Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa.
MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.
Yamamoto
Masaki
M
Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
Ueda
Atsuhisa
A
Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
Kudo
Makoto
M
Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
Matsuo
Yasuhiro
Y
Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
Fukushima
Jun
J
Department of Biotechnology, Akita Prefectural University, Akita 010-0195, Japan.
Nakae
Taiji
T
Antimicrobial Research Center, Kitasato Institute, Kitasato University, Sagamihara 228-8555, Japan.
Kaneko
Takeshi
T
Department of Pulmonary Medicine, Yokohama City University Medical Center, Yokohama 232-0024, Japan.
Ishigatsubo
Yoshiaki
Y
Department of Internal Medicine and Clinical Immunology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D012097:Repressor Proteins / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D015533:Transcriptional Activation
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C402791:MexXY protein, Pseudomonas aeruginosa; C402792:MexZ protein, Pseudomonas aeruginosa; D012097:Repressor Proteins; D013752:Tetracycline