Integrated Omics Analyses Reveal Differential Gene Expression and Potential for Cooperation Between Denitrifying Polyphosphate and Glycogen Accumulating Organisms

Abstract

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The 6 transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA, and IC, and a competing glycogen accumulating organism (GAO) affiliated with Candidatus Competibacter. Strongly elevated expression of nitrite reductase compared to nitrous oxide reductase was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2O accumulation. Surprisingly, while [~]90% of nitrite reductase gene transcripts mapped to the three co-occurring PAO populations, [~]93% of nitric oxide reductase gene transcripts were expressed by the GAO population. This suggests the potential for cooperation between GAOs and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide. Originality-Significance StatementPolyphosphate accumulating organisms (PAOs) affiliated with as-yet-uncultivated Ca. Accumulibacter phosphatis are increasingly employed in enhanced biological phosphorus removal (EBPR) processes, a common environmental biotechnology for removing phosphorus from wastewater and thereby preventing detrimental impacts of nutrient pollution. Under anoxic conditions, PAOs have been associated with unusually high emissions of the potent greenhouse gas and denitrification intermediate nitrous oxide. However, the underlying mechanisms and biological controls on incomplete denitrification by denitrifying Accumulibacter, their ecological interactions with understudied glycogen accumulating organisms (GAOs), and patterns of gene expression under anoxic conditions are all poorly understood. Here, we describe genomic features of a previously unrecognized clade of Accumulibacter that is putatively adapted to high rate P uptake under nitrite-driven denitrification and provide evidence that differential gene expression (namely elevated expression of nitrite reductase compared to nitrous oxide reductase) by Accumulibacter is a key control on nitrous oxide production. Moreover, we document genomic and transcriptional potential for cooperation and crossfeeding of the denitrification intermediate nitric oxide between GAOs and PAOs. This is surprising because GAOs are conventionally considered to be competitors to PAOs, and because nitric oxide is toxic to most microorganisms at low concentrations. Taken together, our work provides significant new understanding of metabolic and ecological interactions in EBPR processes that are critical to environmental protection; demonstrates the potential of previously unrecognized crossfeeding of the denitrification intermediate nitric oxide; and expands our understanding of genomic features and clade level diversity of Accumulibacter.