Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a
Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a
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DOI：
10.1128/jb.185.21.6233-6240.2003
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发表时间：
2003-11-01
影响因子：
3.2
通讯作者：
Foght, JM
中科院分区：
文献类型：
--
作者：
Hearn, EM;Dennis, JJ;Foght, JM
烃降解环境分离物荧光假单胞菌LP 6a具有多环芳烃菲，蒽，荧蒽，但不是萘或甲苯的主动外排机制。PCR用于检测质粒固化衍生物荧光假单胞菌cLP 6a中属于抗性-增殖-细胞分裂（RND）超家族的外排泵基因，所述荧光假单胞菌cLP 6a不能代谢烃。一个RND泵，其基因在荧光假单胞菌cLP 6a中被鉴定并被命名为emhB，显示出与铜绿假单胞菌和恶臭假单胞菌中的多药和溶剂外排泵的同源性。emhB基因位于与emhA和emhC基因的基因簇中，emhA和emhC基因分别编码外排系统的膜融合蛋白和外膜蛋白组分。通过插入抗生素抗性盒破坏emhB表明，相应的基因产物负责多环芳烃的外排。emhB基因破坏不影响荧光假单胞菌cLP 6a对四环素、红霉素、甲氧苄啶或链霉素的抗性，但它确实降低了对氯霉素和萘啶酸的抗性，这表明EmhABC系统也在这些化合物的外排中起作用，并且具有不寻常的选择性。在铜绿假单胞菌、恶臭假单胞菌和洋葱伯克霍尔德菌中观察到菲流出，但在棕色固氮菌中未观察到菲流出。多环芳烃代表了一类新的无毒、高度疏水的化合物，它们是RND外排系统的底物，并且荧光假单胞菌cLP 6a中的EmhABC系统对这些烃和某些抗生素具有窄的底物范围。
The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P.fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.