The microbial H₂-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

微生物H ₂膜相关的CO脱氢酶（CODH）/能量转化的氢酶（ECH）络合物产生（产氢）一氧化碳（CO）的氧化活性是微生物群落中重要的代谢过程。然而，对氢生营养养生类营养的研究由于其罕见的丰富性而不得不依靠固有的培养和分离方法，这是生态学研究的瓶颈。在这里，我们提供了以基因为靶标的测序方法，用于嗜热产氢羧营养生物的多样性估计。我们设计了六对新的简并引物对，这些引物对有效地扩增了Firmicutes中与ECH基因（CODHech基因）形成基因簇的CODH基因的编码区，其中包括陆地热生境中的主要嗜热性产氢营养型。通过这些引物的扩增子测序，使用了来自陆地热液沉积物的DNA和CO-气体培养的样品，可以特异性地检测到多个与Firmictes中的CODHech基因相同或在系统发育上相关的CODH基因。此外，我们发现系统发育上不同的CODHech基因在CO气体培养的样品中富集，这表明我们的引物也检测到未培养的产氢羧基营养族。新的靶向CODH的引物通过扩增子测序为我们提供了嗜热产氢羧化营养菌的细粒度（〜97.9％的核苷酸序列同一性）多样性分析，将有助于对这些微生物进行生态学研究。