Ravel J, Wellington E M, Hill R T
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, USA.
Appl Environ Microbiol. 2000 Feb;66(2):529-34. doi: 10.1128/AEM.66.2.529-534.2000.
The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30 degrees C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 x 10(-4) and 3.6 x 10(-5) CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.
在无菌土壤微观系统中研究了两种巨大线性质粒的种间转移。质粒pRJ3L（322 kb）和pRJ28（330 kb）均编码汞抗性，它们在改良土壤微观系统中分别从链霉菌宿主（分离株CHR3和CHR28）成功转移至无质粒且对汞敏感的菌株天蓝色链霉菌TK24。在30℃孵育2至3天后首次观察到天蓝色链霉菌TK24的接合子，这与土壤中菌丝体形成所需时间一致。pRJ3L和pRJ28的转移频率分别为4.8×10⁻⁴和3.6×10⁻⁵ CFU/供体基因组。通过脉冲场凝胶电泳分析接合子中质粒的存在情况，并通过限制性酶切确认质粒的同一性。全基因组DNA酶切证实接合子为天蓝色链霉菌TK24。通过杂交分析表明汞抗性基因存在于接合子的质粒上且仍具有功能。这是首次在无菌土壤微观系统中证明巨大线性质粒的转移。在从美国波士顿港、澳大利亚汤斯维尔港和阿根廷图库曼省萨利河受汞污染沉积物中分离出的许多链霉菌属中检测到巨大线性质粒。已表明其中一些质粒上存在汞抗性基因。我们的研究结果表明，巨大线性质粒可在链霉菌属之间转移且在环境链霉菌分离株中普遍存在，这表明这些质粒在环境中链霉菌之间的基因转移中很重要。
