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.

Under the Net Zero World Initiative, the United States is mobilizing the capabilities of nine U.S. government agencies, led by the U.S. Department of Energy (DOE), to partner with philanthropies and multiple countries to cocreate and implement tailored technical and investment pathways to accelerate the decarbonization of global energy systems. In addition, 10 of the DOE national laboratories have built a consortium housed in the Net Zero World Action Center to implement this vision by providing the deep analysis and modeling required to carry out the vision. As a whole-of-government program, the Net Zero World Initiative partners with countries committed to raising their climate ambitions by creating and implementing highly tailored, actionable technical and investment strategies that put a net-zero world within reach. The initiative enables country partners to harness the convening power and technical expertise of U.S. agencies and laboratories, international industry, and technical institutions while providing the United States an opportunity to learn from and deepen U.S. technical cooperation with key countries. This report is the first of a series, with future Phase II work being informed by ongoing consultations with the partner countries to address country pathway analysis priorities. This future work will likely include evaluating detailed technological, policy, and investment options for key sectors and for energy systems holistically. This analysis may examine in greater detail the economic and social benefits of net-zero energy transitions, including quality jobs and health outcomes, the impacts of price and supply volatility on energy investments and decisions, the risk of stranded assets, and related issues.