Maider Junkal Echeveste Medrano

195 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress Salinity (%) NeuAc (Pse/Leg)AcAm Neu5Ac sialidase activity (nmol/min/g) 0 0.17 1.5 0.23 A B GDP - GlcNac GDP-4– amino6-deoxyGlcNac GDP-2,4– diNac6-deoxy-Glc GDP-4– keto-6-deoxyGlcNac 2,4–diNac-6deoxy-Man legionaminic acid CMP-legionaminic acid legC legB legH legG legI legF CMP-N-acetyl-neuraminic acid neuC neuB neuA UDP - GlcNac ManNAc N-acetylneuraminic acid UDP – GlcNac UDP-4–keto-6deoxyAltNac UDP-4–amino-6 -deoxyAltNac UDP-4–amino-6deoxyAltNac 2,4-diNAc-6deoxy-Alt pseudaminic acid CMP-pseudaminic acid pseB pseC pseH pseG pseI pseF LEGIONAMINIC ACID N-ACETYLNEURAMINIC ACID PSEUDAMINIC ACID Halorubrum sp. C. jejunii legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm 1.5% 0% legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm 1.5% 0% legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm 0% 1.5% legI legH legG legC pseI pseF pseC pseB legG legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm C. jejunii legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF nueB_p seI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm C. jejunii legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_p neuB_ n pseG pseH pseC pseB pseC pseB pseI fw bck bck fw l le le jejuni hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_ps neuB_p ne pseG pseH pseC pseB pseC pseB pseI OT fw bck bck fw le leg leg jejuni hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT fw bck fw bck bck fw jeju legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legH pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm 0% 1.5% HMM 'Ca. Methanoperedens' TPM 1.5% 0% legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm C. jejunii Halorubrum sp. 0% 1.5% HMM legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm 0% 1.5% HMM KEGG-HMM legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm legI legH legG legC legB pseI pseF pseC pseB legG legF legC legB legI legH legG legC legB pseI pseF pseC pseB neuC neuB neuA legG legF legC legB pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuC neuB neuA pseH pseC pseG pseB legH legC neuA_pseF_legF neuB_pseI_legI neuC_legG neuA_pseF_legF neuB_pseI_legI neuC_legG neuB legH legC legB pseG pseH pseC pseB pseC pseB pseI MT GE DS GA UN OT legC fw bck fw bck bck fw jejuni legI legC legB halorubrum jejuni fw bck hmm/kegg hmm/kegg hmm/kegg hmm/kegg jejuni jejuni halorubrum halorubrum hmm 0% 1.5% C. jejunii HMM KEGG-HMM MT: Ca. Methanoperedens GE: Geobacter DS: Desulfobacterota_GWA25412 GA: Gallionellaceae UN: Unbinned OT: Others BLASTp 0% 1.5% 0% 1.5% zero gene coverage (Pse/Leg)Ac2 2.57E+06 ± 2.28E+04 2.60E+06 ± 4.91E+04 1.98E+04 ± 1.98E+04 1.27E+06 ± 1.21E+05 4.64E+04 ± 1.28E+03 1.65E+06 ± 1.60E+05 normalized 'Ca. Methanoperedens' TPM normalized gene coverage Figure 6. Measured sialic acids/sialidases and reconstructed putative sialic acid production pathways in “Ca. Methanoperedens” enrichment. A Mass spectrometry peak areas of identified sialic acids (nonulosonic acids, NulOs) in both samples (with intensity peak relative to KDN or Deaminated Neuraminic acid/standard) including sialidase activity. 5-N-acetylneuraminic acid (Neu(5)Ac), pseudaminic acid (Pse) and legionaminic acid (Leg). Pse and Leg modifications are indicated as Leg-AcAm(acetamidino) and Leg-Ac2(acetyl). Pse and Leg have the same molecular weight and thus cannot be differentiated. B Metagenomic-based metabolic reconstruction of the production pathways of the three most common NulOs and from which abundant peaks with matching accurate mass could be obtained: NeuAc and Pse/Leg with their respective modifications. Heatmaps refer to “Ca. Methanoperedens” and bacterial side-community normalized gene coverage queried via BLASTp (light grey) or HMM profiles (dark grey). Some BLASTp (against Halorubrum sp. PV6 & Campylobacter jejunii) and HMM sequence hits overlapped. Heatmaps were also employed to depict metatranscriptomic-based normalized gene expression profiles (TPM: transcript per million) calculated 6

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