Chouari Rakia, Le Paslier Denis, Daegelen Patrick, Ginestet Philippe, Weissenbach Jean, Sghir Abdelghani
CNRS-UMR 8030, Genoscope and Université d'Evry Val d'Essonne, 2, rue Gaston Crémieux, 91057 Evry, France.
Environ Microbiol. 2005 Aug;7(8):1104-15. doi: 10.1111/j.1462-2920.2005.00795.x.
A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.
采用一种不依赖培养的分子系统发育方法来研究厌氧污泥消化器中的原核生物多样性。使用总基因组DNA构建了两个16S rRNA基因文库，并使用针对古菌或细菌结构域的引物通过聚合酶链反应（PCR）进行扩增。分别使用ARB软件包对古菌和细菌的246个和579个几乎全长的16S rRNA基因进行系统发育分析。与古菌相关的系统发育类群属于广古菌门和泉古菌门。有趣的是，我们检测到一个由164个克隆组成的新单系群，占古菌文库的66.6%。培养富集和探针杂交表明，该类群在甲酸盐或H2-CO2条件下生长更好。在细菌文库中，95.6%的操作分类单元（OTU）代表了以前从未描述过的新的假定系统发育型，并隶属于8个门类。拟杆菌门是最丰富和最多样化的系统发育类群，占OTU的38.8%，其次是革兰氏阳性菌（27.7%）和变形菌门（21.3%）。与培养代表较少的系统发育门类（如绿弯菌门、互养菌门、栖热袍菌目）或候选门类（如OP9和OP8）相关的序列占总OTU的比例不到5%。使用一套完整的15个针对16S和23S rRNA的寡核苷酸杂交探针，通过点杂交对12个消化器样品中的这些主要类群进行定量。与克隆文库不同，厚壁菌门和放线菌门加起来占21.8±14.9%，是最丰富的门类。令人惊讶的是，其次是绿弯菌门，占总16S rRNA的20.2±4.6%。变形菌门和拟杆菌门分别占14.4±4.9%和14.5±4.3%，一个新的谱系WWE1占11.9±3.1%而浮霉菌门和互养菌门各占不到2%。使用这套新探针，我们将消化器样品中细菌群体的覆盖范围从总rRNA的52%扩展到了85.3%。
