PURIFICATION AND CHARACTERIZATION OF THE PSEUDOMONAS-AERUGINOSA NFXB PROTEIN, THE NEGATIVE REGULATOR OF THE NFXB GENE

The protein NfxB, involved in conferring resistance to quinolones in P seudomonas aeruginosa, has a helix-turn-helix motif which is similar t o that of other DNA-binding proteins, It appears to affect the membran e-associated energy-driven efflux of some antibiotics (H. Nikaido, Sci ence 264:382-388, 1994), We constructed a plasmid that overproduced Nf xB in Escherichia coli and purified the protein, Two species of NfxB ( 23 and 21 kDa), which are probably translated from different initiatio n codons, were isolated, Both proteins are also expressed in vivo in P . aeruginosa, with the 23-kDa NfxB being the major species, NfxB speci fically binds upstream of the nfxB coding region as demonstrated by ge l retardation and DNase I footprinting. Expression of the Phi(nfxB'-la cZ(+)) (Hyb) gene was repressed in the presence of the nfxB gene produ ct provided by a second compatible plasmid in E. coli, In the P. aerug inosa wild-type strain (PAO2142), NfxB was undetectable by immunoblott ing; however, it was detected in the nfxB missense mutant (PK1013E), T hese results suggested that NfxB negatively autoregulates the expressi on of nfxB itself, Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother, 39:645 -649, 1995) was overproduced in nfxB mutants, NfxB may also regulate t he expression of membrane proteins that are involved in the drug efflu x machinery of P. aeruginosa.