The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex.
MfpA(Mt) and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA(Mt) gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA(Mt) on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 microM, MfpA(Mt) inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 microM. We showed that the D87 residue in GyrA has a major role in the MfpA(Mt)-gyrase interaction, as D87H and D87G substitutions abolished MfpA(Mt) inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA(Mt) and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 microM as described for other Qnr proteins, but MfpA(Mt) did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA(Mt) also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA(Mt) and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.
Mérens
Audrey
A
Université Paris, IFR, Bacteriologie, Créteil, France.
Matrat
Stéphanie
S
Aubry
Alexandra
A
Lascols
Christine
C
Jarlier
Vincent
V
Soussy
Claude-James
CJ
Cavallo
Jean-Didier
JD
Cambau
Emmanuelle
E
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D027081:DNA Gyrase / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D024841:Fluoroquinolones / Q000378:metabolism / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology; D013045:Species Specificity; D059005:Topoisomerase II Inhibitors
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; C485384:Qnr protein, E coli; D059005:Topoisomerase II Inhibitors; C445685:MfpA protein, Mycobacterium smegmatis; D020559:Monomeric GTP-Binding Proteins; D027081:DNA Gyrase