Expansion of nitrogenase-like enzymes involved in microbial assimilation of volatile organic sulfur compounds
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Organosulfur compounds are the predominant source of sulfur in terrestrial environments, and bacteria in these environments require enzymes that allow them to assimilate organosulfur compounds. Most described enzymes involved in organosulfur assimilation require oxygen, and understanding of enzymes that function under anoxic conditions is limited. Recently, the enzyme methylthio-alkane reductase (Mar), a nitrogenase-like enzyme that reduces the volatile organic sulfur compounds (VOSCs) methylthio ethanol (MT-EtOH), dimethyl sulfide (DMS), and ethyl methyl sulfide (EMS) in the absence of oxygen was identified in the purple nonsulfur bacterium Rhodospirillum rubrum. However, another purple nonsulfur bacterium, Rhodopseudomonas palustris, has three loci containing nitrogen fixation-like (NFL) genes with high sequence similarity to Mar, suggesting there are other Mar-like enzymes that may serve distinct roles. Here, we tested if these NFL genes in R. palustris are required to assimilate VOSCs. Transcriptomic sequencing (RNA-seq) analysis revealed that all NFL genes in R. palustris are up-regulated in response to sulfur limitation, supporting a role in sulfur assimilation. Only disruption of the NFL genes encoded by RPA2634 - 37, renamed marBHDK1 , resulted in fitness defects when EMS, DMS, and dimethylsulfoniopropionate (DMSP) were provided as a sulfur source, indicating it is a functional Mar enzyme. The NFL genes encoded by RPA2347-48 and RPA2353-54, renamed marKD2 and marHB2 , respectively, were required for activity when MT-EtOH or ethanedithiol was provided as a sulfur source but not DMS, EMS, or DMSP. No activity was observed with the third NFL loci that includes RPA2363 and RPA2364, renamed nflDK . Overall, the results demonstrate that two homologs of Mar in R. palustris are capable of VOSC reduction, with one specialized for simple VOSCs and the other acting preferentially on a substrate with an additional functional group.
IMPORTANCE
VOSCs in freshwater environments play a role in atmospheric processes, impacting global weather patterns. Bacteria are central to cycling sulfur in these environments, driving sulfur transformations even in oxygen-limited environments where sulfate is scarce but organosulfur compounds are abundant. While many oxygen-dependent reactions have been described that contribute to VOSC cycling, anaerobic pathways remain much less understood. Mar enzymes represent a newly characterized mechanism for anoxic VOSC assimilation. Here we find that two Mar homologs in R. palustris are the result of functional specialization of different Mar isozymes. Studying their activity expands our understanding of microbial strategies for sulfur turnover and sheds light on anaerobic sulfur metabolism.
