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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 1,045 records · Page 58
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Cyanobacterial carboxysomes contain an unique rubisco–activase–like protein

In plants, rubisco activase (Rca) regulates rubisco by removing inhibitory molecules such as ribulose–1,5–bisphosphate (RuBP). In cyanobacteria, a homologous protein (activase–like cyanobacterial protein, ALC), contains a distinctive C–terminal fusion resembling the small–subunit of rubisco. Although cyanobacterial rubisco is believed to be less sensitive to RuBP inhibition, the ALC is widely distributed among diverse cyanobacteria. Using microscopy, biochemistry and molecular biology, the cellular localization of the ALC, its effect on carboxysome/cell ultrastructure in Fremyella diplosiphon, and its function in vitro were studied. Bioinformatic analysis uncovered evolutionary relationships between the ALC and rubisco. ALC localizes to carboxysomes and exhibits ATPase activity. Furthermore, the ALC induces rubisco aggregation in a manner similar to that of another carboxysomal protein, M35, and this activity is affected by ATP. An alc deletion mutant showed modified cell morphology when grown under enriched CO 2 and impaired regulation of carboxysome biogenesis, without affecting growth rate. Carbamylation of Fremyella recombinant rubisco was inhibited by RuBP, but this inhibition was not relieved by the ALC. Here, the ALC does not appear to function like a canonical Rca; instead, it exerts an effect on the response to CO 2 availability at the level of a metabolic module, the carboxysome, through rubisco network formation, and carboxysome organization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Increasing thermostability of the key photorespiratory enzyme glycerate 3‐kinase by structure‐based recombination

As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Algae after dark: mechanisms to cope with anoxic/hypoxic conditions

Summary Chlamydomonas reinhardtii is a unicellular, soil‐dwelling (and aquatic) green alga that has significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences hypoxic/anoxic conditions. The diversity of pathways available to ferment sugars is often revealed in mutants in which the activities of specific branches of fermentative metabolism have been eliminated; compensatory pathways that have little activity in parental strains under standard laboratory fermentative conditions are often activated. The ways in which these pathways are regulated and integrated have not been extensively explored. In this review, we primarily discuss the intricacies of dark anoxic metabolism in Chlamydomonas , but also discuss aspects of dark oxic metabolism, the utilization of acetate, and the relatively uncharacterized but critical interactions that link chloroplastic and mitochondrial metabolic networks.

Yang, Wenqiang↗
DOE OSTI2020

Wavelength- and irradiance-dependent changes in intracellular nitric oxide level

Photobiomodulation (PBM) refers to beneficial effects of low energy light absorption. Although there is a large and growing body of literature describing down-stream physiological benefits of PBM, there is limited understanding of the molecular mechanisms that initiate and mediate these effects at the level of photon absorption. At present, the leading hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome c oxidase (COX), allowing it to bind O 2 instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased ATP production. Aim: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to the mechanisms of NO release and subsequent effects on cell signaling. The purpose of this work is to investigate wavelength-specific effects on intracellular NO release in living cells. Approach: We have conducted in depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low energy exposure to one or more wavelengths of laser light. Results: We found statistically significant wavelength-dependent elevations (10-30%) in intracellular NO levels following laser exposures at 447, 532, 635 or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of nitric oxide synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cGMP. Conclusions: This study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multi-wavelength method provides a novel tool for studying immediate and early mechanisms of PBM, as well as exploring intracellular NO signaling networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The structural basis of bacterial manganese import

Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. Here, we present the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The type II transporter has a tightly closed transmembrane channel due to “extracellular gating” residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. Mutagenesis of the extracellular gate perturbs manganese uptake, while coordination site mutagenesis abolishes import. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rnf and Fix Have Specific Roles during Aerobic Nitrogen Fixation in Azotobacter vinelandii

Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, but deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADH's endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the chemiosmotic potential, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different reaction stoichiometries and condition-specific differential gene expression indicate specific roles for the two reactions. This work's complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf is presumed to maintain reduced Fd/Fld production for nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Deletion of Re -citrate synthase allows for analysis of contributions of tricarboxylic acid cycle directionality to the growth of Heliomicrobium modesticaldum

ABSTRACT Heliomicrobium modesticaldum,a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typicalSi-citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodesRe-citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO 2 as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle. IMPORTANCE Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.

Biotechnology & Applied Microbiology↗
DOE OSTI2018

Functional Redundancy in the Hydroxycinnamate Catabolism Pathways of the Salt Marsh Bacterium Sagittula stellata E-37

ABSTRACT The hydroxycinnamates (HCAs) ferulate and p -coumarate are among the most abundant constituents of lignin, and their degradation by bacteria is an essential step in the remineralization of vascular plant material. Here, we investigate the catabolism of these two HCAs by the marine bacterium Sagittula stellata E-37, a member of the roseobacter lineage with lignolytic potential. Bacterial degradation of HCAs is often initiated by the activity of a hydroxycinnamoyl-coenzyme A (hydroxycinnamoyl-CoA) synthase. Genome analysis of S. stellata revealed the presence of two feruloyl-CoA ( fcs ) synthase homologs, an unusual occurrence among characterized HCA degraders. In order to elucidate the role of these homologs in HCA catabolism, fcs-1 and fcs-2 were disrupted using insertional mutagenesis, yielding both single and double fcs mutants. Growth on p -coumarate was abolished in the fcs double mutant, whereas maximum cell yield on ferulate was only 2% of that of the wild type. Interestingly, the single mutants demonstrated opposing phenotypes, where the fcs-1 mutant showed impaired growth (extended lag and ~60% of wild-type rate) on p -coumarate, and the fcs-2 mutant showed impaired growth (extended lag and ~20% of wild-type rate) on ferulate, pointing to distinct but overlapping roles of the encoded fcs homologs, with fcs-1 primarily dedicated to p -coumarate utilization and fcs-2 playing a dominant role in ferulate utilization. Finally, a tripartite ATP-independent periplasmic (TRAP) family transporter was found to be required for growth on both HCAs. These findings provide evidence for functional redundancy in the degradation of HCAs in S. stellata E-37 and offer important insight into the genetic complexity of aromatic compound degradation in bacteria. IMPORTANCE Hydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacterium Sagittula stellata . We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude that S. stellata is uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors.

catalysis (homogeneous), catalysis (heterogeneous)↗
DOE OSTI2019

Understanding and eliminating the detrimental effect of thiamine deficiency on the oleaginous yeast Yarrowia lipolytica

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de novo , the oleaginous yeast, Yarrowia lipolytica , cannot. In this paper, we used proteomics together with physiological characterization to understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica . Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) in the de novo thiamine biosynthesis of Y. lipolytica , and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered the thiamine-prototrophic Y. lipolytica . By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PP i ) to orthophosphate (P i ) are commonly used to accelerate and detect biosynthetic reactions that generate PP i as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PP i -hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a V max of 465 U · mg –1 for PP i hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PP i and Mg 2+ . Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PP i by-product generated in 2 M NaCl by UbaA (a “salt-loving” noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Altogether, we demonstrate HvPPA to be useful for hydrolyzing PP i under conditions of reduced water activity that are a hurdle to current PPA-based technologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Biochemical and Structural Studies of NADH-Dependent FabG Used To Increase the Bacterial Production of Fatty Acids under Anaerobic Conditions

Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ~35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Effects of Sulfate Reduction on Trichloroethene Dechlorination by Dehalococcoides-Containing Microbial Communities

Here, in order to elucidate interactions between sulfate reduction and dechlorination, we systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. Sulfate (up to 5 mM) did not inhibit the growth or metabolism of pure cultures of the dechlorinator Dehalococcoides mccartyi 195, the sulfate reducer Desulfovibrio vulgaris Hildenborough, or the syntroph Syntrophomonas wolfei. In contrast, sulfide at 5 mM exhibited inhibitory effects on growth of the sulfate reducer and the syntroph, as well as on both dechlorination and growth rates of D. mccartyi. Transcriptomic analysis of D. mccartyi 195 revealed that genes encoding ATP synthase, biosynthesis, and Hym hydrogenase were downregulated during sulfide inhibition, whereas genes encoding metal-containing enzymes involved in energy metabolism were upregulated even though the activity of those enzymes (hydrogenases) was inhibited. When the electron acceptor (trichloroethene) was limiting and an electron donor (lactate) was provided in excess to cocultures and enrichments, high sulfate concentrations (5 mM) inhibited reductive dechlorination due to the toxicity of generated sulfide. The initial cell ratio of sulfate reducers to D. mccartyi (1:3, 1:1, or 3:1) did not affect the dechlorination performance in the presence of sulfate (2 and 5 mM). Finally, in contrast, under electron donor limitation, dechlorination was not affected by sulfate amendments due to low sulfide production, demonstrating that D. mccartyi can function effectively in anaerobic microbial communities containing moderate sulfate concentrations (5 mM), likely due to its ability to outcompete other hydrogen-consuming bacteria and archaea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Efficient Metabolic Exchange and Electron Transfer within a Syntrophic Trichloroethene-Degrading Coculture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei

Dehalococcoides mccartyi 195 (strain 195) and Syntrophomonas wolfei were grown in a sustainable syntrophic coculture using butyrate as an electron donor and carbon source and trichloroethene (TCE) as an electron acceptor. The maximum dechlorination rate (9.9 ± 0.1 μmol day -1 ) and cell yield [(1.1 ± 0.3) × 10 8 cells μmol -1 Cl - ] of strain 195 maintained in coculture were, respectively, 2.6 and 1.6 times higher than those measured in the pure culture. The strain 195 cell concentration was about 16 times higher than that of S. wolfei in the coculture. Aqueous H 2 concentrations ranged from 24 to 180 nM during dechlorination and increased to 350 ± 20 nM when TCE was depleted, resulting in cessation of butyrate fermentation by S. wolfei with a theoretical Gibbs free energy of -13.7 ± 0.2 kJ mol -1 . Carbon monoxide in the coculture was around 0.06 μmol per bottle, which was lower than that observed for strain 195 in isolation. The minimum H 2 threshold value for TCE dechlorination by strain 195 in the coculture was 0.6 ± 0.1 nM. Cell aggregates during syntrophic growth were observed by scanning electron microscopy. The interspecies distances to achieve H 2 fluxes required to support the measured dechlorination rates were predicted using Fick's law and demonstrated the need for aggregation. Filamentous appendages and extracellular polymeric substance (EPS)-like structures were present in the intercellular spaces. The transcriptome of strain 195 during exponential growth in the coculture indicated increased ATP-binding cassette transporter activities compared to the pure culture, while the membrane-bound energy metabolism related genes were expressed at stable levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O 2 )-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N 2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O 2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. Here, the energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N 2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO 2 through oxygenic photosynthesis and heterocysts that are specialized in N 2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena , glucoside transporters influence the structure and function of septal junctions. Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO 2 and N 2 . These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena</named-content that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production

Copper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb 3 -type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb 3 -Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb 3 -Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb 3 -Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb 3 -Cox production in bacteria like R. capsulatus.

59 BASIC BIOLOGICAL SCIENCES↗