Lee S, Fuhrman J A
Marine Sciences Research Center, State University of New York, Stony Brook 11794-5000.
Appl Environ Microbiol. 1990 Mar;56(3):739-46. doi: 10.1128/aem.56.3.739-746.1990.
Little is known about the species composition and variability of natural bacterial communities, mostly because conventional identification requires pure cultures, but less than 1% of active natural bacteria are cultivable. This problem was circumvented by comparing species compositions via hybridization of total DNA of natural bacterioplankton communities for the estimation of the fraction of DNA in common between two samples (similarity). DNA probes that were labeled with 35S by nick translation were hybridized to filter-bound DNA in a reciprocal fashion; similarities (in percent) were calculated by normalizing the values to self-hybridizations. In tests with DNA mixtures of pure cultures, the experimentally observed similarities agreed with expectations. However, reciprocal similarities (probe and target reversed) were often asymmetric, unlike those of DNA from single strains. This was due to the relative complexity and G + C content of DNA, which provided a means to interpret the asymmetry that was occasionally observed in natural samples. Natural bacteria were collected by filtration from Long Island Sound (LIS), N.Y., the Caribbean and Sargasso seas, and a coral reef lagoon near Bermuda. The samples showed similarities of less than 10 to 95%. The LIS and Sargasso and Caribbean sea samples were 20 to 50% similar to each other. The coral reef sample was less than 10% similar to the others, indicating its unique composition. Seasonality was also observed; an LIS sample obtained in the autumn was 40% similar to two LIS samples obtained in the summer; these latter two samples were 95% similar. We concluded that total DNA hybridization is a rapid, simple, and unbiased method for investigating the variation of bacterioplankton species composition over time and space, avoiding the need of culturing.
对于自然细菌群落的物种组成和变异性，人们了解甚少，主要是因为传统鉴定需要纯培养物，但活性天然细菌中可培养的不到1%。通过比较天然浮游细菌群落总DNA的杂交物种组成来估计两个样本之间共有DNA的比例（相似度），从而规避了这个问题。通过缺口平移用35S标记的DNA探针以相互的方式与固定在滤膜上的DNA杂交；通过将值归一化为自身杂交来计算相似度（百分比）。在纯培养物DNA混合物的测试中，实验观察到的相似度与预期相符。然而，与单菌株DNA的相似度不同，相互相似度（探针和靶标颠倒）往往不对称。这是由于DNA的相对复杂性和G + C含量，这提供了一种解释在天然样本中偶尔观察到的不对称性的方法。从纽约长岛海峡（LIS）、加勒比海和马尾藻海以及百慕大附近的珊瑚礁泻湖通过过滤收集天然细菌。样本显示相似度低于10%至95%。LIS与马尾藻海和加勒比海样本彼此之间的相似度为20%至50%。珊瑚礁样本与其他样本的相似度低于10%，表明其独特的组成。还观察到了季节性；秋季获得的一个LIS样本与夏季获得的两个LIS样本的相似度为40%；后两个样本的相似度为95%。我们得出结论，总DNA杂交是一种快速、简单且无偏差的方法，用于研究浮游细菌物种组成随时间和空间的变化，无需培养。
