Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time
Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time
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DOI：
10.1073/pnas.0700496104
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发表时间：
2007-05-22
影响因子：
11.1
通讯作者：
Sieracki, Michael E.
中科院分区：
文献类型：
--
作者：
Stepanauskas, Ramunas;Sieracki, Michael E.
由于当前宏基因组学和细胞培养方法的限制，鉴定具有特定代谢能力的优势微生物类群仍然是环境微生物学中最大的挑战之一。我们报告的结果从单个海洋细菌细胞中的多个基因的直接分析，展示了高通量代谢分配尚未培养的类群的潜力。该方案使用高速荧光激活细胞分选，全基因组多重置换扩增（MDA），和随后的PCR筛选。从缅因州湾浮游细菌构建了11个单扩增基因组（SAG）的中试文库作为概念验证。该文库由五种黄杆菌属、一种鞘细菌属、四种α-变形菌属和一种γ-变形菌属组成。除α变形菌外，大多数SAG与现有分离株的遗传距离较远，16 S rRNA基因序列具有88-97%的同一性。因此，单细胞MDA提供了获得数量上占优势但尚未培养的分类群体的基因组材料。SAG文库中5个黄细菌中有2个含有蛋白视紫质基因，表明黄细菌是这种光代谢系统的主要载体之一。pufM和nasA基因在一些100细胞的MIDA产物中被检测到，但在SAG中未检测到，这表明含有细菌叶绿素和同化硝酸还原酶的生物体构成了
The identification of predominant microbial taxa with specific metabolic capabilities remains one the biggest challenges in environmental microbiology, because of the limits of current metagenomic and cell culturing methods. We report results from the direct analysis of multiple genes in individual marine bacteria cells, demonstrating the potential for high-throughput metabolic assignment of yet-uncultured taxa. The protocol uses high-speed fluorescence-activated cell sorting, whole-genome multiple displacement amplification (MDA), and subsequent PCR screening. A pilot library of 11 single amplified genomes (SAGs) was constructed from Gulf of Maine bacterioplankton as proof of concept. The library consisted of five flavobacleria, one sphingobacterium, four alphaproteobacteria, and one gammaproteobacterium. Most of the SAGs, apart from alphaproteobacteria, were phylogenetically distant from existing isolates, with 88-97% identity in the 16S rRNA gene sequence. Thus, single-cell MDA provided access to the genomic material of numerically dominant but yet-uncultured taxonomic groups. Two of five flavobacteria in the SAG library contained proteorhodopsin genes, Suggesting that flavobacteria are among the major carriers of this photometabolic system. The pufM and nasA genes were detected in some 100-cell MIDA products but not in SAGs, demonstrating that organisms containing bacteriochlorophyll and assimilative nitrate reductase constituted