94 Chapter 3 reduction, and a certain degree of sulfur cycling. Pseudomonadales IMCC2047 MAG had potential for partial denitrification, respiring nitrate to nitrous oxide. The Rugosibacter MAG encoded nitrate respiration potential together with sulfide detoxification or thiosulfate oxidation (Figure 5C). Figure 5. Abundance of novel MBAE14-like and Rugosibacter-like MAGs, MAG properties and placement of unusual CuMMO sequences in a phylogenetic tree. A Primary y-axis indicates summed MBAE14 family-like Amplicon Sequencing Variants (ASVs) across time (monthly sampling over the course of the experiment, x-axis). Secondary y-axis indicates Pseudomonadales IMCC2047 normalized mean genome coverage (nmgc) on month 7, 14.5 and 15.5 in %. B Rugosibacter nmgc on month 7, 14.5 and 15.5. C Functional annotation of key genes in Pseudomonadales IMCC2047 and Rugosibacter MAGs. Question marks indicate either not found in MAG/not present (in canonical form) D Copper-dependent monooxygenase (cu-MMO)-A protein tree containing binned sequences (color-coded) in our study and additional selected Pseudomonadales (in pink) IMCC2047 and Rugosibacter (in dark red) GTDB-Tk genomes containing Cu-MMO-A s.For other MAGs the colors refer to current study’s binned AOB Nitrosomonas (orange), pxmA-containing gammaproteobacterial methanotrophs (light purple), pmoA-containing gammaproteobacterial methanotrophs (dark purple) and alphaproteobacterial methanotrophs (green). Tree scale depicts nucleotide substitutions. A 0 10 20 30 40 7 14.5 15.5 Metagenomic coverage Rugosibacter Time (months) 0 60 120 180 0 1000 2000 3000 4000 5000 2 3 5 6 7 8 9 10 11 1214.515.5 Pseudomonadales IMCC2047 (nmgc) MBAE14-like abundance (ASV counts) Time (months) Time (months) Rugosibacter (nmgc) 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high bf high low bf low 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv inoculum 0 10 20 30 40 7 14.5 15.5 coverage Rugosibacter time (months) A B C 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high bf high low bf low 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 60 120 180 1000 2000 3000 4000 5000 rage Pseudomonadales IMCC2047 summedMBAE14 ASVs high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv inoculum 0 10 20 30 40 7 14.5 15.5 coverage Rugosibacter time (months) A B C 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high bf high low bf low 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 1214.515 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv inoculum 0 10 20 30 40 7 14.5 15.5 coverage Rugosibacter time (months) A B C 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high bf high low bf low 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 1214.515 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv inoculum 0 10 20 30 40 7 14.5 15.5 coverage Rugosibacter time (months) A B C 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high bf high low bf low 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 1214.515 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv inoculum 0 10 20 30 40 7 14.5 15.5 coverage Rugosibacter time (months) A B C 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high bf high low bf low 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 60 120 180 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 12 14.515.5 coverage Pseudomonadales IMCC2047 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv 0 1000 2000 3000 4000 5000 sed. 0 2 3 5 6 7 8 9 10 11 1214.515 summedMBAE14 ASVs time (months) high nmc high bfnmc lownmc lowbf nmc high asv high bfasv lowasv lowbf asv inoculum 0 10 20 30 40 7 14.5 15.5 coverage Rugosibacter time (months) A B C eu. eu. bf. ol. ol. bf. B C D 0.1 Verrumicrobia Actinobacteria Nitrospira Methylomirabilota pxMMO Nitrosococcus Nitrosomonas Proteobacteria MBAE14-like Gammaprot. CH4-trophs Alphaprot. CH4-trophs Rugosibacter Rugosibacter Rugosibacter Azoarcus Pseudomonadales IMCC2047 Rugosibacter (p/m)amoCAB (p/m)amoD or ORF4 (amoE) ORF5 (amoD) haoA cyc-B (cM-554) CO2-fixation PQQ-ADH C1-assimilation (Serine/RuMP) cydAB coxAB nrfA napA narG nirS nirK norB nosZ sqr sat soxA soxX soxY soxZ nifH
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