Sakoula Dimitra, Schatteman Arne, Blom Pieter, Jetten Mike S M, van Kessel Maartje A H J, Lehtovirta-Morley Laura, Lücker Sebastian
Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands.
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, United Kingdom.
ISME Commun. 2024 Jul 11;4(1):ycae092. doi: 10.1093/ismeco/ycae092. eCollection 2024 Jan.
Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms.
最近，一种基于活性的标记方案可用于体内检测氨氧化细菌和烷烃氧化细菌。这种功能标记技术能够对这些在环境中广泛存在的功能基团进行靶向研究，但它无法捕获氨氧化古菌（AOA）。自首次发现以来，AOA已成为生物地球化学氮循环中的关键参与者，但我们对它们在自然和工程生态系统中的分布和丰度的了解主要来自基于PCR和宏基因组学的研究。此外，古菌氨单加氧酶与细菌的氨单加氧酶明显不同，人们对其了解仍然很少。在这里，我们报告了一种基于活性的标记方案的开发，用于荧光检测所有氨氧化和烷烃氧化原核生物，包括AOA。在该方案中，1,5-己二炔用作氨和烷烃氧化的抑制剂以及用于细胞荧光标记的双功能酶探针——铜（I）催化的炔烃-叠氮环加成反应。除了对氨氧化和烷烃氧化微生物进行高效的基于活性的标记外，该方法还可与反卷积显微镜结合使用，以确定其氨氧化和烷烃氧化酶系统的亚细胞定位。在多种氨氧化和烷烃氧化微生物中对这些酶进行标记，使它们能够在细胞质膜、氨氧化和甲烷氧化细菌的胞内膜堆叠上可视化，并且有趣的是，在一种AOA物种的囊泡状结构上也能可视化。这种用于氨氧化和烷烃氧化菌的新型基于活性的标记方法的开发，将是可用于硝化和烷烃氧化微生物研究的不断扩展的分子工具箱中的一项有价值的补充。
