Dahlberg C, Bergström M, Hermansson M
Göteborg University, Department of Cell and Molecular Biology, Microbiology, SE-405 30 Göteborg, Sweden.
Appl Environ Microbiol. 1998 Jul 1;64(7):2670-5. doi: 10.1128/AEM.64.7.2670-2675.1998.
Gene transfer of the conjugative plasmid pBF1 from Pseudomonas putida to indigenous bacteria in seawater was investigated with a detection system for gene transfer based on the green fluorescent protein (GFP) (C. Dahlberg et al., Mol. Biol. Evol. 15:385-390, 1998). pBF1 was tagged with the gfp gene controlled by a lac promoter which is down regulated in the donor cell by a chromosomal repressor (lacIq). The plasmid donor cells (Pseudomonas putida KT2442) subsequently do not express gfp. Transfer to recipient strains lacking the repressor results in expression of gfp. The transconjugant can subsequently be detected by epifluorescence microscopy on a single-cell level. By using this method, transfer of pBF1::gfp and expression of the gfp gene were first shown to occur during nutrient-limiting conditions to several defined recipient bacteria in artificial seawater. Second, we measured transfer of pBF1 from P. putida to the marine bacterial community directly in seawater samples, on a single-cell level, without limiting the detection of gene transfer to the culturable fraction of bacteria. Plasmid transfer was detected on surfaces and in bulk seawater. Seawater bacteria with different morphologies were shown to receive the plasmid. Gene transfer frequencies of 2.3 x 10(-6) to 2.2 x 10(-4) transconjugants per recipient were recorded after 3 days of incubation.
利用基于绿色荧光蛋白（GFP）的基因转移检测系统（C. 达尔伯格等人，《分子生物学与进化》15:385 - 390，1998年），研究了接合性质粒pBF1从恶臭假单胞菌向海水中土著细菌的基因转移。pBF1用受lac启动子控制的gfp基因进行标记，该启动子在供体细胞中被染色体阻遏物（lacIq）下调。因此，质粒供体细胞（恶臭假单胞菌KT2442）不表达gfp。转移到缺乏阻遏物的受体菌株会导致gfp表达。随后可通过落射荧光显微镜在单细胞水平上检测转接合子。通过使用这种方法，首次表明pBF1::gfp的转移和gfp基因的表达在营养限制条件下发生在人工海水中的几种特定受体细菌中。其次，我们在单细胞水平上直接测量了pBF1从恶臭假单胞菌向海水样本中海洋细菌群落的转移，而不将基因转移的检测局限于可培养的细菌部分。在海水表面和主体海水中都检测到了质粒转移。具有不同形态的海水细菌被证明接受了该质粒。孵育3天后，记录到每个受体的转接合子基因转移频率为2.3×10⁻⁶至2.2×10⁻⁴。
