Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH₄ and ¹⁵N₂ revealed differences in cellular ¹⁵N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in ¹⁵N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

古菌厌氧甲烷氧化菌（“ANME”）和硫酸盐还原性三角洲变形菌（“SRB”）形成能够进行厌氧甲烷氧化（AOM）的共生多细胞群落，从而调节海洋沉积物中的甲烷通量。然而，ANME 与特定 SRB 伙伴就地联系的特异性却知之甚少。为了表征 ANME-SRB 联盟中的伙伴关系特异性，我们将相关推理技术 SparCC 应用于从哥斯达黎加渗流沉积物样本制备的 310 个 16S rRNA 扩增子文库，发现 ANME-2b 与我们称为 SEEP 的 Deltaproteobacteria 分支成员之间存在强正相关性-SRB1g。我们通过检查使用流式细胞术分选的各个 ANME-SRB 群落中的 16S rRNA 多样性，并使用新设计的针对 SEEP-SRB1g 进化枝的探针，通过荧光原位杂交 (FISH) 显微镜对 ANME-SRB 群落进行成像，证实了这种关联。对属于 SEEP-SRB1g 的基因组箱的分析揭示了固氮营养所需的完整nifHDK操纵子的存在，这在已发表的 ANME 相关 SRB 基因组中不常见。然后，使用杂交链式反应 (HCR-) FISH 靶向nifH转录本，通过显微镜证实了 ANME-2b—SEEP-SRB1g 联合体内 SEEP-SRB1g 中 nifH 的活跃表达，并通过FISH - nanoSIMS 实验记录了固氮活性。对与含有 CH ₄和¹⁵ N ₂的顶空一起孵育的 ANME-2b-SEEP-SRB1g 联合体进行 NanoSIMS 分析，揭示了两个伙伴之间的细胞¹⁵ N 富集差异，该差异在各个联合体之间有所不同，SEEP-SRB1g 细胞相对富集¹⁵ N在一个群落中观察到 ANME-2b，而在其他群落中观察到相反的模式，表明 ANME-2b 和 SEEP-SRB1g 都能够固氮，但在古细菌或细菌伙伴是否是主要固氮菌方面存在群落特异性差异。