Bacterial plasmid conjugation on semi-solid surfaces monitored with green fluorescent protein (GFP) from Aequorea victoria as a marker
Bacterial plasmid conjugation on semi-solid surfaces monitored with green fluorescent protein (GFP) from Aequorea victoria as a marker
复制标题
DOI：
10.1016/0378-1119(95)00707-5
复制
发表时间：
1996-07-01
期刊：
影响因子：
3.5
通讯作者：
Molin, S
中科院分区：
文献类型：
--
作者：
Christensen, BB;Sternberg, C;Molin, S
在恶臭假单胞菌（Pp）KT 2442微菌落中在半固体琼脂表面上检查TOL质粒的水平转移。水平基因转移通常在大群体中进行研究，其中所有信息都基于整个群体中转移事件的平均估计。我们已经使用了绿色荧光蛋白（GFP）从水母Aequorea维多利亚作为质粒标记，结合单细胞观察。这提供了关于接合活性细胞分布的迄今未知的细节。在本研究中，含有从TOL质粒上的噬菌体T7 Phi 10启动子表达的gfp基因的供体细胞和表达相应的噬菌体RNA聚合酶的受体细胞允许我们在用蓝光（470-490 nm）照射时监测作为绿色荧光细胞的前接合物的出现。此外，用luxAB基因标记受体以区分供体细胞的小集落与受体细胞。我们的结论是，接合质粒转移在PP KT 2442细胞的半固体表面上主要发生在一段很短的时间后，最初接触的捐助者和收件人，表明传播的TOL质粒是有限的静态，但可行的文化。
Horizontal transfer of the TOL plasmid was examined in Pseudomonas putida (Pp) KT2442 micro-colonies on semisolid agar surfaces. Horizontal gene transfer is usually studied in large populations where all information is based on average estimates of the transfer events in the entire population. We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a plasmid marker, in combination with single-cell observations. This provided hitherto unknown details on the distribution of cells active in conjugation, In the present study, donor cells containing the gfp gene expressed from the bacteriophage T7 Phi 10 promoter on the TOL plasmid, and recipient cells expressing the corresponding phage RNA polymerase allowed us to monitor the occurrence of ex-conjugants as green fluorescent cells upon illumination with blue light (470-490 nm). Further, the recipients were labeled with the luxAB genes to distinguish micro-colonies of donor cells from recipient cells. We conclude that conjugal plasmid transfer in Pp KT2442 cells on semi-solid surfaces occurs mainly during a short period of time after the initial contact of donors and recipients, indicating that spread of the TOL plasmid is limited in static, but viable cultures.