Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany.
Environ Microbiol. 2010 Nov;12(11):3057-73. doi: 10.1111/j.1462-2920.2010.02281.x.
Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH - a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA-targeted catalysed reporter deposition - fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T(m) of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalysing the oxidation of ammonia.
随着测序技术的进步和新基因的发现，我们对尚未培养的微生物代谢潜力的认识有了极大的提高。另一方面，由于这些序列通常存在于没有细胞身份直接标记物的基因组片段上，例如 rRNA 基因，因此很难可靠地将特定基因分配到特定的进化枝中。因此，本研究的目的是开发基因 FISH——一种将基因存在与环境样品中细胞身份联系起来的方案，其信号可以在单细胞水平上可视化。该方案结合了靶向 rRNA 的催化报告物沉积——荧光原位杂交和原位基因检测。为了测试该方案，将其应用于本格拉上升流系统的海水样本中。为了进行基因检测，使用了多核苷酸探针混合物，该混合物是根据从每个海水样本制备的古菌 amoA 克隆文库设计的。混合物中的每个探针都被选择与具有高达 5%错配的靶标结合。为了确定杂交参数，根据理论计算和体外测量来估计探针、靶标和杂交体的 T(m)。结果表明，这些样品中至少有 30%（但可能是大多数）的古菌携带 amoA 基因，因此很可能催化氨的氧化。
