Selective enrichment of high-affinity clade II NO-reducers in a mixed culture.

Microorganisms encoding for the NO reductase (NosZ) are the only known biological sink of the potent greenhouse gas NO and are central to global NO mitigation efforts. Clade II NosZ populations are of particular biotechnological interest as they usually feature high NO affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different NO-reducers strains and controlling the assembly of NO-respiring communities. Two planktonic NO-respiring mixed cultures were enriched at low dilution rates under limiting and excess dissolved NO availability to assess the impact of substrate affinity and NO cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. Under NO limitation, clade II NO-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched NO-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two and genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess NO, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the NO, outcompeting clade I affiliates. The single dominant NO-reducer (genus ) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic NO concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole remain elusive. We furthermore highlight the potential significance of NO-cobalamin interactions in shaping the composition of NO-respiring microbiomes.