93 Contrasting methane, sulfide and nitrogen regimes in coastal sediment bioreactors essential amino acids for the coordination of the Cu in the active site (Figure 5D and Supplementary Figure 13). The amino acids of the A subunit of the Rugosibacter Cu-MMO was a lone branch between amoA and pxmA sequences in a phylogenetic cluster of genome-derived reference genes (Figure 5D and Supplementary Figure 13). The gene for the A subunit of the Pseudomonadales IMCC2047 Cu-MMO was similar to the gene recovered from the Pseudomonadales IMCC2047 (original; Mori et al., 2019) and to two genes found in MAGs of Gammaprotebacteria (Zhou et al., 2020) (Supplementary Figure 13). In the Pseudomonadales IMCC2047 MAG, a hydroxylamine oxidoreductase (haoA) gene was recovered, without an haoB subunit, though it was accompanied by a single cytochrome c-554 (cycB) in the same gene cluster (Figure 5C and Supplementary Table 6). These genes possibly implicate the Cu-MMO in this MAG for ammonia rather than methane oxidation, however, many MOB also encode haoAB (Campbell et al., 2011; Versantvoort et al., 2020). Both Pseudomonadales and Rugosibacter MAGs contained a PQQ-ADH that did not classify with typical calcium (mxaF) or lanthanide-dependent (xoxF) methanol dehydrogenases suggesting a different function of the respective proteins (Supplementary Figure 14). Furthermore, the Rugosibacter ADH showed the characteristic conserved domain “DXGX(3-4)D” essential for the coordination of the lanthanide cofactor, which is also found in xoxF sequences (Good et al., 2020). Contrasting with AOB and MOB, neither of the MAGs encoded the capacity for carbon dioxide assimilation. The complete large and small Rubisco subunits required for CO2 fixation were missing, and neither of them encoded the WoodLjungdahl pathway nor the reductive tricarboxylic acid (TCA) cycle that AOB utilize to fix CO2 (Figure 5C). Moreover, neither of them appeared to be able of canonical methanotrophic C1 assimilation via formaldehyde using the serine pathway or the ribulose monophosphate pathway (RuMP). They both lacked a formatetetrahydrofolate ligase (FTHFS) encoded by fhs (KEGG: K01938) required for the conversion of formate via formyltetrahydrofolate [EC: 6.3.4.3]. Furthermore, we could not find any 3-hexulose-6-phosphate synthase (hxlA, KEGG: K08093) [EC: 4.1.2.43] of the ribulose monophosphate (RuMP) pathway (Figure 5C). Ultimately, the lack of any apparent CO2 fixation or C1 assimilation pathway suggests that these are heterotrophs, and thus metabolically distinct from characterized AOB and MOB. The two MAGs showed potential metabolic flexibility for nitrogen oxide 3
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