Eilers H, Pernthaler J, Glöckner F O, Amann R
Max-Planck-Institut für marine Mikrobiologie, D-28359 Bremen, Germany.
Appl Environ Microbiol. 2000 Jul;66(7):3044-51. doi: 10.1128/AEM.66.7.3044-3051.2000.
The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.
通过多种培养策略与基于非培养的16S rRNA技术相结合，对北海浮游细菌中大量细菌域成员的可培养性进行了研究。我们从环境DNA中检索16S rRNA基因（rDNA）克隆，并通过荧光原位杂交（FISH）确定不同类群和属的原位丰度。通过在贫营养培养基上平板培养建立了一个包含145株菌株的培养物库。通过FISH、扩增核糖体DNA限制性分析（ARDRA）以及代表性16S rDNA测序对分离株进行筛选。大多数分离株属于假交替单胞菌属、交替单胞菌属和弧菌属。尽管它们易于培养，但在浮游细菌群落中仅占一小部分。在16S rDNA文库中未检测到它们，FISH显示它们作为大型、富含rRNA、与颗粒相关的细菌很少出现（占总细胞数的不到1%）。相反，噬纤维菌-黄杆菌群和γ-变形菌SAR86簇的大量成员，通过FISH分别鉴定为北海浮游细菌中总细胞数的17%至30%和高达10%，很少或根本无法培养。虽然与SAR86相关的克隆在16S rDNA文库中占主导地位（53个克隆中有44个），但未检索到与噬纤维菌-黄杆菌簇相关的克隆。唯一易于培养的大量海洋细菌群与玫瑰杆菌属有关。该群体在夏季占总细胞数的10%，相应序列也存在于我们的克隆文库中。对所有分离株的ARDRA模式进行的稀疏分析表明，我们方法所能培养的总多样性很高，分离菌株的数量尚未涵盖，但对于γ-变形菌来说几乎已饱和。这预示着在没有新的分离技术的情况下，不可培养海洋细菌，特别是γ-变形菌SAR86簇的分离存在局限性，并因此与关于海洋浮游细菌可培养性的更乐观观点形成对比。
