What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH.
What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH.
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DOI：
10.1371/journal.pone.0017769
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发表时间：
2011-03-17
期刊：
影响因子：
3.7
通讯作者：
Emerson D
中科院分区：
文献类型：
--
作者：
Fleming EJ;Langdon AE;Martinez-Garcia M;Stepanauskas R;Poulton NJ;Masland ED;Emerson D
赭纤发菌是淡水铁渗漏和富铁湿地的常见居民。它的定义特征是大量生产的细胞外鞘与铁羟基氧化物。令人惊讶的是，这些鞘中超过90%是空的，因此，看似丰富的铁氧化细菌群体，却由相对较少的细胞组成。因为L.赭石已被证明很难培养，它的识别是完全基于生境偏好和形态。我们利用独立于种植的技术来解决这个长期存在的谜团。通过选择含有纤毛菌的铁垫的活跃生长边缘，获得常规SSU rRNA基因克隆文库，其中有29个克隆（占总文库的42%）与纤毛菌/球衣菌群相关（与培养代表物的一致性≤96%）。从散装垫构建的V4高变区的pyrotagged文库显示，总序列的7.2%也属于发纤菌/球衣菌组。单个L. ochracea鞘，随后通过全基因组扩增（WGA）和PCR鉴定了与推定的发纤菌属克隆和pyrotags紧密聚类的SSU rRNA序列。利用这些数据，荧光原位杂交（FISH）探针，Lepto 175，被设计为绑定到ensheathed细胞。该探针的定量使用表明，在一个活跃增生的铁垫中，高达35%的微生物细胞是L。赭色。L. ochracea与栽培近缘种L. cholodnii，证明L.赭曲霉确实与这组形态相似的丝状鞘状微生物有关。
Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.
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