The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (C_i), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of C_i incorporation into different taxa, mat samples were incubated with [¹³C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of ¹³C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with ¹³C. Most ¹³C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, C_i fixation, and N₂ fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for C_i fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to C_i fixation. Other taxa showed much lower [¹³C]bicarbonate incorporation.

IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven C_i fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in C_i fixation. Because this pathway imparts a lower degree of selection of isotopically heavy C_i than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of ¹³C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the ¹³C/¹²C ratios of mat biomass will help geochemists interpret the ¹³C/¹²C ratios of organic carbon in the fossil record.

与黄石国家公园的碱性硅质温泉相关联的chlorophototrophic微生物席的上绿色层由含氧蓝藻（聚球藻属物种），不产氧Roseiflexus属，和其他一些不产氧chlorophototrophs。聚球藻属。被认为是无机碳（C _i）的主要固定剂，但一些证据表明Roseiflexus spp。在弱光，缺氧的早晨，也有助于固定无机碳。尚未研究其他光养类群的贡献。为了遵循C _i掺入不同类群的途径，将垫子样品与[ ¹³在早间，弱光缺氧期间，将C]碳酸氢盐放置3小时。提取的蛋白用胰蛋白酶处理，并通过质谱分析，从而得到包含¹³ C标签掺入证据的肽鉴定和肽同位素轮廓特征。通过与宏基因组组装序列比较，从光谱特征中鉴定出总共25,483个肽段，对应于7,221个蛋白，并与垫分类群相关联。用¹³ C可检测到总共1,417个来自720个蛋白的肽。大多数¹³ C标记的肽来自Synechococcus spp蛋白。和Roseiflexusspp。伴侣和碳水化合物代谢的蛋白质标记最丰富。在Synchococcus spp中也标记了参与光合作用，C _i固定和N ₂固定的蛋白质。重要的是，3-羟基丙酸酯的双循环对C的大多数蛋白质_我固定在Roseiflexus属。被标记，证明该紫杉烷的成员有助于固定C _i。其他分类单元显示[ ¹³ C]碳酸氢盐的掺入量低得多。

重要信息黄石温泉垫已被研究为理解微生物群落生态的自然模型，并被作为地球上最早的群落化石-叠层石的现代类似物。蛋白质的稳定同位素探测（Pro-SIP）可以短期询问类群，该类群参与了这个高度活跃社区中光驱动C _i固定的重要过程，将有助于将其他代谢过程与垫类群联系起来。在这里，证据表明使用3-羟基丙酸酯双环的Roseiflexus spp。在C _i固定中具有活性。因为此途径赋予同位素重C _i较低的选择度与Calvin-Benson-Bassham循环相比，该结果提出了一种机制来解释为什么如果仅涉及后者，则垫层生物量中¹³ C的自然丰度比预期的要大。了解垫群落成员如何影响垫生物量的¹³ C / ¹² C比值将有助于地球化学家解释化石记录中有机碳的¹³ C / ¹² C比值。