193 Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress 0.00 0.03 0.05 0.08 0.10 0 1.5 3 Relative PHA content Salinity (%) A B C distance (microns) (R)-3-hydroxybutanoyl-CoA (3-HB) acetoacetyl-CoA reductase (PhbB) Acetoacetyl-CoA Poly-3-hydroxybutyrate (PHB) (S)-3-hydroxybutanoyl-CoA (3-HB) poly[R]-3-hydroxyalkanoate polymerase sub.E (PhaE) polyhydroxyalkanoate synthase (PhaC) 3-hydroxyacyl-CoA dehydrogenase (FadJ) 3-hydroxybutyryl-CoA dehydratase (CroR) enoyl-CoA hydratase (Crt) Crotonyl-CoA (PHB derivative) 1 5 6 4 3 1 2 4 3 5 6 acetyl-CoA C-acetyltransferase (AtoB) Acetyl-CoA 2 among most transcribed genes in the ‘Ca. Methanoperedens’ dataset * * * Intensity value 0 5 10 15 20 2530 20 40 60 80 DAMOARCH 641 Alexa350 DAMOARCH 641 Alexa350 DAMOARCH 641 Alexa350 0 5 1015 202530 20 40 60 Intensity value 80 PHA Nile Red PHA Nile Red PHA Nile Red 0 distance (microns) 1 2 1 2 1 2 Figure 5. Linking “Ca. Methanoperendes” to polyhydroxyalkanoate (PHA) production and decrease upon salt-stress. A From left to right: 1.5% salinity “Ca. Methanoperedens” biomass labelled with Alexa350-fluorophore-labelled DAMO-ARCH-641 probe signal coupled to Red Nile (PHA stain) signal. Intensity plots from color-coded cross section (yellow line) displaying the Alexa350 signal (blue) and Nile Red signal (pink). Separate imaging from the overlapped signals of Alexa350-fluorophore-labelled N-DAMO-ARCH 641 and Nile Red for the same image. The micrographs are representatives of two independent experiments with 2-5 granules per well (n=3) at two salinities (0% and 1.5%) per experiment. See Supplementary Fig. 6 for additional micrographs. All scale bars represent 20 µm. B Reconstruction of the poly-3-hydroxybutyrate (PHB) and crotonyl-CoA (PHB derivative) production pathways, highlighting the top abundant transcript found on the “Ca. Methanoperedens” metatranscriptomes for the studied culture. Genes indicated with an asterisk refer to transcripts with top transcript per million (TPM) (relative abundance) within the “Ca. Methanoperedens” total transcripts. C Quantification/ratio results of the summed area peaks of PHB and crotonyl-CoA identified by Gas Chromatography/Mass spectrometry (GC/MS), normalized with an internal standard (IS) and dry weight (in mg). Relative PHA content refers to PHA derivatives area normalized by internal standard and weight (mg). 6
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