Microbial communities in their natural habitats consist of closely related populations that may exhibit phenotypic differences and inhabit distinct niches. However, connecting genetic diversity to ecological properties remains a challenge in microbial ecology due to the lack of pure cultures across the microbial tree of life. ‘Candidatus Accumulibacter phosphatis’ is a polyphosphate-accumulating organism that contributes to the Enhanced Biological Phosphorus Removal (EBPR) biotechnological process for removing excess phosphorus from wastewater and preventing eutrophication from downstream receiving waters. Distinct Accumulibacter clades often co-exist in full-scale wastewater treatment plants and lab-scale enrichment bioreactors, and have been hypothesized to inhabit distinct ecological niches. However, since individual strains of the Accumulibacter lineage have not been isolated in pure culture to date, these predictions have been made solely on genome-based comparisons and enrichments with varying strain composition. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of co-existing Accumulibacter clades in an engineered bioreactor environment. We obtained four high-quality genomes of Accumulibacter strains that were present in the bioreactor ecosystem, one of which is a completely contiguous draft genome scaffolded with long reads. We identified core and accessory genes to investigate how gene expression patterns differ among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may differ between Accumulibacter clades and provide key functional insights into this biotechnologically significant microbial lineage.IMPORTANCE‘Candidatus Accumulibacter phosphatis’ is a model polyphosphate accumulating organism that has been studied using genome-resolved metagenomics, metatranscriptomics, and metaproteomics to understand the EBPR process. Within the Accumulibacter lineage, several similar but diverging clades are defined by the polyphosphate kinase (ppk1) locus sequence identity. These clades are predicted to have key functional differences in acetate uptake rates, phage defense mechanisms, and nitrogen cycling capabilities. However, such hypotheses have largely been made based on gene-content comparisons of sequenced Accumulibacter genomes, some of which were obtained from different systems. Here, we performed time-series genome-resolved metatranscriptomics to explore gene expression patterns of co-existing Accumulibacter clades in the same bioreactor ecosystem. Our work provides an approach for elucidating ecologically relevant functions based on gene expression patterns between closely related microbial populations.
Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acIActinobacteria. Members of this cosmopolitan lineage are proposed to bolster heterotrophic growth using phototrophy because their genomes encode actino-opsins (actR). This model has been difficult to validate experimentally because acIActinobacteriaare not consistently culturable. Based primarily on genomes from single cells and metagenomes, we provide a detailed biosynthetic route for members of acI clades A and B to synthesize retinal and its carotenoid precursors. Consequently, acI cells should be able to natively assemble light-driven actinorhodopsins (holo-ActR) to pump protons, unlike many bacteria that encode opsins but may need to exogenously obtain retinal because they lack retinal machinery. Moreover, we show that all acI clades contain genes for a secondary branch of the carotenoid pathway, implying synthesis of a complex carotenoid. Transcription analysis of acIActinobacteriain a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and thatactRis among the most highly transcribed of all acI genes. Furthermore, heterologous expression of acI retinal pathway genes showed that lycopene, retinal, and ActR can be made using the genes encoded in these organisms. Model cells producing ActR and the key acI retinal-producing β-carotene oxygenase formed holo-ActR and acidified solution during illumination. Taken together, our results prove that acIActinobacteriacontaining both ActR and acI retinal production machinery have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin.IMPORTANCEMicrobes play critical roles in determining the quality of freshwater ecosystems, which are vital to human civilization. Because acIActinobacteriaare ubiquitous and abundant in freshwater lakes, clarifying their ecophysiology is a major step in determining the contributions that they make to nitrogen and carbon cycling. Without accurate knowledge of these cycles, freshwater systems cannot be incorporated into climate change models, ecosystem imbalances cannot be predicted, and policy for service disruption cannot be planned. Our work fills major gaps in microbial light utilization, secondary metabolite production, and energy cycling in freshwater habitats.
The metabolic activity of uncultivated microorganisms contributes to numerous ecosystem processes, ranging from nutrient cycling in the environment to influencing human health and disease. Advances in sequencing technology have enabled the assembly of genomes for these microorganisms, but our ability to generate reference genomes far outstrips our ability to analyze them. Common approaches to analyzing microbial metabolism require reconstructing the entirety of an organism’s metabolic pathways or performing targeted searches for genes involved in a specific process. This paper presents a third approach, in which draft metabolic reconstructions are used to identify compounds through which an organism may interact with its environment. These compounds can then guide more-intensive metabolic reconstruction efforts and can also provide new hypotheses about the specific contributions that microbes make to ecosystem-scale metabolic processes.
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