Establishment of a gene transfer system for Rhodothermus marinus
Establishment of a gene transfer system for Rhodothermus marinus
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DOI：
10.1007/s00253-004-1730-3
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发表时间：
2005-03-01
影响因子：
5
通讯作者：
Eggertsson, G
中科院分区：
文献类型：
--
作者：
Bjornsdottir, SH;Thorbjarnardottir, SH;Eggertsson, G
由于缺乏基因转移的选择系统，海洋红栖热菌的遗传操作受到阻碍。我们报道了一个Rhodothermus/ Escherichia coli穿梭质粒的构建。marinustrpB基因是基于pUC 18和隐性R. marinus质粒pRM 21。无质粒R.根据生长特性和限制酶活性的缺乏选择marinus受体菌株。利用电穿孔法将穿梭质粒pRM 100成功地导入受体菌株的TrpB（-）突变体中，并发现其转化为原养型。在非选择性培养基中生长80代后，未观察到pRM 100的丢失或重排。pRM 100和亲本质粒pRM 21的相对拷贝数分别测定为7 +/-1和42 +/-4。穿梭质粒用于优化电穿孔方案，并且在22.5 kV/cm、200 Ω和25 μ F下获得的转化体的最大数目为4.3 +/- 0.7 x 10（6）cfu/mug DNA。然而，在根据几种方案化学制备细胞后，转化失败。这是在Rhodothermus属中首次报道的遗传转化。
Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/ Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB(-) mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7 +/- 1 and 42 +/- 4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3 +/- 0.7 x 10(6) cfu/mug DNA at 22.5 kV/cm, 200 Omega and 25 muF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.