its mechanism of action is uncharacterized. Here we investigated the V. cholerae parDE2 locus. We found that this locus encodes a functional proteic TA pair that is active in Escherichia coli as well as V. cholerae. ParD2 co-purified with ParE2 and interacted with it directly. Unlike many other antitoxins, ParD2 could prevent but not reverse ParE2 toxicity. ParE2, like the unrelated F-encoded toxin CcdB and quinolones, targeted the GyrA subunit and stalled the DNA-gyrase cleavage complex. However, in contrast to other gyrase poisons, ParE2 toxicity required ATP, and it interfered with gyrase-dependent DNA supercoiling but not DNA relaxation. ParE2 did not bind GyrA fragments bound by CcdB and quinolones, and a set of strains resistant to a variety of known gyrase inhibitors all exhibited sensitivity to ParE2. Together, our findings suggest that ParE2 and presumably its many plasmid- and chromosome-encoded homologues inhibit gyrase in a different manner than previously described agents.

FIGURE 1.

Organization of the V. cholerae parDE2 locus. A, shown is a schematic representation of the parDE2 region in the V. cholerae N16961 chromosome. The white arrow represents the original vca0360 ORF as previously annotated (26), and the light gray arrow represents the vca0360.1 ORF as annotated in Bocs et al. (27). The thin arrow represents the predicted parD2 promoter based on the 5′ rapid amplification of cDNA ends. B: lane M, 1-kb plus marker; lane 1, specific PCR product from the 5′ rapid amplification of cDNA ends experiment. C, the start of the parD2 transcript, represented by the arrow, was determined by sequencing the PCR shown in B. The parD2 translational start site, as predicted by Bocs et al. (27), is shown with the box around the ATG. D, alignments of the predicted amino acid sequences of vca0360.1 (V. cholerae ParD2) with the indicated chromosome- and plasmid (RK2)-encoded ParD sequences are shown.

FIGURE 2.

Effect of ParE2 and ParD2 on V. cholerae and E. coli viability. V. cholerae N16961 (A) and E. coli BW27784 (B) contained a plasmid-borne, arabinose-inducible parE2 (pBAD-E2) and either an empty vector (pGZ, diamonds and squares) or the vector harboring parD2 under control of its native promoter (pGZ-D2, circles and triangles). All cultures were grown in LB supplemented with 0.2% glucose at 37 °C until an A₆₀₀ of ∼0.3, washed and resuspended in either LB plus 0.2% glucose (glu) (black squares and triangles) or LB plus 0.02% arabinose (ara) (red diamonds and circles). CFU were enumerated at the indicated time points. C, E. coli BL21 (pBAD-E2, pET-D2), (JY307), harbors plasmids with arabinose-inducible parE2 and IPTG-inducible parD2. Cultures were grown in LB supplemented with 0.2% glucose until an A₆₀₀ of ∼0.3, washed and resuspended in either LB plus 0.2% glucose (glu) or glucose plus 50 μm IPTG (black squares and circles) or LB plus 0.02% arabinose (ara) (red shapes). IPTG (50 μm) was added at the indicated time points after the addition of arabinose. CFU were enumerated at the indicated time points.

FIGURE 3.

Co-purification of Myc tagged ParE2 with His-tagged ParD2. A, His-ParD2 from JY307, E. coli BL21 co-expressing His-ParD2 and ParE2-Myc (lysate) was affinity-purified using Ni-NTA resin. The starting lysate flowthrough (FT), washes, and eluted fractions were analyzed by Western blotting with anti-His and anti-Myc antibodies. B, cell lysate from JY281, E. coli DH5α-expressing ParE2-Myc were processed as in A and then analyzed using anti-Myc antibody. C, His-tagged ParD2 (∼50 relative units (R.U.) was coupled to a Ni-NTA chip, and native ParE2 was injected at the indicated concentrations, expressed in terms of monomer.

FIGURE 4.

Co-purification of GyrA and GyrB with His-ParE2. Cell lysates from E. coli BL21(DE3) expressing His-ParE2 (JY302) (A) or expressing His-ParD2(JY262) (B) or E. coli BL21 co-expressing His-ParD2 and ParE2-Myc (JY307) (C) were incubated with Ni-NTA resin, washed, and then eluted with imidazole. The initial lysates flowthrough (FT), washes, and eluted fractions were electrophoresed on polyacrylamide gels and then analyzed by Western blotting with anti-GyrA, anti-GyrB anti-His, and anti-ParE2 antibodies as indicated. Purified E. coli gyrase was used as the control. The positions of GyrA or GyrB are indicated with arrows.

FIGURE 5.

SPR measurements of interactions between V. cholerae ParE2 or V. fischeri CcdB and gyrase. GyrBA (∼200 relative units (RU)) (A), GyrA (∼150 RU) (C), GyrA59 (∼600 RU) (D), and GyrA14 (∼400 RU) (E) were non-covalently coupled to a Ni-NTA chip, whereas GyrB (∼1800 RU) (B) was covalently coupled to a CM5 chip. The analytes were injected over the immobilized ligands at the indicated concentrations, expressed in terms of monomer. F, V. fischeri CcdB was injected at a concentration of 50 μm to saturate the CcdB binding site on GyrA. After a dissociation time of 200 s, ParE2 was injected at a concentration of 10 μm. A control experiment with a second CcdB (10 μm) injection showed that all CcdB binding sites are saturated after the first CcdB injection. G, shown is kinetic analysis of the interaction between V. cholerae ParE2 and E. coli GyrA59. The top graph displays the sensorgrams at different ParE2 concentrations (0 nm, 3.9 nm, 7.8 nm, 15.6 nm, 31.25 nm, 62.5 nm, 125 nm, 250 nm, 500 nm, 1 μm, 2 μm), which were collected in duplicate and are shown in black. The red lines represent the best fit of the model function (heterogeneous ligand model) to the experimental curves. The residuals of the fitting procedure are shown in the bottom graph. Model-based analyses of the sensorgrams recorded during the multicycle analysis of the ParE2-GyrA59 interaction indicates that the binding of ParE2 to GyrA59 is not monophasic and that two binding events occur in parallel. Fitting with a simple 1:1 Langmuir binding model does not result in acceptable residuals (χ² = 22.4). The simplest model providing a reasonable fit to the data is a heterogeneous ligand model, resulting in a χ² of 1.84. In aggregate, analysis of this data set reveals a very high affinity for the ParE2-GyrA59 interaction with a K_D between ∼20 pm and 10 nm.

FIGURE 6.

Effect of ParE2 and ParD2 on cleavage of chromosomal DNA by gyrase. In vitro reactions with chromosomal DNA, gyrase, ParE2, and ParD2 were carried out as described under “Experimental Procedures.” M indicates 1 kb plus DNA ladder.

FIGURE 7.

The effect of ParE2 on gyrase supercoiling (A) and relaxation (B) of plasmid DNA substrates. The reactions were performed as described under “Experimental Procedures” except in B the reaction was stopped with SDS and proteinase K. In A, the substrate was relaxed pBR322 DNA, and in B the substrate was negatively supercoiled pCB182. M indicates 1Kb plus DNA ladder.

FIGURE 8.

Requirement for ATP hydrolysis in ParE2 stabilization of gyrase-DNA cleavages. The reactions were carried out as described under “Experimental Procedures” using V. cholerae chromosomal DNA as substrate. 1 mm ATP, ATPγS, or GTP were used in these reactions. 5 μm ParE2 or 1 mm nalidixic acid were included as indicated. M indicates 1 kb plus DNA ladder.

FIGURE 9.

Strains bearing gyrase alleles conferring resistance to several gyrase toxins remain sensitive to ParE2. In A, the strains indicated were tested for their resistance to ParE2 after the toxin was expressed from pBAD-E2. Strains were scored as sensitive if there was attenuated growth after induction of ParE2 expression with arabinose. In B, the mutations listed in A were mapped onto the crystal structure of E. coli GyrA59 colored in green (PDB entry 1AB4 (52). GyrA14 is shown in dark gray, and the red patches correspond to the residues involved in the interaction between CcdB and GyrA (Gln-456, Asp-460, Arg-462, Gln-464). The residues conferring resistance to quinolones when mutated are colored blue. This figure was generated using MacPyMOL (DeLano Scientific, Ltd.).