古菌在环境中广泛存在,在不同的生态系统中发挥着重要作用;然而,他们独特的生物学特征需要先进的工具。这在表征基因功能时尤其具有挑战性。这里,我们在产甲烷古细菌甲烷球菌模型中生成随机条形码转座子文库,并使用高通量生长方法在超过100种独特的生长条件下进行适应性测定(RB-TnSeq)。用我们的方法,我们发现了与营养利用和氧化应激反应相关的新基因.我们确定了在M.maripaludis中使用多种氮源的新基因,包括推定的丙氨酸脱氨调节剂和对固氮重要的钼酸盐转运蛋白。此外,利用健身数据,我们推断M.maripaludis可以利用额外的氮源,包括β-谷氨酰胺,-葡糖醛酸酰胺,和腺苷。在自养生长条件下,我们确定了一个编码未知功能域(DUF166)的基因,该基因对适应度很重要,并假设它在二氧化碳同化中具有辅助作用.最后,比较氧气和亚硫酸盐压力的健身成本,我们确定了以前未表征的一类异化亚硫酸盐还原酶样蛋白(Dsr-LP;IIId组),在亚硫酸盐存在下的生长过程中很重要。当过度表达时,DSr-LP赋予了亚硫酸盐抗性,并且使得能够使用亚硫酸盐作为唯一的硫源。此处采用的高通量方法允许生成大规模数据集,该数据集可以用作进一步了解古细菌域中基因功能和代谢的资源。重要古细菌在环境中普遍存在,然而,他们生物学的基本方面仍未得到充分的探索。为了解决这个问题,我们将随机条形码转座子文库(RB-TnSeq)应用于模型古细菌甲烷球菌。RB-TnSeq与在超过100个独特条件下的高通量生长测定相结合,确定了以前未表征的基因的作用。包括几种具有未知功能结构域(DUFs)的编码蛋白。我们还扩展了对碳和氮代谢的理解,并将IIId族异化亚硫酸盐还原酶样蛋白表征为功能性亚硫酸盐还原酶。此数据集涵盖了广泛的附加条件,包括压力,固氮,补充氨基酸,和自力更生,从而为古细菌群落提供了广泛的数据集,以用于表征未知功能的其他基因。
Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.