Ecosystems biology of the hidden sulfur cycle in rice paddy soil - phylogenetic, functional, and proteogenomic analysis of key players with focus on sulfate reducers

稻田是人工湿地，对主食生产至关重要。这些淹水土壤中普遍存在的缺氧条件使它们成为温室气体甲烷的最重要来源之一。因此，了解它们的生物地球化学和微生物生态学对于预测它们对全球变化的反应具有普遍的重要性和不可或缺的作用。该项目旨在阐明水稻土中硫酸盐还原微生物(SRM)的特性和生理潜力。这些微生物驱动着一个高度活跃但隐藏的硫循环，这一循环在低水位的硫酸盐池中并不明显，因此到目前为止一直被严重研究不足。因此，人们对SRM本身知之甚少。尤其是因为在水稻土壤中可以发现大量新的深分枝dsrAB基因，作为SRM的功能标记。由于硫酸盐还原和有机质的产甲烷降解有效竞争，因此SRM对稻田甲烷的产生具有控制作用。为了在微生物参与平行生物地球化学途径的背景下识别活跃的SRM，我们将应用多相方法，在温室水稻植株生长到生殖早期之前，刺激根际和周围土壤中隐藏的硫循环。这将通过对稻田土壤进行石膏(CaSO4)修正来实现，其数量与水稻农业有关，并随后与未修正的控制措施进行比较。根际活跃的SRM将被稳定同位素探测(SIP)瞄准，该探测使用13C标记的根分泌物，这些分泌物是由水稻自身从提供的13CO2中合成的。平行高谷产量扩增序列将识别散装土壤中额外的活性SRM，那里可能没有足够数量的13C标记底物用于SIP，以及根际中不使用或太少使用13C标记底物进行合成代谢的SRM。在这两种方法中，除了16S rRNA基因外，还将针对功能标记基因dsrB和/或它们的转录本来区分SRM和共同靶标的硫氧化剂。为了初步了解SRM和硫氧化剂的基因组含量和最活跃的表达蛋白质，我们将分析所有样本的元基因组和元蛋白质组。元蛋白质组分析将与蛋白质-sip相结合，以扩展标准sip获得的结果。这种生态系统方法将伴随着在整个项目中有针对性地培养SRM，以获得以深分支dsrAB谱系为代表的可能新颖的SRM的稳定丰富或纯培养。我们推测，含有新的dsrAB的微生物以及以前通过分子方法或培养鉴定的SRM，其生态功能尚未被证实，两者都在推动硫酸盐还原，从而助长了水稻土中隐藏的硫循环。

Rice paddies are man-made wetlands that are essential for staple food production. The prevailing anoxic conditions in these water-logged soils make them one of the most important sources of the greenhouse gas methane. Understanding their biogeochemistry and microbial ecology is therefore of general importance and indispensable to foresee their response to global change. The proposed project targets to elucidate the identity and physiological potential of sulfate reducing microorganisms (SRM) in rice paddy soil. These microorganisms drive a highly active but hidden sulfur cycle that is not apparent from the low standing pools of sulfate and thus has been severely understudied so far. As a consequence little is known about SRM themselves. This is especially true since a large diversity of novel deep-branching dsrAB genes, which serve as functional marker for SRM, can be found in rice paddy soil. Since sulfate reduction effectively competes with the methanogenic degradation of organic matter, SRM have a control function on methane production in rice paddies. To identify active SRM against the background of microorganism involved in parallel biogeochemical pathways, we will apply a multiphasic approach under conditions that stimulate the hidden sulfur cycle in the rhizosphere and bulk soil around rice plants grown in a greenhouse until the early reproductive phase. This will be achieved by gypsum (CaSO4) amendment to rice paddy soil in amounts relevant for rice agriculture and subsequent comparison to unamended controls. SRM active in the rhizosphere will be targeted by stable isotope probing (SIP) using 13C-labeled root exudates that were synthesized by the rice plant itself from provided 13CO2. Parallel high-troughput amplicon sequencing will identify additional active SRM in the bulk soil, where insufficient amounts of 13C-labeled substrates for SIP can be expected, and SRM in the rhizosphere that use no or too little 13C-labeled substrates for anabolism. In both approaches, besides the 16S rRNA gene also the functional marker gene dsrB and/or their transcripts will be targeted to differentiate between SRM and co-targeted sulfur oxidizers. To get initial insights into the genomic content and most actively expressed proteins of SRM and sulfur oxidizers, we will analyze the metagenomes and metaproteomes of all samples. The metaproteome analysis will be conducted in combination with Protein-SIP to extend results obtained by standard SIP. This ecosystems approach will be flanked by targeted cultivation of SRM throughout the project to get stable enrichment or pure cultures of putatively novel SRM represented by deep-branching dsrAB lineages. We hypothesize that both, microorganism harboring novel dsrAB as well as SRM previously identified by molecular methods or cultivation but whose ecological function has yet to be demonstrated, are driving sulfate reduction and thus fuel the hidden sulfur cycle in rice paddy soil.