151 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea (Wang et al., 2024; Wang et al., 2023; Zuo et al., 2024). Therefore, the response “Ca. Methanoperedens BLZ2” might vary depending on the enrichment cultures containing different active bacterial nitrite scavengers (Echeveste Medrano et al., 2024b, Chapter 6; Ouboter et al., 2024) or, N-DAMO (Arshad et al., 2017; Wissink et al., 2024). Genes encoding core metabolic enzymes of “Ca. Methanoperedens” are upregulated at the first sulfide spike but get downregulated after longterm exposure The general response of “Ca. Methanoperedens” was assessed by its genes and comparing them between the three time points: T0 vs T1, T0 vs T2 and T0 vs T3. A total of 202, 30 and 321 genes were significantly upregulated and 265, 67 and 479 genes downregulated (Padj<0.05) with more than 2 log2FC between T0 vs T1, T0 vs T2 and T0 vs T3, respectively. We then assessed the response of the transcripts belonging to the methaneoxidizing complex methyl coenzyme-M reductase (MCR) and nitrate reductase to the different sulfide exposures (Table 1 and Supplementary Table 3). The mcr genes were upregulated (mcrA, log2FC 2.31) only during the first sulfide spike but were downregulated during long-term exposure (mcrA, log2FC -0.59) and after the third pulse (mcrA, log2FC -2.31) (Table 1). The “Ca. Methanoperedens” nitrate reductase (narG) gene showed similar transcriptional trends to the mcr genes, although at higher log2FC values (+3.96 -1.46 & -5.65, respectively; Table 1). Genes encoding for Mcr and nitrate reductase of “Ca. Methanoperedens” were upregulated from T0 to T1 congruent with less severe inhibition of methane oxidation activity after the first pulse of sulfide addition. In contrast, the loss of activity over long-term sulfide exposure is reflected in the downregulation of key metabolic genes at that time point (Figure 2 and Table 1). Although the genes of the MCR complex and narG show reduced expression, this does not indicate decreased survivability. Instead, it likely reflects a metabolic shift for adaptation, similar to what was observed in a salt-stress experiment with “Ca. Methanoperedens” (Echeveste Medrano et al., 2024b, Chapter 6) where the metabolism shifted toward osmoregulation to help the culture adapt to marine salinities. 5
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