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Grand Challenges for Biological and Environmental Research: Progress and Future Vision. A Report from the Biological and Environmental Research Advisory Committee

The Biological and Environmental Research (BER) program within the U.S. Department of Energy (DOE) Office of Science supports research focusing on the interconnections between energy production and the living environment. This fundamental research, conducted at universities, DOE national laboratories, and research institutions across the country, explores organisms and ecosystems that can influence the U.S. energy system and advances understanding of the relationships between energy and environment from local to global scales. Research within BER can be categorized into biological systems and Earth and environmental systems. Biological systems research seeks to characterize and predictively understand microbial and plant systems using genomic science, computational analyses, and experimental approaches. Foundational knowledge of the structure and function of these systems underpins the ability to leverage natural processes for energy production, including the sustainable development of biofuels and other bioproducts. Earth and environmental systems research seeks to characterize and predictively understand feedbacks between Earth and energy systems and includes studies on atmospheric physics and chemistry, ecosystem ecology and biogeochemistry, and development and validation of Earth system models extending from regional to global scales. To promote world-class research in these areas, BER supports user facilities that enable observation and measurement of atmospheric, biological, and biogeochemical processes using the latest technologies.

54 ENVIRONMENTAL SCIENCES↗

Electron transport across the cell envelope via multiheme c -type cytochromes in Geobacter sulfurreducens

Extracellular electron transfer (EET) enables certain microorganisms to respire using soluble and insoluble extracellular electron acceptors by transporting electrons across the cell envelope. Among these, G. sulfurreducens serves as a model organism for understanding direct EET pathways, where multiheme c-type cytochromes mediate electron transport from intracellular redox carriers to extracellular acceptors such as Fe(III) oxides and electrodes. This review focuses on heme-dependent electron transfer in Geobacter sulfurreducens, detailing the roles of inner membrane cytochromes, periplasmic carriers, outer membrane conduits, and recently characterized extracellular nanowires formed by polymerized multiheme c-type cytochromes, including OmcS, OmcE, and OmcZ. We examine the state of understanding of their physiological function, their structural features, expression patterns, and essentiality under various respiratory conditions. These insights advance our understanding of microbial anaerobic respiration and have implications for biogeochemical cycling, bioenergy generation, and bioremediation. The molecular architecture, assembly mechanisms, and secretion pathways of multiheme c-type cytochrome nanowires remain active areas of investigation, offering promising directions for future research and biotechnological innovation in engineered microbial systems.

Chemistry↗

Comparative Genomic Analysis of Three Pseudomonas Species Isolated from the Eastern Oyster (Crassostrea virginica) Tissues, Mantle Fluid, and the Overlying Estuarine Water Column

The eastern oysters serve as important keystone species in the United States, especially in the Gulf of Mexico estuarine waters, and at the same time, provide unparalleled economic, ecological, environmental, and cultural services. One ecosystem service that has garnered recent attention is the ability of oysters to sequester impurities and nutrients, such as nitrogen (N), from the estuarine water that feeds them, via their exceptional filtration mechanism coupled with microbially-mediated denitrification processes. It is the oyster-associated microbiomes that essentially provide these myriads of ecological functions, yet not much is known on these microbiota at the genomic scale, especially from warm temperate and tropical water habitats. Among the suite of bacterial genera that appear to interplay with the oyster host species, pseudomonads deserve further assessment because of their immense metabolic and ecological potential. To obtain a comprehensive understanding on this aspect, we previously reported on the isolation and preliminary genomic characterization of three Pseudomonas species isolated from minced oyster tissue (P. alcaligenes strain OT69); oyster mantle fluid (P. stutzeri strain MF28) and the water collected from top of the oyster reef (P. aeruginosa strain WC55), respectively. In this comparative genomic analysis study conducted on these three targeted pseudomonads, native to the eastern oyster and its surrounding environment, provided further insights into their unique functional traits, conserved gene pools between the selected pseudomonads, as well as genes that render unique characteristics in context to metabolic traits recruited during their evolutionary history via horizontal gene transfer events as well as phage-mediated incorporation of genes. Moreover, the strains also supported extensively developed resistomes, which suggests that environmental microorganisms native to relatively pristine environments, such as Apalachicola Bay, Florida, have also recruited an arsenal of antibiotic resistant gene determinants, thus posing an emerging public health concern.

Pathak, Ashish↗

The Phylogenomic Diversity of Herbivore-Associated Fibrobacter spp. Is Correlated to Lignocellulose-Degrading Potential

The herbivore gut microbiome is incredibly diverse, and a functional understanding of this diversity is needed to more reliably manipulate this community for specific gain, such as increased production in ruminant livestock. Microbial degraders of plant cell wall polysaccharides in the herbivore gut, particularly Fibrobacter spp., are of fundamental importance to their hosts for digestion of a diet consisting primarily of recalcitrant plant fibers. Considerable phylogenetic diversity exists among members of the genus Fibrobacter , but much of this diversity remains cryptic. Here, we used comparative genomics, applied to a diverse collection of recently isolated Fibrobacter strains, to identify a robust association between carbohydrate-active enzyme gene content and the Fibrobacter phylogeny. Our results provide the strongest evidence reported to date for functional differences among Fibrobacter phylotypes associated with either the rumen or the hindgut and emphasize the general significance of carbohydrate-active enzymes in the evolution of fiber-degrading bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Antarctic lake viromes reveal potential virus associated influences on nutrient cycling in ice-covered lakes

The McMurdo Dry Valleys (MDVs) of Antarctica are a mosaic of extreme habitats which are dominated by microbial life. The MDVs include glacial melt holes, streams, lakes, and soils, which are interconnected through the transfer of energy and flux of inorganic and organic material via wind and hydrology. For the first time, we provide new data on the viral community structure and function in the MDVs through metagenomics of the planktonic and benthic mat communities of Lakes Bonney and Fryxell. Viral taxonomic diversity was compared across lakes and ecological function was investigated by characterizing auxiliary metabolic genes (AMGs) and predicting viral hosts. Our data suggest that viral communities differed between the lakes and among sites: these differences were connected to microbial host communities. AMGs were associated with the potential augmentation of multiple biogeochemical processes in host, most notably with phosphorus acquisition, organic nitrogen acquisition, sulfur oxidation, and photosynthesis. Viral genome abundances containing AMGs differed between the lakes and microbial mats, indicating site specialization. Using procrustes analysis, we also identified significant coupling between viral and bacterial communities (p = 0.001). Finally, host predictions indicate viral host preference among the assembled viromes. Collectively, our data show that: (i) viruses are uniquely distributed through the McMurdo Dry Valley lakes, (ii) their AMGs can contribute to overcoming host nutrient limitation and, (iii) viral and bacterial MDV communities are tightly coupled.

Microbiology↗

Defining the Minimal Set of Microbial Genes Required for Valorization of Lignin Biomass (Final Report)

Project Goals: Lignin is the second most abundant biopolymer on earth and represents a critically underutilized biomass resource for hydrocarbon feedstocks. Despite substantial effort, there is still no efficient process to convert lignin to useable carbon-based platform chemicals and materials. The goal of this project is identify a minimal set of microbial enzymes necessary for lignin breakdown and sufficient for the synthesis of valuable chemical intermediates from lignin isolated as a byproduct of lignocellulosic ethanol production. These genes will be then used to engineer functional whole cell biocatalysts for tunable lignin metabolism. To date, although a number of enzymes have been associated with lignin degradation, most have been tested in isolation (as individual enzymes) and on drastically different substrates -- often dyes that are not related to lignin. In contrast, lignin utilization in nature likely occurs by microbial consortia with multiple enzymes acting synergistically. We propose to examine two separate stages of lignin breakdown carried out by the microbes that do it best: (1) early breakdown of native polymeric lignin into soluble fragments by a set of sequenced wood-rotting fungal species, and (2) downstream metabolism of these soluble lignin fragments to useful chemical intermediates by a panel of sequenced soil saprophytes. Our approach involves testing sets of genes that will be assayed combinatorially in the context of a heterologous expression host. The resulting engineered strains will be systematically assayed using soluble lignin fragments, synthetic defined polymeric lignin, and finally lignin directly sourced from lignocellulosic processing streams. In addition to resulting in a functional whole cell biocatalyst for lignin utilization, we anticipate that this approach will allow us to address key unanswered questions about lignin metabolism in nature, including: (1) Why does the Trametes versicolor genome contain 25 different class II peroxidases? (2) What is the role of laccases in lignin metabolism? Why do some aggressive lignin degraders have many laccases (e.g. >7 in T. versicolor) while others have none (e.g. P. chrysosporium)? (3) How is peroxide provided in a controlled manner to drive peroxidase activity without causing the enzyme inhibition that is so often observed in vitro? (4) What strategies do microbial lignin degraders use to avoid the problem of repolymerization during active lignin degradation? and (5) Can microbial lignin metabolism be diverted for high level production of defined aromatics? A final critical question is whether combining key minimal sets of enzymes from a wide range of organisms will result in engineered strains capable of highly efficient, streamlined pathways for lignin utilization that can be tuned for a specific carbon output. This effort will leverage DOE investments in microbial genome sequencing, and secure a critical channel for lignin biomass utilization that will also help to render lignocellulosic a viable feedstock for the production of renewable liquid biofuels.

59 BASIC BIOLOGICAL SCIENCES↗

Gut microbiota and their putative metabolic functions in fragmented Bengal tiger population of Nepal

Bengal tigers (Panthera tigris tigris) serve a pivotal role as an apex predator in forest ecosystems. To increase our knowledge on factors impacting the viability and health of this endangered species, we studied the gut microbiota in 32 individual Bengal tigers from three geographically separated areas (Chitwan National Park (CNP), Bardia National Park (BNP) and Suklaphanta Wildlife Reserve (SWR)) in Nepal, using noninvasive genetic sampling methods. Gut microbiota influence the immune system, impact various physiological functions, and modulates metabolic reactions, that ultimately impact the host health, behavior and development. Across the tiger populations in Nepal, we found significant differences in the composition of microbial communities based on their geographic locations. Specifically, we detected significant differences between CNP and the other two protected areas (CNP vs BNP: pseudo t = 1.944, P = 0.006; CNP vs SWR: pseudo t = 1.9942, P = 0.0071), but no differences between BNP and SWR. This mirrors what has been found for tiger gene flow in the same populations, suggesting gut microbiota composition and host gene flow may be linked. Furthermore, predictive metagenome functional content analysis (PICRUSt) revealed a higher functional enrichment and diversity for significant gut microbiota in the Chitwan tiger population and the lowest enrichment and diversity in Suklaphanta. The CNP tiger population contained higher proportions of microbiota that are associated with predicted functions relevant for metabolism of amino acid, lipid, xenobiotics biodegradation, terpenoides and polyketides than the SWR population. We conclude the tiger population structure, gut microbiota profile and associated functional metabolic categories are correlated, with geographically most separated CNP and SWR tiger population having the most distinct and different host genotype and microbiota profiles. Our work dramatically expands the understanding of tiger microbiota in wild populations and provides a valuable case study on how to investigate genetic diversity at different hierarchical levels, including hosts as well as their microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

Exploring structure-function relationships governing transport by and regulation of the major cyanobacterial bicarbonate transporters SbtA and BicA

Cyanobacteria play a large and essential role in the global carbon cycle, while also representing promising microbial platforms for the direct bioproduction of fuels and chemicals from solar energy and carbon dioxide (CO 2 ). In both cases, a critical step in the overall photosynthetic process is the transport of inorganic carbon (Ci) across the cell membrane, which occurs via different mechanisms and associated transporter proteins that target either CO 2 or bicarbonate (HCO 3 - ). In the latter case, BicA (constitutively-expressed, high-flux/low-affinity Na + /HCO 3 - symporter) and SbtA (low-flux/high-affinity Na + /HCO 3 - symporter, expressed only under low CO 2 conditions) have been identified as the two main cyanobacterial HCO 3 - transporters. This project entailed combining detailed functional and high-resolution structural studies to help fill key knowledge gaps regarding the mechanisms of HCO 3 - transport by BicA and SbtA. Specifically, major goals of this project included: i) identifying key residues and structural features of BicA and SbtA that are responsible for modulating their uptake flux and affinity behaviors, and ii) obtaining high-resolution structures of full-length BicA and SbtA.

59 BASIC BIOLOGICAL SCIENCES↗

Flux balance modeling to predict bacterial survival during pulsed-activity events

Abstract. Desert biological soil crusts (BSCs) are cyanobacteria-dominated surface soil microbial communities common to plant interspaces in arid environments. The capability to significantly dampen their metabolism allows them to exist for extended periods in a desiccated dormant state that is highly robust to environmental stresses. However, within minutes of wetting, metabolic functions reboot, maximizing activity during infrequent permissive periods. Microcoleus vaginatus, a primary producer within the crust ecosystem and an early colonizer, initiates crust formation by binding particles in the upper layer of soil via exopolysaccharides, making microbial dominated biological soil crusts highly dependent on the viability of this organism. Previous studies have suggested that biopolymers play a central role in the survival of this organism by powering resuscitation, rapidly forming compatible solutes, and fueling metabolic activity in dark, hydrated conditions. To elucidate the mechanism of this phenomenon and provide a basis for future modeling of BSCs, we developed a manually curated, genome-scale metabolic model of Microcoleus vaginatus (iNJ1153). To validate this model, gas chromatography–mass spectroscopy (GC–MS) and liquid chromatography–mass spectroscopy (LC–MS) were used to characterize the rate of biopolymer accumulation and depletion in in hydrated Microcoleus vaginatus under light and dark conditions. Constraint-based flux balance analysis showed agreement between model predictions and experimental reaction fluxes. A significant amount of consumed carbon and light energy is invested into storage molecules glycogen and polyphosphate, while β-polyhydroxybutyrate may function as a secondary resource. Pseudo-steady-state modeling suggests that glycogen, the primary carbon source with the fastest depletion rate, will be exhausted if M. vaginatus experiences dark wetting events 4 times longer than light wetting events.

59 BASIC BIOLOGICAL SCIENCES↗

Co-occurrence networks reveal more complexity than community composition in resistance and resilience of microbial communities

Plant response to drought stress involves fungi and bacteria that live on and in plants and in the rhizosphere, yet the stability of these myco- and micro-biomes remains poorly understood. We investigate the resistance and resilience of fungi and bacteria to drought in an agricultural system using both community composition and microbial associations. Here we show that tests of the fundamental hypotheses that fungi, as compared to bacteria, are (i) more resistant to drought stress but (ii) less resilient when rewetting relieves the stress, found robust support at the level of community composition. Results were more complex using all-correlations and co-occurrence networks. In general, drought disrupts microbial networks based on significant positive correlations among bacteria, among fungi, and between bacteria and fungi. Surprisingly, co-occurrence networks among functional guilds of rhizosphere fungi and leaf bacteria were strengthened by drought, and the same was seen for networks involving arbuscular mycorrhizal fungi in the rhizosphere. We also found support for the stress gradient hypothesis because drought increased the relative frequency of positive correlations.

59 BASIC BIOLOGICAL SCIENCES↗

Decrypting bacterial polyphenol metabolism in an anoxic wetland soil

Abstract Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

Lake Arrowhead Microbial Genomics Conference

The Lake Arrowhead Microbial Genomics Conference (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) was held September 16-20, 2018, at the UCLA Conference Center at Lake Arrowhead, California. This conference is part of a yearly meeting initiated in 1991 to bring together genome sequencers, bioinformatics specialists, biologists, and geneticists, to forge interactions that would result in meaningful functional genomics. The goal of the meeting was to translate the influx of new genome sequencing information into useful biological studies. The Lake Arrowhead 2018 meeting had a major focus on microbial communities, the human microbiome, pathogens, bioenergetics, and environmental microbiology. The field of genomics has reached the point where deriving the sequence of an organism’s entire genome is now seen as a beginning rather than an endpoint. Moreover, understanding the sequences of whole communities, and particularly those that make up different microbiomes, is now a central point of the field. The 2018 meeting covered microorganisms for which extensive analyses exist, and those for which new biological and technical strategies are being developed. The focus on biodiversity, the human microbiome, pathogenic organisms, and bioenergetics added special significance to this meeting. This meeting was designed to have a mix of 34 invited presentations and poster sessions with 80 posters, and had 160 participants. This meeting has become established as a major annual microbial genomics conference during the last 26 years and has assured status and quality. It offers a major opportunity for young investigators and students to attend, present posters, and give talks. In fact, 22 of the 34 invited speakers were early career stage scientists. An important impact of the conference was the forging of new collaborations between scientists and particularly young scientists with the array of multidisciplinary researchers at the meeting.

Miller, Jeffrey H.↗

Lake Arrowhead Microbial Genomics Conference

The Lake Arrowhead Microbial Genomics Conference (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) was held September 11-15, 2022, at the UCLA Conference Center at Lake Arrowhead, California. This conference is part of a yearly meeting initiated in 1991 to bring together genome sequencers, bioinformatics specialists, biologists, and geneticists, to forge interactions that would result in meaningful functional genomics. The goal of the meeting was to translate the influx of new genome sequencing information into useful biological studies. The Lake Arrowhead 2022 meeting had a major focus on microbial communities, the human microbiome, pathogens, phage therapy, bioinformatic methods of analysis, bioenergenics, and environmental microbiology. The field of genomics has reached the point where deriving the sequence of an organism’s entire genome is now seen as a beginning rather than an endpoint. Moreover, understanding the sequences of whole communities, and particularly those that make up different microbiomes, is now a central point of the field. The 2022 meeting covered microorganisms for which extensive analyses exist, and those for which new biological and technical strategies are being developed. The focus on biodiversity, the human microbiome, pathogenic organisms and methods of countering them, and bioenergetics added special significance to this meeting. This meeting was designed to have a mix of 36 invited presentations and poster sessions with more than 80 posters, and had 147 participants. This meeting has become established as a major annual microbial genomics conference during the last 29 years and has assured status and quality. It offers a major opportunity for young investigators and students to attend, present posters, and give talks. In fact, 50% of the invited speakers were early career stage scientists. An important impact of the conference was the forging of new collaborations between scientists and particularly young scientists with the array of multidisciplinary researchers at the meeting.

Miller, Jeffrey H.↗

Log-normal model for microbial survival in heat sterilization

An analytical model is developed for the survival times of organisms in heat sterilization in which the probability of inactivation as a function of exposure time is log-normally distributed. Experimental data is examined relative to this model and it is concluded that the model is valid except during the initial period of heating when an additional interaction between the organisms and their surrounding medium appears to be present. At long heating times, the log-normal model appears more accurate for extrapolating to low survival probabilities than the usual logarithmic survivor curves and is generally more conservative.

SURVIVAL↗

Geochemical and Microbial Community Attributes in Relation to Hyporheic Zone Geological Facies

The hyporheic zone (HZ) is the active ecotone between the surface stream and groundwater, where exchanges of water, nutrients, and organic matter occur in response to variations in discharge and riverbed properties. Within this region, a confluence of surface-derived organic carbon and subsurface nitrogen (in the form of nitrate) has been shown to stimulate microbial activity and transformations of carbon and nitrogen species. For example, production of gases such as CO 2 , N 2 and N 2 O indicate hyporheic zones might have a significant effect on energy and nutrient flows between the atmosphere and the subsurface. Managed and seasonal river stage changes and geomorphology-controlled sediment texture drive water flow within the HZ of the Columbia River. To examine the relationship between sediment texture, biogeochemistry, and biological activity in the HZ, the grain size distributions for sediment samples taken across 320 m of shoreline were characterized to define geological facies, and the relationships among physical properties of the facies, physicochemical attributes of the local environment, and the structure and activity of associated microbial communities were examined. Mud and sand content and the presence of carbon and nitrogen oxidizers were found to explain the variability in many biogeochemical attributes. Microbial community analysis revealed a high relative abundance of putative ammonia-oxidizing Thaumarchaeota and nitrite-oxidizing Nitrospirae, together comprising ~20% of the total community across all samples, but scant ammonia-oxidizing Bacteria. Network analysis of operational taxonomic units and the measured geophysical, chemical, and functional parameters showed negative relationships between abundance-based modules of organisms and sand and mud contents, and positive relationships with total organic carbon. The relationships identified in this work indicate grain size distribution is a good predictor of biogeochemical properties, and that subsets of the overall microbial community respond to different sediment texture. Some member populations of these sub-communities appear to respond directly to environmental conditions, while others may be dependent on the function of the first group. For example, nitrification is a strong primary response to the observed conditions, and this activity appears to support a larger heterotrophic community. Relationships between facies and hydrobiogeochemical properties enables facies-based conditional simulation/mapping of these properties to inform multiscale modeling of hyporheic exchange and biogeochemical processes.

54 ENVIRONMENTAL SCIENCES↗

Microbial Biomass in Soils Receiving Varying Levels of Organic Inputs - A Data Compilation

This dataset contains soil microbial biomass data compiled from published laboratory studies and field manipulations of organic inputs to soils. Each study included at least three levels of a given type of organic input applied to a given type of soil. Studies were selected from the literature that had experimentally manipulated organic input rates to soils using unburned, non-synthetic material (e.g. plant litters, manures, and composts). Fifty-five (55) studies, conducted from 1997 to October 2019, were chosen and compiled into a dataset with 358 observations from 96 combinations of a given organic material applied to a given soil in agricultural systems, laboratory incubations, forests, or grasslands. • Studies were selected when biomass measurements used the chloroform fumigation-extraction procedure followed by carbon analysis, or, in two instances, studies estimated microbial biomass using phospholipid fatty acid analysis. • Studies that used fewer than three input rates or characterized microbial biomass via substrate induced respiration were excluded. • The temporal and spatial aspects of the laboratory and field studies were not essential to the analyses (Craig et al., 2021) and were not included with the measurement data. These data were used to assess the relationship between organic input amounts and microbial biomass. Complete reference citations are provided if additional information is needed. The compiled data are provided in two .csv files: (1) soil treatments and microbial biomass data and (2) literature citations. The “Study” field is the cross reference between files. Also provided is the R code for reproducing the results in the related publication.

54 ENVIRONMENTAL SCIENCES↗

Microscale spatiotemporal patterns of water, soil organic carbon, and enzymes in plant litter detritusphere

Understanding biophysical and biochemical processes in detritusphere is critical to quantifying and modeling plant residue decomposition dynamics and subsequent soil organic carbon (C) accrual. The objectives of the study were to explore (i) micro-environmental conditions within the detritusphere formed around decomposing corn and soybean leaves, and (ii) the relationships between moisture distribution, enzyme activity, and C dynamics during decomposition. We assessed spatial and temporal dynamics of moisture distribution using X-ray and neutron computed tomography, and activities of β-glucosidase and chitinase, two enzymes involved in soil C and N processing, using zymography. We used 13 C labeled residue to track residue contribution to atmospheric CO 2 and soil organic C. Moisture redistribution pattern varied depending on the residue type. While the water was immediately absorbed by the corn leaves and maintained afterward, switchgrass leaves absorbed water more slowly and created water-deficient zones within ~1mm from the residue. This initial moisture depletion led to lower chitinase activity and residue-derived CO 2 emissions in switchgrass. In contrast to chitinase, β-glucosidase activity was influenced by a combination of vegetation history and residue type, and it was higher when the origin of the residue matched the vegetation history of the soil. Pore size had an opposite impact on the studied enzymes, supporting the notion that contrasting soil pore architecture can stimulate activities of different enzymes through a selection of dominant enzyme producers. We concluded that the decomposition dynamics of plant residues is not only a simple function of residue chemistry, but rather a combined effect of the vegetation history, in part through its effect on microbial community composition, the plant residue chemical and likely physical characteristics, and the soil pore structure in the detritusphere. Together, they create temporally dynamic micro-environmental conditions influencing decomposition. Specifically, our study demonstrated that the initial micro-environment formulated in detritusphere can play an important role in enzyme activities and consequent C dynamics.

59 BASIC BIOLOGICAL SCIENCES↗

Mutant phenotypes for thousands of bacterial genes of unknown function

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Lastly, our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

59 BASIC BIOLOGICAL SCIENCES↗