嗜水气单胞菌acrA缺失菌株的构建及其生理功能的测定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0380

摘要/Abstract

摘要：

AcrA是革兰氏阴性菌中的一种膜融合蛋白,现今关于AcrA蛋白的研究,以AcrA蛋白在细菌中药物外排等膜转运过程中的调节作用为主。为了阐明细菌中acrA基因敲除后,该菌在溶血性、毒理性、压力胁迫性及抗生素敏感性等生理功能的变化,以嗜水气单胞菌（Aeromonas hydrophila）为研究对象,利用无痕敲除等分子生物学手段构建acrA（AHA_2911）的缺失菌株,并对野生菌株和缺失菌株的溶血性、胞外蛋白酶活、抗生素敏感性、高温胁迫、生长趋势以及生物膜形成能力等生理特性进行了测定。结果显示,敲除菌株的溶血性和胞外蛋白酶活并未受到影响,但acrA基因敲除后增加了该菌对卡那霉素、罗红霉素、巴罗沙星抗生素及吖啶黄素染料的敏感度并且显著提高了嗜水气单胞菌的生物膜形成能力。但是,∆acrA 在42℃下的生长趋势及其生物膜形成能力明显弱于野生株。以上研究证明嗜水气单胞菌acrA基因在药物外排泵中有一定的功能,并且提示该基因可能在细菌的高温耐受性及生物膜形成中起重要作用。

关键词: acrA, 嗜水气单胞菌, 同源重组, 生理表型, 生物被膜, 高温

Abstract:

AcrA is a membrane fusion protein in Gram-negative bacteria,and scientists mainly study the regulation of AcrA protein in the process of drug efflux and other membrane transport in bacteria. To elucidate the effects of acrA deficient strain ΔacrA in the bacteria on physiological functions such as hemolytic,toxicological,stress and antibiotic sensitivity,acrA（AHA_2911）deleted strain was constructed in A. hydrophila using traceless based homologous recombination technology. The physiological phenotypes,such as hemolytic,extracellular protease activity,antibiotics susceptibility,high temperature stress,growth trends,and biofilm formation ability for both wild strain and ΔacrA were measured. The results showed that the hemolytic and extracellular protease activities in the ΔacrA were not affected,while the acrA deletion increased the antibiotics sensitivity of the ΔacrA to kanamycin,erythromycin,balofloxacin and acriflavinium chloride,and significantly improved the biofilm formation. But,the growth trend of ∆acrA and its biofilm formation ability at 42^oC were significantly lower than wild strain. The above studies proved that acrA has a certain function in drug efflux pumps and suggested that it may play an important role in the high temperature tolerance and biofilm formation.

Key words: acrA, Aeromonas hydrophila, homologous recombination, physiological phenotype, biofilm, high temperature

李小艳, 李泽琦, 汪玉倩, 于晶, 林镇平, 林向民. 嗜水气单胞菌acrA缺失菌株的构建及其生理功能的测定[J]. 生物技术通报, 2020, 36(11): 63-69.

LI Xiao-yan, LI Ze-qi, WANG Yu-qian, YU Jing, LIN Zhen-ping, LIN Xiang-min. Construction of Aeromonas hydrophila acrA Deficient Strain and Determination of Its Physiological Function[J]. Biotechnology Bulletin, 2020, 36(11): 63-69.

图/表 7

表1 嗜水气单胞菌acrA基因缺失以及验证引物列表

引物名称	序列（5'-3'）	用途
P1	AATTCCCGGGAGAGCTCAACATCGCATGG	左臂上游引物（Left arm upstream primer）
P2	TACTCCATCGGAGAACCTGAGCCTCTGTCTATC	左臂下游引物（Left arm downstream primer）
P3	GCTCAGGTTCTCCGATGGAGTAGTCGCCTCAT	右臂上游引物（Right arm upstream primer）
P4	CAAGCTTCTTCTAGAGTTGGCCACCACGTAG	右臂下游引物（Right arm downstream primer）
P5	ATGCATAAACATATTCTCGCACG	目的基因验证引物上游引物（Target gene verification primer upstream primer）
P6	CTACTTGTTGGCCGGGGCATTGG	目的基因验证引物下游引物（Target gene verification primer downstream primer）
P7	GTGCAGATGACCCCGGGTGACG	左臂外侧引物上游引物（Left arm outer primer upstream primer）
P8	CGTCACCCGGGTCATCTGCAC	右臂外侧引物上游引物（Right arm lateral primer upstream primer）

图1 acrA敲除菌株PCR验证 M：DL2000 Marker;1：P5P6（1 194 bp）;2：P5P6 阳性对照;3：P7P8（1 000 bp）;4：P7P8（2 194 bp）阳性对照

图2 野生菌株与敲除菌株溶血性测定 A：野生菌株和敲除菌株在血平板上的透明圈生长情况;B：野生菌株和敲除菌株在血平板上的透明圈直经结果柱状图

图3 野生菌株与敲除菌株胞外蛋白酶活测定 A：野生菌株和敲除菌株在脱脂牛奶平板上的牛乳蛋白水解圈生长情况;B：野生菌株和敲除菌株在牛奶琼脂平板上的水解圈直经结果柱状图

图4 野生菌株与敲除菌株对Kanamycin、Erythromycin、Balofloxacin抗生素及Acriflavinium chloride染料敏感性的测定（10-2-10-8）为菌液梯度稀释倍数,Kanamycin、Erythromycin、Balofloxacin、Acriflavinium chloride分别是卡那霉素、罗红霉素、巴洛沙星和吖啶黄素

图5 野生菌株与敲除菌株生长趋势测定 A：30℃环境下野生菌株与敲除菌株的生长趋势图;B：42℃环境下野生菌株与敲除菌株的生长趋势图

图6 野生菌株与敲除菌株生物膜形成能力测定 A：30℃下野生菌株与敲除菌株的生物膜测定结果柱状图;B：42℃下野生菌株与敲除菌株的生物膜测定结果柱状图

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