Sulfur Metabolites Play Key System-Level Roles in Modulating Denitrification.

Anne E Otwell,Erica L W Majumder,Gary Siuzdak,Alex V Carr,Bill Webb,David A Stahl,Nitin S Baliga,Serdar Turkarslan,Dwayne A Elias,Sean M Gibbons,Regina L Wilpiszeski,Maryann K Ruiz,Linh T Hoang,Laura A Hug

doi:10.1128/msystems.01025-20

Abstract

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. It is now recognized that intermediates of nitrogen and sulfur cycling (e.g., hydrogen sulfide, nitrite, etc.) can also directly impact NRB and SRB activities in freshwater, wastewater, and sediment and therefore may play important roles in competitive interactions. Here, through comparative transcriptomic and metabolomic analyses, we have uncovered mechanisms of hydrogen sulfide- and cysteine-mediated inhibition of nitrate respiratory growth for the NRB Intrasporangium calvum C5. Specifically, the systems analysis predicted that cysteine and hydrogen sulfide inhibit growth of I. calvum C5 by disrupting distinct steps across multiple pathways, including branched-chain amino acid (BCAA) biosynthesis, utilization of specific carbon sources, and cofactor metabolism. We have validated these predictions by demonstrating that complementation with BCAAs and specific carbon sources relieves the growth inhibitory effects of cysteine and hydrogen sulfide. We discuss how these mechanistic insights give new context to the interplay and stratification of NRB and SRB in diverse environments.IMPORTANCE Nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) colonize diverse anoxic environments, including soil subsurface, groundwater, and wastewater. NRB and SRB compete for resources, and their interplay has major implications on the global cycling of nitrogen and sulfur species, with undesirable outcomes in some contexts. For instance, the removal of reactive nitrogen species by NRB is desirable for wastewater treatment, but in agricultural soils, NRB can drive the conversion of nitrates from fertilizers into nitrous oxide, a potent greenhouse gas. Similarly, the hydrogen sulfide produced by SRB can help sequester and immobilize toxic heavy metals but is undesirable in oil wells where competition between SRB and NRB has been exploited to suppress hydrogen sulfide production. By characterizing how reduced sulfur compounds inhibit growth and activity of NRB, we have gained systems-level and mechanistic insight into the interplay of these two important groups of organisms and drivers of their stratification in diverse environments.

Highlights

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics
To characterize growth effects of reduced sulfur compounds, we first investigated the ability of I. calvum to grow anaerobically in the absence of cysteine, which is typically used as a reducing agent in anoxic culture medium [3, 20]
We found that varying the carbon-to-nitrogen ratio in the medium led to increased dissimilatory nitrate reduction to ammonia (DNRA) activity when nitrate was limited

Summary

Introduction

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. Microbial activities are impacted by factors that include resource concentration, pH, metal availability, and interactions between microorganisms in the environment [3,4,5] Knowing how these factors differentially impact subsurface microbial nitrogen cycling is essential for developing a predictive understanding of the fate of different nitrogen species in natural and engineered systems. Recent work has shown that groundwater nitrate levels are associated with nitrous oxide production at the FRC and enrichment of denitrification pathway genes [6, 8] Taken together, these observations suggest that dissimilatory nitrate reduction by nitrate-reducing bacteria (NRB) is a major metabolic process in nitrate-contaminated regions of the subsurface. The activity of denitrifiers can lead to significant losses of soil nitrogen through reduction of nitrate to gaseous forms, including nitrous oxide or dinitrogen gas [12]

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