Dissecting the black box of microbially mediated pyrite formation from FeS and H2S

FeS与H_2S的剧烈反应生成了FeS_2(黄铁矿)和H_2，被认为是原始地球能量代谢的早期形式。太古代以来，沉积黄铁矿建造在全球铁、硫循环中起主要作用，直接影响大气的氧化还原状态。然而，沉积黄铁矿的形成机制和微生物对这一过程的贡献仍在争论中。今年，我们发表了第一个浓缩培养物的工作，它能够以FeS、H2S和CO2作为唯一底物生长，以产生FeS2和CH4(Thiel等人，2019年，PNAS)。这一建议旨在阐明微生物介导的黄铁矿形成与产甲烷作用的机制。将检验两个假设，以揭示浓缩J5中的黄铁矿形成是否直接涉及能源节约。假设I(H-I)将解决这样一个问题，即参与黄铁矿形成的微生物是否有可能利用Wächtershäuser反应，即从FeS和H2S直接形成FeS2和H2以实现能量守恒。这将需要从细胞表面到细胞膜或细胞质的长距离电子转移，例如涉及多血红素细胞色素c复合体。假设II(H-II)将测试非产甲烷伙伴的能量代谢是否限于硫呼吸的逆转(硫化物转化为零价硫和氢)，随后的黄铁矿形成是由形成的零价硫与FeS的(可能是非生物的)反应所介导的。这两个假设将在三个互补的工作包(WP)中进行检验。在WP1中，浓缩J5成员的高质量草稿基因组将通过元基因组学获得，并根据其潜在的能量代谢进行注释，包括指示H-I或H-II的标志蛋白。随后的转译将跟随编码这些标志蛋白的基因的表达。在WP2中，将通过荧光原位杂交和单细胞拉曼显微光谱相结合的方法来鉴定浓缩J5中的单个群落成员。后者将用于识别拉曼信号，表明过表达的多血红素细胞色素C氧化还原复合体(H-I)或微生物细胞内或与微生物细胞相关的零价硫的形成(H-II)。在WP3中，浓缩J5将暴露在不同的生长条件下，据推测，这些条件需要与H-I和H-II中列出的类似的酶复合体。这将包括元素铁在硫酸盐还原条件下的氧化(H-I)和氢与元素硫一起氧化成硫化氢(H-II)。在正生长的情况下，表达的基因之后将进行后转录。该项目对于建立富集型J5作为微生物黄铁矿形成模式具有重要意义，这将对我们理解生物地球化学硫和铁的循环以及生命起源假说产生影响。

The exergonic reaction of FeS with H2S to form FeS2 (pyrite) and H2 was postulated to have operated as an early form of energy metabolism on primordial Earth. Since the Archean, sedimentary pyrite formation played a major role in the global iron and sulfur cycles, with direct impact on the redox state of the atmosphere. However, the mechanism of sedimentary pyrite formation and the way microorganisms contribute to this process is still being debated. This year, we published work on the first enrichment culture, which is capable to grow with FeS, H2S, and CO2 as sole substrates to produce FeS2 and CH4 (Thiel et al., 2019, PNAS). This proposal aims to elucidate the mechanism behind microbially mediated pyrite formation coupled to methanogenesis. Two hypotheses will be tested to unravel whether pyrite formation in enrichment J5 is directly involved in energy conservation or not. Hypothesis I (H-I) will address the question whether microorganisms involved in pyrite formation could potentially utilize the Wächtershäuser reaction, i.e. the direct formation of FeS2 and H2 from FeS and H2S for energy conservation. This would necessitate long-distance electron transport from the cell surface into the cytoplasmic membrane or cytoplasm, e.g. involving multiheme cytochrome c complexes. Hypothesis II (H-II) will test whether the energy metabolism of the non-methanogenic partner is restricted to a reversal of sulfur respiration (sulfide conversion to zero-valent sulfur and H2), with subsequent pyrite formation being mediated by the (possibly abiotic) reaction of the formed zero-valent sulfur with FeS. The two hypotheses will be tested in three complementary work packages (WP). In WP1, high quality draft genomes of enrichment J5 members will be obtained by metagenomics and annotated in respect to their potential energy metabolism including hallmark proteins indicative of H-I or H-II. Subsequent metatranscriptomics will follow expression of genes encoding such hallmark proteins. In WP2, individual community members in enrichment J5 will be identified by fluorescent in situ hybridization in combination with single-cell RAMAN microspectroscopy. The latter will serve the identification of RAMAN signals indicative of overexpressed multiheme cytochrome c redox complexes (H-I) or the formation of zero-valent sulfur within or associated with microbial cells (H-II). In WP3, enrichment J5 will be exposed to alternative growth conditions, which are postulated to require similar enzyme complexes as laid out in H-I and H-II. This will include the oxidation of elemental Fe under sulfate-reducing conditions (H-I) and the oxidation of hydrogen with elemental sulfur to H2S (H-II). Upon positive growth, expressed genes will be followed by metatranscriptomics. The proposed project will be important to establish enrichment J5 as a model for microbial pyrite formation, which has impact for our understanding of biogeochemical sulfur and iron cycling and origin-of-life-hypotheses.