ABSTRACTThe sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (H2S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce H2S when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced H2S. To understand the genomic and genetic basis for cysteine degradation and H2S production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and H2S levels over their growth ranges:Stenotrophomonas maltophilia(Gammaproteobacteria),S. bentonitica(Gammaproteobacteria), andChryseobacterium piscium(Bacteroidota). Cysteine decreased and H2S increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce H2S under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H2S production from the degradation of organosulfur compounds.IMPORTANCEHydrogen sulfide (H2S), a naturally occurring gas with both biological and abiotic origins, can be toxic to living organisms. In aquatic environments, H2S production typically originates from anoxic (lacking oxygen) environments, such as sediments, or the bottom layers of thermally stratified lakes. However, the degradation of sulfur-containing amino acids such as cysteine, which all cells and life forms rely on, can be a source of ammonia and H2S in the environment. Unlike other approaches for biological H2S production such as dissimilatory sulfate reduction, cysteine degradation can occur in the presence of oxygen. Yet, little is known about how cysteine degradation influences sulfur availability and cycling in freshwater lakes. In our study, we identified diverse bacteria from a freshwater lake that can produce H2S in the presence of O2. Our study highlights the ecological importance of oxic H2S production in natural ecosystems and necessitates a change in our outlook on sulfur biogeochemistry.