Sekar Raju, Fuchs Bernhard M, Amann Rudolf, Pernthaler Jakob
Max-Planck-Institut für Marine Mikrobiologie, Celsiusstrasse 1, D-28359 Bremen, Germany.
Appl Environ Microbiol. 2004 Oct;70(10):6210-9. doi: 10.1128/AEM.70.10.6210-6219.2004.
We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.
我们描述了一种方法，即在与靶向rRNA的辣根过氧化物酶标记寡核苷酸探针原位杂交并进行催化荧光报告沉积（CARD-FISH）后，通过流式细胞术对北海沿岸浮游细菌中的细胞进行分选。在2003年春季的一个样本中，使用细菌探针（EUB338）通过CARD-FISH检测到超过90%的细胞。大约30%的微生物群落属于拟杆菌门的噬纤维菌-黄杆菌谱系（CFB组）（探针CF319a），近10%的细胞被β-变形菌探针（探针BET42a）靶向。优化了一个方案，以从膜过滤器上分离与EUB338、BET42a和CF319a杂交的细胞（回收率为70%），并通过流式细胞术对细胞进行分选。分选细胞的纯度>95%。从杂交和分选的细胞（S-EUB、S-BET和S-CF文库）以及未杂交和未分选的细胞（UNHYB文库）构建了16S rRNA基因克隆文库。与CFB组相关的序列在S-CF文库中（66%）比在UNHYB文库中（13%）明显更频繁。在S-BET文库中未发现β-变形菌序列类型的富集，但仅在该文库中发现了与亚硝化螺菌相关的新序列。如通过使用特异性探针的CARD-FISH所确定的，这些细菌与海洋进化枝OM43的成员一起，占水样中β-变形菌的>9%。这表明CARD-FISH和流式分选相结合可能是研究水生栖息地中不同细菌种群的多样性、潜在活性和基因组的有力方法。
