Department of Civil and Environmental Engineering, 4105 Seamans Center, The University of Iowa, Iowa City, IA 52242, USA.
Department of Civil and Environmental Engineering, 4105 Seamans Center, The University of Iowa, Iowa City, IA 52242, USA.
J Microbiol Methods. 2021 Feb;181:106147. doi: 10.1016/j.mimet.2021.106147. Epub 2021 Jan 22.
Fluorescence in situ hybridization (FISH) can provide information on the morphology, spatial arrangement, and local environment of individual cells enabling the investigation of intact microbial communities. GeneFISH uses polynucleotide probes and enzymatic signal amplification to detect genes that are present in low copy numbers. Previously, this technique has only been applied in a small number of closely related organisms. However, many important functional genes, such as those involved in xenobiotic degradation or pathogenesis, are present in diverse microbial strains. Here, we present a geneFISH method for the detection of the functional gene etnC, which encodes the alpha subunit of an alkene monooxygenase used by aerobic ethene and vinyl chloride oxidizing bacteria (etheneotrophs). The probe concentration was optimized and found to be 100 pg/μl, similar to previous geneFISH reports. Permeabilization was necessary for successful geneFISH labeling of Mycobacteria; sequential treatment with lysozyme and achromopeptidase was the most effective treatment. This method was able to detect etnC in several organisms including Mycobacteria and Nocardioides, demonstrating for the first time that a single geneFISH probe can detect a variety of alleles (>80% sequence similarity) across multiple species. Detection of etnC with geneFISH has practical applications for bioremediation. This method can be readily adapted for other functional genes and has broad applications for investigating microbial communities in natural and engineered systems.
荧光原位杂交（FISH）可以提供有关单个细胞的形态、空间排列和局部环境的信息，从而能够研究完整的微生物群落。GeneFISH 使用多核苷酸探针和酶促信号放大来检测低拷贝数存在的基因。以前，该技术仅应用于少数密切相关的生物体。然而，许多重要的功能基因，如参与异生物质降解或发病机制的基因，存在于不同的微生物菌株中。在这里，我们提出了一种用于检测功能基因 etnC 的 GeneFISH 方法，该基因编码用于好氧乙烯和氯乙烯氧化细菌（乙烯营养菌）的烯烃单加氧酶的α亚基。优化了探针浓度，发现其为 100 pg/μl，与以前的 GeneFISH 报告相似。需要透化才能成功地对分枝杆菌进行 GeneFISH 标记；溶菌酶和无色素肽酶的顺序处理是最有效的处理方法。该方法能够检测到包括分枝杆菌和诺卡氏菌在内的几种生物体中的 etnC，这首次证明了单个 GeneFISH 探针可以检测多种等位基因（>80%的序列相似性）在多个物种中。使用 GeneFISH 检测 etnC 具有生物修复的实际应用。该方法可以很容易地适应其他功能基因，并广泛应用于研究自然和工程系统中的微生物群落。
