Pilhofer Martin, Pavlekovic Marko, Lee Natuschka M, Ludwig Wolfgang, Schleifer Karl-Heinz
Technische Universität München, Lehrstuhl für Mikrobiologie, Am Hochanger 4, 85350 Freising, Germany.
Syst Appl Microbiol. 2009 May;32(3):186-92. doi: 10.1016/j.syapm.2008.12.007. Epub 2009 Feb 12.
Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.
尽管环境微生物学的一个主要目标是将功能/活性与生物体的身份联系起来，但关于细菌原位mRNA检测的报道却很少。本研究报告了一种基于先前描述的pmoA方案，使用荧光杂交原位检测nifH mRNA的可靠方法。nifH编码二氮酶还原酶，这是固氮过程中的一种关键酶。nifH mRNA与地高辛标记的多核苷酸探针杂交。用辣根过氧化物酶标记的抗地高辛抗体检测杂交体。随后，通过用荧光染料标记的酪胺进行催化报告沉积（CARD）来放大信号。此外，使用rRNA的标准荧光原位杂交来鉴定成像的生物体。因此，该方法使我们能够特别地将生物体固氮活性的原位信息与其身份联系起来。出乎意料的是，nifH mRNA杂交产生的信号在细胞内呈现出明显的不均匀模式。这表明所使用的方法甚至可以深入了解检测到的mRNA在细胞内的定位，这是一种尚未见报道的细菌细胞mRNA荧光原位杂交（FISH）的潜在用途。
