Secondary-site mutation restores the transport defect caused by the transmembrane domain mutation of the xenobiotic transporter MexB in Pseudomonas aeruginosa.
It has been suggested that the MexB subunit of the MexAB-OprM efflux transporter of Pseudomonas aeruginosa exports xenobiotics in an energy-dependent manner. To investigate the role of the transmembrane segments (TMS) of MexB in the transporter activity, we isolated 24 spontaneous mutants showing hypersusceptibility to antibiotics. Among them, three mutations were located at TMS-3, TMS-4, and TMS-10 having amino acid substitution Leu376vPro, Gly397vVal, and Val928vGly, respectively. A secondary mutation, which suppressed the defect caused by the Val928vGly mutation in TMS-10, was found at the 403rd amino acid residue in TMS-4 with a change of glycine to serine, suggesting that TMS-4 and TMS-10 may be in close proximity. This result provided strong support for the recent notion that negatively charged residues in TMS-4 might form a salt-bridge with a positive charge in TMS-10 (Guan, L., and Nakae, T. (2001) J. Bacteriol. 183, 1734-1739). The transporter function impaired by the Gly397vVal mutation in TMS-4 was recovered by the secondary mutation, Gln998vHis, in the loop between TMS-11 and TMS-12, thereby suggesting that TMS-4 and TMS-11 or TMS-12 might also be in close proximity. Thus, it is most likely that TMS-4, TMS-10, and TMS-11 or TMS-12 are packed close three dimensionally.
Yoneyama
Hiroshi
H
Department of Molecular Life Science, Tokai University School of Medicine, Isehara, 259-1193, Japan
Maseda
Hideaki
H
Yamabayashi Ta
Taka-aki
TA
Izumi
Shotaro
S
Nakae
Taiji
T
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001398:Aztreonam / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002701:Chloramphenicol / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D007700:Kinetics; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009154:Mutation; D015242:Ofloxacin / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial; D015262:Xenobiotics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C118597:MexB protein, Pseudomonas aeruginosa; D015262:Xenobiotics; D002701:Chloramphenicol; D015242:Ofloxacin; D001398:Aztreonam