Béjà O, Suzuki M T, Koonin E V, Aravind L, Hadd A, Nguyen L P, Villacorta R, Amjadi M, Garrigues C, Jovanovich S B, Feldman R A, DeLong E F
Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039-0628, USA.
Environ Microbiol. 2000 Oct;2(5):516-29. doi: 10.1046/j.1462-2920.2000.00133.x.
Cultivation-independent surveys of ribosomal RNA genes have revealed the existence of novel microbial lineages, many with no known cultivated representatives. Ribosomal RNA-based analyses, however, often do not provide significant information beyond phylogenetic affiliation. Analysis of large genome fragments recovered directly from microbial communities represents one promising approach for characterizing uncultivated microbial species better. To assess further the utility of this approach, we constructed large-insert bacterial artificial chromosome (BAC) libraries from the genomic DNA of planktonic marine microbial assemblages. The BAC libraries we prepared had average insert sizes of 80 kb, with maximal insert sizes > 150 kb. A rapid screening method assessing the phylogenetic diversity and representation in the library was developed and applied. In general, representation in the libraries agreed well with previous culture-independent surveys based on polymerase chain reaction (PCR)amplified rRNA fragments. A significant fraction of the genome fragments in the BAC libraries originated from as yet uncultivated microbial species, thought to be abundant and widely distributed in the marine environment. One entire BAC insert, derived from an uncultivated, surface-dwelling euryarchaeote, was sequenced completely. The planktonic euryarchaeal genome fragment contained some typical archaeal genes, as well as unique open reading frames (ORFs) suggesting novel function. In total, our results verify the utility of BAC libraries for providing access to the genomes of as yet uncultivated microbial species. Further analysis of these BAC libraries has the potential to provide significant insight into the genomic potential and ecological roles of many indigenous microbial species, cultivated or not.
对核糖体RNA基因进行的非培养法调查揭示了新型微生物谱系的存在，其中许多谱系尚无已知的培养代表菌株。然而，基于核糖体RNA的分析通常除了系统发育归属外，无法提供重要信息。直接从微生物群落中回收的大片段基因组分析是一种更深入了解未培养微生物物种特征的有前景的方法。为了进一步评估这种方法的实用性，我们从浮游海洋微生物群落的基因组DNA构建了大插入片段细菌人工染色体（BAC）文库。我们制备的BAC文库平均插入片段大小为80 kb，最大插入片段大小>150 kb。我们开发并应用了一种快速筛选方法来评估文库中的系统发育多样性和代表性。总体而言，文库中的代表性与先前基于聚合酶链反应（PCR）扩增的rRNA片段的非培养法调查结果吻合良好。BAC文库中的很大一部分基因组片段源自尚未培养的微生物物种，这些物种被认为在海洋环境中数量丰富且分布广泛。对一个源自未培养的、生活在海洋表层的广古菌的完整BAC插入片段进行了全序列测定。该浮游广古菌基因组片段包含一些典型的古菌基因以及一些暗示新功能的独特开放阅读框（ORF）。总的来说，我们的结果证实了BAC文库在获取尚未培养的微生物物种基因组方面的实用性。对这些BAC文库的进一步分析有可能为许多本地微生物物种（无论是否已培养）的基因组潜力和生态作用提供重要见解。
