Abstract
Plasmid toxins CcdB and ParE are part of addiction systems promoting plasmid maintenance. Both target host DNA gyrase, as do quinolones and plasmid-determined Qnr proteins that protect gyrase from quinolone inhibition. We cloned qnrB4, qnrS1, ccdB, parE, and the antitoxin-encoding genes ccdA and parD on compatible plasmids and tested them in combination. CcdB and ParE had no specific effect on quinolone susceptibility or Qnr protection, and Qnr did not act as a CcdB or ParE antitoxin.
TEXT
Many low-copy-number plasmids ensure their stable maintenance by encoding a toxin-antitoxin pair. The antitoxin has a shorter half-life than the toxin, so that if both were no longer synthesized following plasmid loss, the cell would die, thus enforcing plasmid stability. Plasmid toxins have various targets, with CcdB encoded by IncF plasmids (1) and ParE encoded by IncP plasmids (2) known to poison host bacterial DNA gyrase. CcdB binds to the dimerization domain of the GyrA subunit at Arg462, where replacement with cysteine blocks binding and prevents gyrase inhibition (1, 3). Quinolones also interact with the GyrA subunit but at sites different from those of the plasmid toxins, with mutations at selected amino acid positions between 51 and 106 lowering susceptibility (4). Quinolone inhibition is blocked by plasmid-mediated Qnr, a pentapeptide repeat protein that binds to both gyrase subunits (5, 6). Whether Qnr interferes with CcdB or ParE toxicity is not known, nor is the effect of these toxins on quinolone susceptibility in the presence of Qnr (7). We investigated the interaction of quinolone and ccdB, parE, and qnr genes cloned into compatible vectors in Escherichia coli.
Toxin genes were cloned after PCR amplification with primers carrying terminal restriction sites, allowing ligation into EcoRI and KpnI sites of expression plasmid pBAD24 (Ampr, 4.5 kb) (8). The ccdB gene was amplified from E. coli clinical isolate 6-74 (9), which is positive by PCR for both qnrS1 and ccdAB, and the parE gene was amplified from plasmid RP4 (10). After ligation, plasmids were chemically transformed into XL1-Blue competent cells (Agilent) with selection on Luria-Bertani (LB; Difco) agar plates containing ampicillin (100 μg/ml) plus 0.2% glucose to repress expression of the toxin insert. Antitoxin genes ccdA (from strain 6-74) and parD (from plasmid RP4) were cloned by using BamHI and PstI into pACYCDuet-1 (Chlr, 4.0 kb; Novagen), a vector compatible with pBAD24, by using selection on chloramphenicol at 34 μg/ml. qnrB4 from plasmid pMG319 (11) and qnrS1 from plasmid pMG306 (12) were cloned similarly. The accuracy of cloning was confirmed by sequencing. Strain DH10B (Life Technologies) with various plasmid combinations was constructed by transformation with these recombinant plasmids by using ampicillin, chloramphenicol, or both for selection.
MICs of ciprofloxacin (Sigma-Aldrich) were determined at least in duplicate by agar dilution in accordance with the guidelines of the Clinical and Laboratory Standards Institute (13), except that LB agar containing ampicillin (50 μg/ml) or ampicillin plus chloramphenicol (25 μg/ml) was used to prevent plasmid loss. The ciprofloxacin MIC for recA1 mutant strain DH10B was 0.008 μg/ml and was unchanged by the addition of pBAD24ccdB or pBAD24parE with glucose present in the medium to minimize toxin expression. With toxin expression stimulated by 0.0025 or 0.005% arabinose, MICs decreased by 1 dilution but a comparable change was seen with toxin-free strain DH10B, indicating that the slight effect was not toxin specific (Table 1). QnrS1 increased the ciprofloxacin MIC 4-fold compared to that for plasmid-free DH10B. The MIC elevation was 1 dilution less in DH10B with ccdB or parE present as well as qnrS1 but was unchanged with arabinose stimulation, suggesting that the toxins had little, if any, effect on the ability of QnrS1 to protect gyrase from quinolone inhibition.
TABLE 1.
Ciprofloxacin MICs
| Strain | Ciprofloxacin MICa (μg/ml) at arabinose concn (%) of: |
|||
|---|---|---|---|---|
| 0 | 0.00125 | 0.0025 | 0.005 | |
| DH10B | 0.008 | 0.008 | 0.004 | 0.004 |
| DH10B/pBAD24ccdB | 0.008 | 0.008 | 0.004 | 0.004 |
| DH10B/pBAD24parE | 0.008 | 0.008 | 0.004 | 0.004 |
| DH10B/pACycDuet-1 qnrS1 | 0.032 | 0.032 | 0.032 | 0.016 |
| DH10B/pBAD24ccdB/pACYCDuet-1qnrS1 | 0.016 | 0.016 | 0.016 | 0.016 |
| DH10B/pBAD24parE/pACYCDuet-1qnrS1 | 0.016 | 0.016 | 0.016 | 0.016 |
Determined by agar dilution on LB agar plates containing ampicillin and/or chloramphenicol with 0.2% glucose and 0% arabinose or the arabinose concentration indicated and 0% glucose.
DH10B containing pBAD24ccdB or pBAD24parE could grow in LB broth with 0.01 or 0.02% arabinose but not higher concentrations, unless the CcdA or ParD antitoxin was supplied via plasmid pACYCDuet-1 derivatives (Table 2). Growth of strain CSH501/pBAD24ccdB in arabinose at up to 0.8% occurred since CSH501 (14) has a gyrA462 mutation blocking ccdB toxicity. Neither pACYCDuet-1qnrS1 nor pACYCDuet-1qnrB4 could substitute for ccdA or parD, indicating that the qnr genes lack an antitoxin effect. The same result was obtained by measuring growth rates by colony counting. The growth rate of DH10B with pBAD24ccdB or pBAD24parE was the same whether pACYC-Duet-1qnrS1 was present in the strain or not and the medium was LB plus 0.2% glucose or LB with 0.005% arabinose, where the growth rate of the toxin-expressing strains was reduced 60%. We also found no plasmid instability in strains like clinical isolate 6-74 containing both qnrS1 and ccdAB.
TABLE 2.
Viability of CcdB and ParE toxin-containing clones
| Strain | Growth at arabinose concn (%) of: |
||||||
|---|---|---|---|---|---|---|---|
| 0.01 | 0.02 | 0.05 | 0.1 | 0.2 | 0.4 | 0.8 | |
| DH10B/pBAD24ccdB | +a | + | − | − | − | − | − |
| DH10B/pBAD24ccdB/pACYCDuet-1qnrS1 | + | + | − | − | − | − | − |
| DH10B/pBAD24ccdB/pACYCDuet-1qnrB4 | + | + | − | − | − | − | − |
| DH10B/pBAD24ccdB/pACYCDuet-1ccdA | + | + | + | + | − | − | − |
| CSH501 pBAD24ccdB | + | + | + | + | + | + | + |
| DH10B/pBAD24parE | + | + | − | − | − | − | − |
| DH10B/pBAD24parE/pACYCDuet-1qnrS1 | + | + | − | − | − | − | − |
| DH10B/pBAD24parE/pACYCDuet-1qnrB4 | + | + | − | − | − | − | − |
| DH10B/pBAD24parE/pACYCDuet-1parD | + | + | + | + | + | − | − |
+, growth; −, no growth (in LB broth containing the concentration of arabinose indicated).
Qnr-encoding genes are widely distributed on bacterial chromosomes, as well as plasmids, and predated the therapeutic use of quinolones. Their native role is not known, but a function as antidotes to plasmid maintenance systems mediated by gyrase toxins CcdB and ParE can be eliminated.
ACKNOWLEDGMENTS
We thank R. Varadarajan (Indian Institute Science, Bangalore) for supplying strain CHS501.
This work was supported by the Inje Research and Scholarship Foundation in 2011 (Y.G.K.) from the Inje University College of Medicine and by grant R01 AI057576 (to D.C.H. and G.A.J.) from the National Institutes of Health, U.S. Public Health Service.
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