Enhancing phosphorus removal of photogranules by incorporating polyphosphate accumulating organisms.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat water with lower energy input and have great potential for nutrient recovery applications. They have been proven to efficiently remove nitrogen and carbon but show lower conversion rates for phosphorus compared to established treatment systems, such as aerobic granular sludge. In this study, we successfully introduced polyphosphate accumulating organisms (PAOs) to an established photogranular culture. We operated photobioreactors in sequencing batch mode with six cycles per day and alternating anaerobic (dark) and aerobic (light) phases. We were able to increase phosphorus removal/recovery by 6 times from 5.4 to 30 mg/L/d while maintaining similar nitrogen and carbon removal compared to photogranules without PAOs. To maintain PAOs activity, alternating anaerobic feast and aerobic famine conditions were required. In future applications, where aerobic conditions are dependent on in-situ oxygenation via photosynthesis, the process will rely on sunlight availability. Therefore, we investigated the feasibility of the process under diurnal cycles with a 12-h anaerobic phase during nighttime and six short cycles during the 12 h daytime. The 12-h anaerobic phase had no adverse effect on the PAOs and phototrophs. Due to the extension of one anaerobic phase to 12 h the six aerobic phases were shortened by 47% and consequently decreased the light hours per day. This resulted in a decrease of phototrophs, which reduced nitrogen removal and biomass productivity up to 30%. Finally, we discuss and suggest strategies to apply PAO-enriched photogranules at large-scale.

Diversity, distribution, and expression of opsin genes in freshwater lakes.

Microbial rhodopsins are widely distributed in aquatic environments and may significantly contribute to phototrophy and energy budgets in global oceans. However, the study of freshwater rhodopsins has been largely limited. Here, we explored the diversity, ecological distribution, and expression of opsin genes that encode the apoproteins of type I rhodopsins in humic and clearwater lakes with contrasting physicochemical and optical characteristics. Using metagenomes and metagenome-assembled genomes, we recovered opsin genes from a wide range of taxa, mostly predicted to encode green light-absorbing proton pumps. Viral opsin and novel bacterial opsin clades were recovered. Opsin genes occurred more frequently in taxa from clearwater than from humic water, and opsins in some taxa have nontypical ion-pumping motifs that might be associated with physicochemical conditions of these two freshwater types. Analyses of the surface layer of 33 freshwater systems revealed an inverse correlation between opsin gene abundance and lake dissolved organic carbon (DOC). In humic water with high terrestrial DOC and light-absorbing humic substances, opsin gene abundance was low and dramatically declined within the first few meters, whereas the abundance remained relatively high along the bulk water column in clearwater lakes with low DOC, suggesting opsin gene distribution is influenced by lake optical properties and DOC. Gene expression analysis confirmed the significance of rhodopsin-based phototrophy in clearwater lakes and revealed different diel expressional patterns among major phyla. Overall, our analyses revealed freshwater opsin diversity, distribution and expression patterns, and suggested the significance of rhodopsin-based phototrophy in freshwater energy budgets, especially in clearwater lakes.

Disentangling the genetic basis of rhizosphere microbiome assembly in tomato.

Microbiomes play a pivotal role in plant growth and health, but the genetic factors involved in microbiome assembly remain largely elusive. Here, we map the molecular features of the rhizosphere microbiome as quantitative traits of a diverse hybrid population of wild and domesticated tomato. Gene content analysis of prioritized tomato quantitative trait loci suggests a genetic basis for differential recruitment of various rhizobacterial lineages, including a Streptomyces-associated 6.31 Mbp region harboring tomato domestication sweeps and encoding, among others, the iron regulator FIT and the water channel aquaporin SlTIP2.3. Within metagenome-assembled genomes of root-associated Streptomyces and Cellvibrio, we identify bacterial genes involved in metabolism of plant polysaccharides, iron, sulfur, trehalose, and vitamins, whose genetic variation associates with specific tomato QTLs. By integrating 'microbiomics' and quantitative plant genetics, we pinpoint putative plant and reciprocal rhizobacterial traits underlying microbiome assembly, thereby providing a first step towards plant-microbiome breeding programs.

Metabolic Differentiation of Co-occurring Accumulibacter Clades Revealed through Genome-Resolved Metatranscriptomics.

Natural microbial communities consist of closely related taxa 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" (Accumulibacter) 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 coexist in full-scale wastewater treatment plants and laboratory-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 compositions. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of coexisting Accumulibacter strains 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 Nanopore reads. We identified core and accessory genes to investigate how gene expression patterns differed among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may confer distinct functions to Accumulibacter strains 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 shared sequence identity of the polyphosphate kinase (ppk1) locus. 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 coexisting 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.

Genome-Resolved Metagenomics of a Photosynthetic Bioreactor Performing Biological Nutrient Removal.

Enhanced biological phosphorus removal (EBPR) is an economically and environmentally significant wastewater treatment process for removing excess phosphorus by harnessing the metabolic physiologies of enriched microbial communities. We present a genome-resolved metagenomic data set consisting of 86 metagenome-assembled genome sequences from a photosynthetically operated lab-scale bioreactor simulating EBPR.

Successive plant growth amplifies genotype-specific assembly of the tomato rhizosphere microbiome.

Plant microbiome assembly is a spatial and dynamic process driven by root exudates and influenced by soil type, plant developmental stage and genotype. Genotype-dependent microbiome assembly has been reported for different crop plant species. Despite the effect of plant genetics on microbiome assembly, the magnitude of host control over its root microbiome is relatively small or, for many plant species, still largely unknown. Here we cultivated modern and wild tomato genotypes for four successive cycles and showed that divergence in microbiome assembly between the two genotypes was significantly amplified over time. Also, we show that the composition of the rhizosphere microbiome of modern and wild plants became more dissimilar from the initial bulk soil and from each other. Co-occurrence analyses further identified amplicon sequence variants (ASVs) associated with early and late successions of the tomato rhizosphere microbiome. Among the members of the Late Successional Rhizosphere microbiome, we observed an enrichment of ASVs belonging to the genera Acidovorax, Massilia and Rhizobium in the wild tomato rhizosphere, whereas the modern tomato rhizosphere was enriched for an ASV belonging to the genus Pseudomonas. Collectively, our approach allowed us to study the dynamics of rhizosphere microbiome over successional cultivation as well as to categorize rhizobacterial taxa for their ability to form transient or long-term associations with their host plants.

Restoring degraded microbiome function with self-assembled communities.

The natural microbial functions of many soils are severely degraded. Current state-of-the-art technology to restore these functions is through the isolation, screening, formulation and application of microbial inoculants and synthetic consortia. These approaches have inconsistent success, in part due to the incompatibility between the biofertilizer, crop, climate, existing soil microbiome and physicochemical characteristics of the soils. Here, we review the current state of the art in biofertilization and identify two key deficiencies in current strategies: the difficulty in designing complex multispecies biofertilizers and the bottleneck in scaling the production of complex multispecies biofertilizers. To address the challenge of producing scalable, multispecies biofertilizers, we propose to merge ecological theory with bioprocess engineering to produce 'self-assembled communities' enriched for particular functional guilds and adapted to a target soil and host plant. Using the nitrogen problem as an anchor, we review relevant ecology (microbial, plant and environmental), as well as reactor design strategies and operational parameters for the production of functionally enriched self-assembled communities. The use of self-assembled communities for biofertilization addresses two major hurdles in microbiome engineering: the importance of enriching microbes indigenous to (and targeted for) a specific environment and the recognized potential benefits of microbial consortia over isolates (e.g. functional redundancy). The proposed community enrichment model could also be instrumental for other microbial functions such as phosphorus solubilization, plant growth promotion or disease suppression.

Extracting the GEMs: Genotype, Environment, and Microbiome Interactions Shaping Host Phenotypes.

One of the fundamental tenets of biology is that the phenotype of an organism (Y) is determined by its genotype (G), the environment (E), and their interaction (GE). Quantitative phenotypes can then be modeled as Y = G + E + GE + e, where e is the biological variance. This simple and tractable model has long served as the basis for studies investigating the heritability of traits and decomposing the variability in fitness. The importance and contribution of microbe interactions to a given host phenotype is largely unclear, nor how this relates to the traditional GE model. Here we address this fundamental question and propose an expansion of the original model, referred to as GEM, which explicitly incorporates the contribution of the microbiome (M) to the host phenotype, while maintaining the simplicity and tractability of the original GE model. We show that by keeping host, environment, and microbiome as separate but interacting variables, the GEM model can capture the nuanced ecological interactions between these variables. Finally, we demonstrate with an in vitro experiment how the GEM model can be used to statistically disentangle the relative contributions of each component on specific host phenotypes.

Integrated Omic Analyses Provide Evidence that a "Candidatus Accumulibacter phosphatis" Strain Performs Denitrification under Microaerobic Conditions.

The ability of "Candidatus Accumulibacter phosphatis" to grow and remove phosphorus from wastewater under cycling anaerobic and aerobic conditions has also been investigated as a metabolism that could lead to simultaneous removal of nitrogen and phosphorus by a single organism. However, although phosphorus removal under cyclic anaerobic and anoxic conditions has been demonstrated, clarifying the role of "Ca. Accumulibacter phosphatis" in this process has been challenging, since (i) experimental research describes contradictory findings, (ii) none of the published "Ca. Accumulibacter phosphatis" genomes show the existence of a complete respiratory pathway for denitrification, and (iii) some genomes lacking a complete respiratory pathway have genes for assimilatory nitrate reduction. In this study, we used an integrated omics analysis to elucidate the physiology of a "Ca. Accumulibacter phosphatis" strain enriched in a reactor operated under cyclic anaerobic and microaerobic conditions. The reactor's performance suggested the ability of the enriched "Ca. Accumulibacter phosphatis" strain (clade IC) to simultaneously use oxygen and nitrate as electron acceptors under microaerobic conditions. A draft genome of this organism was assembled from metagenomic reads ("Ca. Accumulibacter phosphatis" UW-LDO-IC) and used as a reference to examine transcript abundance throughout one reactor cycle. The genome of UW-LDO-IC revealed the presence of a full pathway for respiratory denitrification. The observed transcript abundance patterns showed evidence of coregulation of the denitrifying genes along with a cbb 3 cytochrome, which has been characterized as having high affinity for oxygen. Furthermore, we identified an FNR-like binding motif upstream of the coregulated genes, suggesting transcription-level regulation of both denitrifying and respiratory pathways in UW-LDO-IC. Taking the results together, the omics analysis provides strong evidence that "Ca. Accumulibacter phosphatis" UW-LDO-IC uses oxygen and nitrate simultaneously as electron acceptors under microaerobic conditions. IMPORTANCE "Candidatus Accumulibacter phosphatis" is widely found in full-scale wastewater treatment plants, where it has been identified as the key organism for biological removal of phosphorus. Since aeration can account for 50% of the energy use during wastewater treatment, microaerobic conditions for wastewater treatment have emerged as a cost-effective alternative to conventional biological nutrient removal processes. Our report provides strong genomics-based evidence not only that "Ca. Accumulibacter phosphatis" is the main organism contributing to phosphorus removal under microaerobic conditions but also that this organism simultaneously respires nitrate and oxygen in this environment, consequently removing nitrogen and phosphorus from the wastewater. Such activity could be harnessed in innovative designs for cost-effective and energy-efficient optimization of wastewater treatment systems.

acI Actinobacteria Assemble a Functional Actinorhodopsin with Natively Synthesized Retinal.

Freshwater lakes harbor complex microbial communities, but these ecosystems are often dominated by acI Actinobacteria 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 acI Actinobacteria are 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 acI Actinobacteria in a eutrophic lake shows that all retinal and carotenoid pathway operons are transcribed and that actR is 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 ß-carotene oxygenase formed holo-ActR and acidified solution during illumination. Taken together, our results prove that acI Actinobacteria containing both ActR and acI retinal production machinery have the capacity to natively synthesize a green light-dependent outward proton-pumping rhodopsin.IMPORTANCE Microbes play critical roles in determining the quality of freshwater ecosystems, which are vital to human civilization. Because acI Actinobacteria are 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.

Stripping Away the Soil: Plant Growth Promoting Microbiology Opportunities in Aquaponics.

As the processes facilitated by plant growth promoting microorganisms (PGPMs) become better characterized, it is evident that PGPMs may be critical for successful sustainable agricultural practices. Microbes enrich plant growth through various mechanisms, such as enhancing resistance to disease and drought, producing beneficial molecules, and supplying nutrients and trace metals to the plant rhizosphere. Previous studies of PGPMs have focused primarily on soil-based crops. In contrast, aquaponics is a water-based agricultural system, in which production relies upon internal nutrient recycling to co-cultivate plants with fish. This arrangement has management benefits compared to soil-based agriculture, as system components may be designed to directly harness microbial processes that make nutrients bioavailable to plants in downstream components. However, aquaponic systems also present unique management challenges. Microbes may compete with plants for certain micronutrients, such as iron, which makes exogenous supplementation necessary, adding production cost and process complexity, and limiting profitability and system sustainability. Research on PGPMs in aquaponic systems currently lags behind traditional agricultural systems, however, it is clear that certain parallels in nutrient use and plant-microbe interactions are retained from soil-based agricultural systems.

Road MAPs to engineer host microbiomes.

Microbiomes contribute directly or indirectly to host health and fitness. Thus far, investigations into these emergent traits, referred to here as microbiome-associated phenotypes (MAPs), have been primarily qualitative and taxonomy-driven rather than quantitative and trait-based. We present the MAPs-first approach, a theoretical and experimental roadmap that involves quantitative profiling of MAPs across genetically variable hosts and subsequent identification of the underlying mechanisms. We outline strategies for developing 'modular microbiomes'-synthetic microbial consortia that are engineered in concert with the host genotype to confer different but mutually compatible MAPs to a single host or host population. By integrating host and microbial traits, these strategies will facilitate targeted engineering of microbiomes to the benefit of agriculture, human/animal health and biotechnology.

Metabolic Network Analysis and Metatranscriptomics Reveal Auxotrophies and Nutrient Sources of the Cosmopolitan Freshwater Microbial Lineage acI.

An explosion in the number of available genome sequences obtained through metagenomics and single-cell genomics has enabled a new view of the diversity of microbial life, yet we know surprisingly little about how microbes interact with each other or their environment. In fact, the majority of microbial species remain uncultivated, while our perception of their ecological niches is based on reconstruction of their metabolic potential. In this work, we demonstrate how the "seed set framework," which computes the set of compounds that an organism must acquire from its environment (E. Borenstein, M. Kupiec, M. W. Feldman, and E. Ruppin, Proc Natl Acad Sci U S A 105:14482-14487, 2008, https://doi.org/10.1073/pnas.0806162105), enables computational analysis of metabolic reconstructions while providing new insights into a microbe's metabolic capabilities, such as nutrient use and auxotrophies. We apply this framework to members of the ubiquitous freshwater actinobacterial lineage acI, confirming and extending previous experimental and genomic observations implying that acI bacteria are heterotrophs reliant on peptides and saccharides. We also present the first metatranscriptomic study of the acI lineage, revealing high expression of transport proteins and the light-harvesting protein actinorhodopsin. Putative transport proteins complement predictions of nutrients and essential metabolites while providing additional support of the hypothesis that members of the acI are photoheterotrophs. IMPORTANCE 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.

Community Assembly and Ecology of Activated Sludge under Photosynthetic Feast-Famine Conditions.

Here, we demonstrate that photosynthetic oxygen production under light-dark and feast-famine cycles with no mechanical aeration and negligible oxygen diffusion is able to maintain phosphorus cycling activity associated with the enrichment of polyphosphate accumulating organisms (PAOs). We investigate the ecology of this novel system by conducting a time series analysis of prokaryotic and eukaryotic biodiversity using the V3-V4 and V4 regions of the 16S and 18S rRNA gene sequences, respectively. In the Eukaryotic community, the initial dominant alga observed was Desmodesmus. During operation, the algal community became a more diverse consortium of Desmodesmus, Parachlorella, Characiopodium, and Bacillariophytina. In the Prokaryotic community, there was an initial enrichment of the PAO Candidatus Accumulibacter phosphatis (Accumulibacter) Acc-SG2, and the dominant ammonia-oxidizing organism was Nitrosomonas oligotropha; however, these populations decreased in relative abundance, becoming dominated by Accumulibacter Acc-SG3 and Nitrosomonas ureae. Furthermore, functional guilds that were not abundant initially became enriched including the putative Cyanobacterial PAOs Obscuribacterales and Leptolyngbya and the H2-oxidizing denitrifying autotroph Sulfuritalea. After a month of operation, the most-abundant prokaryote belonged to an uncharacterized clade of Chlorobi classified as Chlorobiales;SJA-28 Clade III, the first reported enrichment of this lineage. This experiment represents the first investigation into the ecological interactions and community assembly during photosynthetic feast-famine conditions. Our findings suggest that photosynthesis may provide sufficient oxygen to drive polyphosphate cycling.

Transformation and speciation of typical heavy metals in soil aquifer treatment system during long time recharging with secondary effluent: Depth distribution and combination.

Soil aquifer treatment (SAT) systems rely on extensive physical and biogeochemical processes in the vadose zone and aquifer for water quality improvement. In this study, the distribution, quantitative changes, as well as the speciation characteristics of heavy metals in different depth of soils of a two-year operated lab-scale SAT was explored. A majority of the heavy metals in the recharged secondary effluent were efficiently trapped by the steady-state operated SAT (removal efficiency ranged from 74.7% to 98.2%). Thus, significant accumulations of 31.7% for Cd, 15.9% for Cu, 15.3% for Zn and 8.6% for Cr were observed for the top soil after 730 d operation, leading to the concentration (in µg g-1) of those four heavy metals of the packed soil increased from 0.51, 46.7, 61.0 and 35.7 to 0.66, 54.2, 70.4 and 38.8, respectively. By contrast, the accumulation of Mn and Pb were quite low. The residual species were the predominant fraction of the six heavy metals (ranged for 59.8-82.4%), followed by oxidisable species. Although the Zn, Cr, Cd, Cu and Mn were efficiently bounded onto the oxide components within the soil, the percentage of the labile metal fractions (water-, acid-exchangeable and reducible metal fractions) exhibited a slight increasing after 2 Y operation. Significantly heavy metals accumulation and slightly decreasing of the proportion of the stable fractions indicated a potentially higher environmental hazard for those six heavy metals after long-term SAT operation (especially for Cu, Zn and Cd). Finally, a linear relationship between the accumulation rate of metal species and the variation of soil organic carbon concentration and water extractable organic carbon was demonstrated.

Ancestral genome reconstruction identifies the evolutionary basis for trait acquisition in polyphosphate accumulating bacteria.

The evolution of complex traits is hypothesized to occur incrementally. Identifying the transitions that lead to extant complex traits may provide a better understanding of the genetic nature of the observed phenotype. A keystone functional group in wastewater treatment processes are polyphosphate accumulating organisms (PAOs), however the evolution of the PAO phenotype has yet to be explicitly investigated and the specific metabolic traits that discriminate non-PAO from PAO are currently unknown. Here we perform the first comprehensive investigation on the evolution of the PAO phenotype using the model uncultured organism Candidatus Accumulibacter phosphatis (Accumulibacter) through ancestral genome reconstruction, identification of horizontal gene transfer, and a kinetic/stoichiometric characterization of Accumulibacter Clade IIA. The analysis of Accumulibacter's last common ancestor identified 135 laterally derived genes, including genes involved in glycogen, polyhydroxyalkanoate, pyruvate and NADH/NADPH metabolisms, as well as inorganic ion transport and regulatory mechanisms. In contrast, pathways such as the TCA cycle and polyphosphate metabolism displayed minimal horizontal gene transfer. We show that the transition from non-PAO to PAO coincided with horizontal gene transfer within Accumulibacter's core metabolism; likely alleviating key kinetic and stoichiometric bottlenecks, such as anaerobically linking glycogen degradation to polyhydroxyalkanoate synthesis. These results demonstrate the utility of investigating the derived genome of a lineage to identify key transitions leading to an extant complex phenotype.

Metatranscriptomic insights on gene expression and regulatory controls in Candidatus Accumulibacter phosphatis.

Previous studies on enhanced biological phosphorus removal (EBPR) have focused on reconstructing genomic blueprints for the model polyphosphate-accumulating organism Candidatus Accumulibacter phosphatis. Here, a time series metatranscriptome generated from enrichment cultures of Accumulibacter was used to gain insight into anerobic/aerobic metabolism and regulatory mechanisms within an EBPR cycle. Co-expressed gene clusters were identified displaying ecologically relevant trends consistent with batch cycle phases. Transcripts displaying increased abundance during anerobic acetate contact were functionally enriched in energy production and conversion, including upregulation of both cytoplasmic and membrane-bound hydrogenases demonstrating the importance of transcriptional regulation to manage energy and electron flux during anerobic acetate contact. We hypothesized and demonstrated hydrogen production after anerobic acetate contact, a previously unknown strategy for Accumulibacter to maintain redox balance. Genes involved in anerobic glycine utilization were identified and phosphorus release after anerobic glycine contact demonstrated, suggesting that Accumulibacter routes diverse carbon sources to acetyl-CoA formation via previously unrecognized pathways. A comparative genomics analysis of sequences upstream of co-expressed genes identified two statistically significant putative regulatory motifs. One palindromic motif was identified upstream of genes involved in PHA synthesis and acetate activation and is hypothesized to be a phaR binding site, hence representing a hypothetical PHA modulon. A second motif was identified ~35 base pairs (bp) upstream of a large and diverse array of genes and hence may represent a sigma factor binding site. This analysis provides a basis and framework for further investigations into Accumulibacter metabolism and the reconstruction of regulatory networks in uncultured organisms.

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage.

Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di- and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits.