Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae
Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae
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DOI：
10.1046/j.1365-2958.1998.00657.x
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发表时间：
1998-01-01
影响因子：
3.6
通讯作者：
Hamood, AN
中科院分区：
文献类型：
--
作者：
Colmer, JA;Fralick, JA;Hamood, AN
霍乱弧菌（肠道疾病霍乱的病原体）的多药耐药菌株最近已被描述。为了鉴定大肠杆菌TolC在霍乱弧菌中的同源物，我们分离了一个DNA片段（pVC），该片段使大肠杆菌TolC在霍乱弧菌中的同源性增加。colitolC突变体在0.05%脱氧胆酸盐（DOG）存在下生长。然而，其他TolC缺陷没有得到补充。核苷酸序列分析表明，该片段存在两个开放阅读框架（ORF 1和ORF 2），它们之间相隔9 bp，分别编码42.4和55.8 kDa的蛋白。这两个ORF的翻译产物与预测蛋白的分子量密切相关。ORF1和ORF2的氨基酸序列与E.大肠杆菌外排泵蛋白EmrA和EmrB。pVC 2在E.在emrAB或acrAB基因中缺陷的大肠杆菌外排泵突变体提供了对几种抗生素具有抗性的突变体。通过基因置换构建了霍乱弧菌ORF 2缺陷的同基因突变体。该突变株对CCCP、PMA、PCP、萘啶酸和DOC的敏感性均高于亲本菌株。这些结果表明，ORF1和ORF2构成一个操纵子编码的两个组件的一个假定的多药耐药泵在霍乱弧菌。此外，VceAB和EmrAB之间存在结构和功能相似性，表明VceAB是EmrAB的同源物。
Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V. cholerae, we isolated a DNA fragment (pVC) that enabled an E. coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOG). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E. coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E. coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V. cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V. cholerae. In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.