Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry
Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry
复制标题
DOI：
10.1007/s00248-002-2028-6
复制
发表时间：
2003-03-01
影响因子：
3.6
通讯作者：
Sorensen, SJ
中科院分区：
文献类型：
--
作者：
Burmolle, M;Hansen, LH;Sorensen, SJ
群体感应使细菌能够根据种群密度调节某些基因的表达。已知基于N-酰基高丝氨酸内酯（阿勒）的群体感应在革兰氏阴性细菌中广泛存在。已经开发了几种用于阿勒检测的细菌全细胞生物传感器，其中一些用于阿勒生产的原位研究。通过这些研究，我们对群体感应在各种环境中的意义的认识得到了提高。然而，对土壤环境中AHLs的产生知之甚少。在本研究中，一种方法来检测阿勒产生的散装土壤。构建了一种基于来自费氏弧菌的lux操纵子的调节区融合的全细胞生物传感器，导致luxR-P-luxI-gfpmut 3 *-融合在高拷贝质粒pAHL-GFP中。携带pAHL-GFP的大肠杆菌MC 4100通过表达绿色荧光响应AHL化合物N-辛酰基高丝氨酸内酯（OHL）。原位应用E. coliMC 4100/pAHL-GFP的转化率。E. coliMC 4100/pAHL-GFP在土壤微宇宙中培养，用改良的Nycodenz-extraction法进行提取。然后通过流式细胞术的单细胞分析验证诱导细胞的存在。OHL浓度在0.5和50纳摩尔每克土壤之间进行检测。当将产AHL的产酶沙雷氏菌引入土壤微宇宙中时，绿色荧光蛋白在E. coli MC4100/pAHL-GFP。因此，该菌株检测S.在无菌土壤中的芽孢杆菌。使用改进的提取方法和全细胞生物传感器结合流式细胞仪分析被证明是有前途的工具，在未来的研究阿勒生产的微生物种群在土壤环境中。
Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to & was constructed, resulting in a luxR-P-luxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.