122 Chapter 4 Figure 4. Metabolic reconstruction of six genomes of interest. Only genes with TPM > 0 in metatranscriptomic analyses are shown in selected time points in which the highest number of genes were transcribed (T5 for MAGs 36, 60 and 65, T4 for MAG 38, and T0 for MAGs 32 and 39). Gene abbreviations, loci and annotations are provided in Supplementary Table 4. The gene nrfH was absent in MAG 39 Nitrobium versatile (in grey) but it was previously identified in the first MAG representing this organism (Arshad et al., 2017). In MAG 32 Kuenenia stuttgartiensis, the internal compartment represents the anammoxosome. The Nitrospirota MAG 39 Nitrobium versatile, which was the most abundant microbial community member based on genome coverage before the ammonium removal experiment (Figure 3, G0), was estimated to be a 100% complete genome with 5% contamination (Figure 2). Based on metatranscriptomic analyses, we infer that the most likely metabolism performed by this organism was sulfide oxidation coupled to denitrification (Figure 4). A previously unidentified sulfide:quinone oxidoreductase sqr gene in “Ca. Nitrobium versatile” was one of the mostly highly transcribed functional genes (TPM = 0.2 ± 0.05) of this organism in a thriving period (T0, Supplementary Table 2), along with cytochrome c-oxidizing nitric oxide reductase genes norBC (respectively, TPM = 1.9 ± 0.7 and 2.8 ± 1.2). Sulfide could thus be oxidized to elemental sulfur, which might be the substrate for a dissimilatory sulfite reductase (dsrABC) operating in the oxidative direction, despite the two dsrA copies in the genome being phylogenetically related to reductive dsrA genes (Supplementary Figure 3) and the presence and transcription of a dsrD gene. Interestingly, both dsrABC copies in the genome were transcribed, one approximately 3 times more than the other (Supplementary Table 2, average TPM 0.3-0.4 vs. 0.1). Sulfite could be oxidized to sulfate via transcribed adenosinephosphosulfate (APS) reductase (aprAB) and sulfate adenylyltransferase (sat) or via a sulfite:cytochrome c oxidoreductase (sorAB) (Figure 4). Although we did not provide reduced sulfur compounds to the reactor other than sulfide, two genes encoding putative sulfide-generating enzymes were identified and transcribed: a sulfhydrogenase polysulfide reductase (hydB subunit beta only, TPM = 0.1 ± 0.06), and a thiosulfate reductase / polysulfide reductase (chain A only, phsA, present twice in the genome, TPMs = 0.09 ± 0.05 and 0.016 ± 0.018). This organism was severely affected by the ammonium removal experiment, as indicated by genome coverage (Figure 3). TPM values suggest that “Ca. Nitrobium versatile” became a rare community member but remained transcriptionally active across all time points. When the reactor was primed for the sulfide and NO toxicity experiment
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