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Aligning theoretical and empirical representations of soil carbon-to-nitrogen stoichiometry with process-based terrestrial biogeochemistry models

Soil carbon-nitrogen (C:N) stoichiometry acts as a control over decomposition and soil organic matter formation and loss, making it a key soil property for understanding ecosystem dynamics and projected ecosystems responses to global environmental change. However, the controls of soil C:N and how they respond to increasing pressures from global change agents are not fully understood. The “foundational” controls on soil C:N, namely plant and microbial C:N, have been used to predict soil C:N, but fail to accurately simulate all ecosystems and may be insufficient for predictions under global environmental change. Here we present an “emerging” representation of controls of soil C:N that includes plant-microbe-mineral feedbacks that have been shown to regulate soil C:N. We argue that including representation of these emerging drivers in process-based terrestrial biogeochemistry models, which include biological N fixation, mycorrhizae, priming, root exudation of organic acids, and mineralogy (including soil texture, mineral composition, and aggregation), will improve mechanistic representation of soil C:N and associated processes. Such improvements will produce models that will better simulate a variety of ecological states and predict soil C:N when global changes modify plant-microbe-mineral interactions. Here, we align our empirical understanding of controls of soil C:N with those controls represented in models, identifying contexts where emerging drivers might be particularly important to represent (e.g., priming and root exudation in nutrient-limited conditions) and areas of future work. Additionally, we show that implementing emerging drivers of soil C:N results in different simulated outcomes at steady state and in response to elevated atmospheric CO2. Our review and preliminary simulations support the need to incorporate emerging drivers of soil C:N into process-based terrestrial biogeochemistry models, allowing for both theoretical exploration of mechanisms and potentially more accurate predictions of land biogeochemical responses to global change.

54 ENVIRONMENTAL SCIENCES↗

Influence of sulfide on diazotrophic growth of the methanogen Methanococcus maripaludis and its implications for the origin of nitrogenase

Abstract Methanogens inhabit euxinic (sulfide-rich) or ferruginous (iron-rich) environments that promote the precipitation of transition metals as metal sulfides, such as pyrite, reducing metal or sulfur availability. Such environments have been common throughout Earth’s history raising the question as to how anaerobes obtain(ed) these elements for the synthesis of enzyme cofactors. Here, we show a methanogen can synthesize molybdenum nitrogenase metallocofactors from pyrite as the source of iron and sulfur, enabling nitrogen fixation. Pyrite-grown, nitrogen-fixing cells grow faster and require 25-fold less molybdenum than cells grown under euxinic conditions. Growth yields are 3 to 8 times higher in cultures grown under ferruginous relative to euxinic conditions. Physiological, transcriptomic, and geochemical data indicate these observations are due to sulfide-promoted metal limitation, in particular molybdenum. These findings suggest that molybdenum nitrogenase may have originated in a ferruginous environment that titrated sulfide to form pyrite, facilitating the availability of sufficient iron, sulfur, and molybdenum for cofactor biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗

Microbial functional genes commonly respond to elevated carbon dioxide

Atmospheric CO 2 concentration is increasing, largely due to anthropogenic activities. Previous studies of individual free-air CO 2 enrichment (FACE) experimental sites have shown significant impacts of elevated CO 2 (eCO 2 ) on soil microbial communities; however, no common microbial response patterns have yet emerged, challenging our ability to predict ecosystem functioning and sustainability in the future eCO 2 environment. Here we analyzed 66 soil microbial communities from five FACE sites, and showed common microbial response patterns to eCO 2 , especially for key functional genes involved in carbon and nitrogen fixation (e.g., pcc/acc for carbon fixation, nifH for nitrogen fixation), carbon decomposition (e.g., amyA and pulA for labile carbon decomposition, mnp and lcc for recalcitrant carbon decomposition), and greenhouse gas emissions (e.g., mcrA for methane production, norB for nitrous oxide production) across five FACE sites. Also, the relative abundance of those key genes was generally increased and directionally associated with increased biomass, soil carbon decomposition, and soil moisture. In addition, a further literature survey of more disparate FACE experimental sites indicated increased biomass, soil carbon decay, nitrogen fixation, methane and nitrous oxide emissions, plant and soil carbon and nitrogen under eCO 2 . A conceptual framework was developed to link commonly responsive functional genes with ecosystem processes, such as pcc/acc vs. soil carbon storage, amyA/pulA/mnp/lcc vs. soil carbon decomposition, and nifH vs. nitrogen availability, suggesting that such common responses of microbial functional genes may have the potential to predict ecosystem functioning and sustainability in the future eCO 2 environment.

59 BASIC BIOLOGICAL SCIENCES↗

Discovery of a novel filamentous prophage in the genome of the Mimosa pudica microsymbiont Cupriavidus taiwanensis STM 6018

Integrated virus genomes (prophages) are commonly found in sequenced bacterial genomes but have rarely been described in detail for rhizobial genomes. Cupriavidus taiwanensis STM 6018 is a rhizobial Betaproteobacteria strain that was isolated in 2006 from a root nodule of a Mimosa pudica host in French Guiana, South America. Here we describe features of the genome of STM 6018, focusing on the characterization of two different types of prophages that have been identified in its genome. The draft genome of STM 6018 is 6,553,639 bp, and consists of 80 scaffolds, containing 5,864 protein-coding genes and 61 RNA genes. STM 6018 contains all the nodulation and nitrogen fixation gene clusters common to symbiotic Cupriavidus species; sharing >99.97% bp identity homology to the nod / nif / noeM gene clusters from C. taiwanensis LMG19424 T and “ Cupriavidus neocalidonicus” STM 6070. The STM 6018 genome contains the genomes of two prophages: one complete Mu-like capsular phage and one filamentous phage, which integrates into a putative dif site. This is the first characterization of a filamentous phage found within the genome of a rhizobial strain. Further examination of sequenced rhizobial genomes identified filamentous prophage sequences in several Beta-rhizobial strains but not in any Alphaproteobacterial rhizobia.

59 BASIC BIOLOGICAL SCIENCES↗

Chapter 4: What We Can Learn from Measuring Metabolic Fluxes in Cyanobacteria

Cyanobacteria with the capability of oxygen-evolving photosynthesis play a vital role in the global carbon, oxygen, and nitrogen cycles. From a biochemical perspective, all phenotypic traits and functions of cyanobacteria are derived from the potential of their metabolism and mirrored in metabolic fluxes through an integrated metabolic network. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is therefore of great importance for an in-depth understanding of the role of cyanobacteria in the biosphere. Recent interest in cyanobacteria metabolic fluxes is also motivated by the prospect to utilize their light-driven CO2 fixation for sustainable production of valuable chemicals and biofuels. Valorization of cyanobacterial metabolism will require engineering of the native metabolic network - a challenge that can benefit from a quantitative understanding of metabolic reactions taking place intracellularly. In this chapter, we will discuss some intriguing questions relevant to metabolic fluxes in cyanobacteria: How are metabolic fluxes predicted or measured? What do we know about the fluxome inside various cyanobacteria thus far? How are they different between strains as well as in different conditions? And how can we use fluxomics information to guide metabolic engineering and synthetic biology? This chapter will highlight updated knowledge on cyanobacterial metabolism. Specifically, we will focus on providing readers a quantitative vision to understand how this photosynthetic model works.

BASIC BIOLOGICAL SCIENCES,INORGANIC, ORGANIC, PHYS↗

Contrasting Community Assembly Forces Drive Microbial Structural and Potential Functional Responses to Precipitation in an Incipient Soil System

Microbial communities in incipient soil systems serve as the only biotic force shaping landscape evolution. However, the underlying ecological forces shaping microbial community structure and function are inadequately understood. We used amplicon sequencing to determine microbial taxonomic assembly and metagenome sequencing to evaluate microbial functional assembly in incipient basaltic soil subjected to precipitation. Community composition was stratified with soil depth in the pre-precipitation samples, with surficial communities maintaining their distinct structure and diversity after precipitation, while the deeper soil samples appeared to become more uniform. The structural community assembly remained deterministic in pre- and post-precipitation periods, with homogenous selection being dominant. Metagenome analysis revealed that carbon and nitrogen functional potential was assembled stochastically. Sub-populations putatively involved in the nitrogen cycle and carbon fixation experienced counteracting assembly pressures at the deepest depths, suggesting the communities may functionally assemble to respond to short-term environmental fluctuations and impact the landscape-scale response to perturbations. We propose that contrasting assembly forces impact microbial structure and potential function in an incipient landscape; in situ landscape characteristics (here homogenous parent material) drive community structure assembly, while short-term environmental fluctuations (here precipitation) shape environmental variations that are random in the soil depth profile and drive stochastic sub-population functional dynamics.

16S amplicon sequencing↗

Inoculated biocrust cover and functions diverged over a gradient of soil textures and water availability

Restoring biological crust (biocrust) in disturbed drylands is challenging due to the difficult environmental conditions, such as limited soil moisture, low soil nutrients, and extreme temperatures, that impede growth. Understanding how the key components of biocrust—mosses, lichens, and cyanobacteria—react to different environmental factors informs the optimal timing, locations, and species composition for biocrust reintroduction, thereby increasing the likelihood of establishment. Here, we inoculated soils with a diverse range of biocrust organisms, analogous to seeding an area with diverse vascular plant seeds, and varied environmental conditions to observe how these changes influenced the development and functions of reintroduced biocrust. We found that by manipulating soil texture and time spent wet, we can change the proportional cover of biocrust within a restoration‐like setting. Specifically, we found that 4 months after inoculation, finer textured soils that received more water become dominated by moss cover, while coarser textured soils with less water remained dominated by cyanobacteria cover, and the interactions between texture and time spent wet strongly influenced cover. We found biocrust morphological group cover had a small, but detectable, effect on ecosystem functions (soil stability and nitrogenase activity, a proxy for nitrogen fixation), but that environmental conditions had a stronger impact on the functions we measured. Manipulative experiments in controlled environments, like this one, can help elucidate the mechanisms underlying the establishment rate and patterns of biocrusts post‐inoculation, and inform implementation of inoculations in the field.

Environmental Sciences & Ecology↗

Mechanistic Studies of a Primitive Homolog of Nitrogenase Involved in Coenzyme F430 Biosynthesis (Final Report)

Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel and other high-value chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory recently elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction. This remarkable transformation, which involves a 6-electron reduction, the formation of a γ-lactam ring, and the generation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the adenosine triphosphate (ATP)-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, specific aims were pursued for the 1) identification of physiological electron donors and in vivo coenzyme F430 synthesis, 2) analysis of the iron sulfur centers, structure, and oligomerization state changes, and 3) characterization of transient intermediates and the intercomponent electron transfer.

03 NATURAL GAS↗

The phototrophic purple non‐sulfur bacteria Rhodomicrobium spp. are novel chassis for bioplastic production

Abstract Petroleum‐based plastics levy significant environmental and economic costs that can be alleviated with sustainably sourced, biodegradable, and bio‐based polymers such as polyhydroxyalkanoates (PHAs). However, industrial‐scale production of PHAs faces barriers stemming from insufficient product yields and high costs. To address these challenges, we must look beyond the current suite of microbes for PHA production and investigate non‐model organisms with versatile metabolisms. In that vein, we assessed PHA production by the photosynthetic purple non‐sulfur bacteria (PNSB) Rhodomicrobium vannielii and Rhodomicrobium udaipurense. We show that both species accumulate PHA across photo‐heterotrophic, photo‐hydrogenotrophic, photo‐ferrotrophic, and photo‐electrotrophic growth conditions, with either ammonium chloride (NH 4 Cl) or dinitrogen gas (N 2 ) as nitrogen sources. Our data indicate that nitrogen source plays a significant role in dictating PHA synthesis, with N 2 fixation promoting PHA production during photoheterotrophy and photoelectrotrophy but inhibiting production during photohydrogenotrophy and photoferrotrophy. We observed the highest PHA titres (up to 44.08 mg/L, or 43.61% cell dry weight) when cells were grown photoheterotrophically on sodium butyrate with N 2 , while production was at its lowest during photoelectrotrophy (as low as 0.04 mg/L, or 0.16% cell dry weight). We also find that photohydrogenotrophically grown cells supplemented with NH 4 Cl exhibit the highest electron yields – up to 58.89% – while photoheterotrophy demonstrated the lowest (0.27%–1.39%). Finally, we highlight superior electron conversion and PHA production compared to a related PNSB, Rhodopseudomonas palustris TIE‐1. This study illustrates the value of studying non‐model organisms like Rhodomicrobium for sustainable PHA production and indicates future directions for exploring PNSB metabolisms.

59 BASIC BIOLOGICAL SCIENCES↗

Ecosystem consequences of a nitrogen-fixing proto-organelle

Microscale symbioses can be critical to ecosystem functions, but the mechanisms of these interactions in nature are often cryptic. Here, we use a combination of stable isotope imaging and tracing to reveal carbon (C) and nitrogen (N) exchanges among three symbiotic primary producers that fuel a salmon-bearing river food web. Bulk isotope analysis, nanoSIMS (secondary ion mass spectrometry) isotope imaging, and density centrifugation for quantitative stable isotope probing enabled quantification of organism-specific C- and N-fixation rates from the subcellular scale to the ecosystem. After winters with riverbed-scouring floods, the macroalga Cladophora glomerata uses nutrients in spring runoff to grow streamers up to 10 m long. During summer flow recession, riverine N concentrations wane and Cladophora becomes densely epiphytized by three species of Epithemia, diatoms with N-fixing endosymbionts (proto-organelles) descended from a free-living Crocosphaera cyanobacterium. Over summertime epiphyte succession on Cladophora, N-fixation rates increased as Epithemia spp. became dominant, Cladophora C-fixation declined to near zero, and Epithemia C-fixation increased. Carbon transfer to caddisflies grazing on Cladophora with high densities of Epithemia was 10-fold higher than C transfer to caddisflies grazing Cladophora with low Epithemia loads. In response to demand for N, Epithemia allocates high levels of newly fixed C to its endosymbiont. Consequently, these endosymbionts have the highest rates of C and N accumulation of any taxon in this tripartite symbiosis during the biologically productive season and can produce one of the highest areal rates of N-fixation reported in any river ecosystem.

Marks, Jane C. [Northern Arizona Univ., Flagstaff,↗

Photosynthetic modulation during the diurnal cycle in a unicellular diazotrophic cyanobacterium grown under nitrogen-replete and nitrogen-fixing conditions

Abstract Cyanobacteria are the only oxygenic photosynthetic organisms that can fix nitrogen. In diazotrophic cyanobacteria, the regulation of photosynthesis during the diurnal cycle is hypothesized to be linked with nitrogen fixation and involve the D1 protein isoform PsbA4. The amount of bioavailable nitrogen has a major impact on productivity in aqueous environments. In contrast to low- or nitrogen-fixing (−N) conditions, little data on photosynthetic regulation under nitrogen-replete (+ N) conditions are available. We compared the regulation of photosynthesis under −N and + N conditions during the diurnal cycle in wild type and a psbA4 deletion strain of the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. We observed common changes to light harvesting and photosynthetic electron transport during the dark in + N and −N conditions and found that these modifications occur in both diazotrophic and non-diazotrophic cyanobacteria. Nitrogen availability increased PSII titer when cells transitioned from dark to light and promoted growth. Under −N conditions, deletion of PsbA4 modified charge recombination in dark and regulation of PSII titer during dark to light transition. We conclude that darkness impacts the acceptor-side modifications to PSII and photosynthetic electron transport in cyanobacteria independently of the nitrogen-fixing status and the presence of PsbA4.

59 BASIC BIOLOGICAL SCIENCES↗

Proximity proteomics in a marine diatom reveals a putative cell surface-to-chloroplast iron trafficking pathway

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum . Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

59 BASIC BIOLOGICAL SCIENCES↗

Nitrogen and phosphorous acquisition strategies drive coexistence patterns among archaeal lineages in soil

Soil represents the largest reservoir of Archaea on Earth. Present-day archaeal diversity in soils globally is dominated by members of the class Nitrososphaeria. The evolutionary radiation of this class is thought to reflect adaptations to a wide range of temperatures, pH, and other environmental conditions. However, the mechanisms that govern competition and coexistence among Nitrososphaeria lineages in soil remain poorly understood. Here we show that predominant soil Nitrososphaeria lineages compose a patchwork of gene inventory and expression profiles for ammonia, urea, and phosphate utilization. In contrast, carbon fixation, respiration, and ATP synthesis genes are conserved and expressed consistently among predominant phylotypes across 12 major evolutionary lineages commonly found in soil. In situ gene expression profiles closely resemble pure culture reference strains under optimal growth conditions. Together, these results reveal resource-based coexistence patterns among Nitrososphaeria lineages and suggest complementary ecophysiological niches associated with differential nutrient acquisition strategies among globally predominant archaeal lineages in soil.

59 BASIC BIOLOGICAL SCIENCES↗

Mechanistic Studies of a Primitive Homolog of Nitrogenase Involved in Coenzyme F430 Biosynthesis

Methyl-coenzyme M reductase (MCR) is the key enzyme in the biological formation and anaerobic oxidation of methane (AOM). Methane is a potent greenhouse gas and the major component of natural gas. Given the abundance of natural gas reserves in remote areas, there is great current interest in a scalable bio-based process for the conversion of methane to liquid fuel or other high-value commodity chemicals. MCR holds much promise for use in such a methane bioconversion strategy. However, MCR cannot currently be produced in an active form in a heterologous host, due in large part to the lack of genetic and biochemical information about the production of holo MCR. In an effort to overcome this deficiency, our laboratory elucidated the biosynthetic pathway of the unique nickel-containing coenzyme of MCR, F430. The key step in coenzyme F430 biosynthesis (Cfb) was found to involve an unprecedented reductive cyclization reaction that converts Ni-sirohydrochlorin a , c -diamide to 15,17 3 -seco-F430-17 3 -acid. This remarkable transformation, which involves a 6-electron reduction of the isobacteriochlorin ring system, cyclization of the c -acetamide side chain to form a γ-lactam ring, and the formation of 7 stereocenters, is catalyzed by a primitive homolog of nitrogenase (CfbCD). Nitrogenase is a two-component metalloenzyme that catalyzes the ATP-dependent reduction of dinitrogen to ammonia (nitrogen fixation). Homologs of nitrogenase are also involved in the biosynthesis of the photosynthetic pigments chlorophyll and bacteriochlorophyll. Phylogenetic analysis of the CfbCD complex suggests that it is representative of a more ancient lineage of the nitrogenase superfamily, and a thorough investigation of its structure and function is likely to shed light on the mechanisms and evolution of these important metalloenzymes that catalyze multi-electron redox reactions. Moreover, a detailed understanding of the mechanism of the CfbCD complex may aid in the development of specific inhibitors to help reduce natural greenhouse gas emissions and can be exploited for the heterologous production of MCR for methane bioconversion. Towards these goals, the following Specific Aims will be pursued to determine the: 1) Identity of the CfbCD reaction product. The exact reaction catalyzed by CfbCD, including the number of electrons transferred and whether it involves enzymatic cyclization, is unclear. Several approaches, including reaction stoichiometry measurements, spectroelectrochemistry, and magnetic resonance spectroscopy will be applied to elucidate the structure of the reaction product and establish whether CfbCD is a reductase or reductive cyclase. 2) Structure, conformational dynamics, and oligomerization state changes of CfbCD. Significant insight into the mechanism and allosteric regulation of CfbCD can be obtained by assessing changes in the structure and dynamics of the complex during the catalytic cycle. To accomplish this, a combination of size-exclusion chromatography, hydrogen-deuterium exchange mass spectrometry, molecular dynamics simulations, and high-resolution structural methods will be employed. 3) Source, order, and stereochemistry of proton additions during CfbCD catalysis. Details regarding the order and stereochemistry of proton additions during the CfbCD reaction will be uncovered using a combined spectroscopic and computational approach. Complementary mechanistic studies employing site-directed mutagenesis and substrate analogs will establish the identity of active site acid residues and the possible involvement of substrate-assisted catalysis during the CfbCD reaction.

09 BIOMASS FUELS↗

Cyanobacterial Biofertilizer Production by Guanidine-Producing Enzymes

Cyanobacterial production of a biofertilizer shows promise as an environmentally benign alternative to conventional nitrogen fertilizers, reducing environmental and energy burdens through light-driven nitrogen and carbon fixation. One route to realizing the potential for a nitrogen-rich, slow-releasing biofertilizer involves the genetic engineering of cyanobacteria to produce guanidine. Recent advances have demonstrated enzymatic guanidine production in cyanobacteria, but an understanding of cyanobacterial guanidine metabolism is still limited. This Perspective highlights strategies and opportunities for cyanobacterial guanidine production in a Design−Build−Test−Learn cycle. Exploring new guanidine-producing enzymes via phylogenetics could expand candidate enzymes, while understanding the metabolism of substrates can identify constraints and opportunities in substrate utilization. Additionally, guanidine sensing and export are crucial areas of study to enable continuous fertilizer production and stable nitrogen flux. These strategies will guide the development of advanced nitrogen biofertilizer strategies for the agricultural sector.

09 BIOMASS FUELS↗

A Genome-Scale Metabolic Model of Anabaena 33047 to Guide Genetic Modifications to Overproduce Nylon Monomers

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, iAnC892, for this organism using annotations and content obtained from multiple databases. iAnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. iAnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of iAnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to iAnC892. iAnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N2 fixation. When used alongside the strain design algorithm, OptForce, iAnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

59 BASIC BIOLOGICAL SCIENCES↗

Carbon-Nitrogen Coupling Under Three Schemes of Model Representation: A Traceability Analysis

The interaction between terrestrial carbon (C) and nitrogen (N) cycles has been incorporated into more and more land surface models. However, the scheme of C-N coupling differs greatly among models, and how these diverse representations of C-N interactions will affect C-cycle modeling remains unclear. In this study, we explored how the simulated ecosystem C storage capacity in the terrestrial ecosystem (TECO) model varied with three different commonly used schemes of C-N coupling. The three schemes (SM1, SM2, and SM3) have been used in three different coupled C-N models (i.e., TECO-CN, CLM 4.5, and O-CN, respectively). They differ mainly in the stoichiometry of C and N in vegetation and soils, plant N uptake strategies, down regulation of photosynthesis, and the pathways of N import. We incorporated the three C-N coupling schemes into the C-only version of the TECO model and evaluated their impacts on the C cycle with a traceability framework. Our results showed that all three of the C-N schemes caused significant reductions in steady-state C storage capacity compared with the C-only version with magnitudes of −23%, −30%, and −54% for SM1, SM2, and SM3, respectively. This reduced C storage capacity was mainly derived from the combined effects of decreases in net primary productivity (NPP; −29%, −15%, and −45%) and changes in mean C residence time (MRT; 9%, −17%, and −17%) for SM1, SM2, and SM3, respectively. The differences in NPP are mainly attributed to the different assumptions on plant N uptake, plant tissue C : N ratio, down regulation of photosynthesis, and biological N fixation. In comparison, the alternative representations of the plant vs. microbe competition strategy and the plant N uptake, combined with the flexible C : N ratio in vegetation and soils, led to a notable spread in MRT. These results highlight the fact that the diverse assumptions on N processes represented by different C-N coupled models could cause additional uncertainty for land surface models. Understanding their difference can help us improve the capability of models to predict future biogeochemical cycles of terrestrial ecosystems.

Du, Zhenggang↗

A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI 1 I 2 DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD. Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2. Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. Furthermore, these results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans.

59 BASIC BIOLOGICAL SCIENCES↗