Structural and biophysical properties of a [4Fe4S] ferredoxin-like protein from Synechocystis sp. PCC 6803 with a unique two domain structure.

Electron carrier proteins (ECPs), binding iron-sulfur clusters, are vital components within the intricate network of metabolic and photosynthetic reactions. They play a crucial role in the distribution of reducing equivalents. In Synechocystis sp. PCC 6803, the ECP network includes at least nine ferredoxins. Previous research, including global expression analyses and protein binding studies, has offered initial insights into the functional roles of individual ferredoxins within this network. This study primarily focuses on Ferredoxin 9 (slr2059). Through sequence analysis and computational modeling, Ferredoxin 9 emerges as a unique ECP with a distinctive two-domain architecture. It consists of a C-terminal iron­sulfur binding domain and an N-terminal domain with homology to Nil-domain proteins, connected by a structurally rigid 4-amino acid linker. Notably, in contrast to canonical [2Fe2S] ferredoxins exemplified by PetF (ssl0020), which feature highly acidic surfaces facilitating electron transfer with photosystem I reaction centers, models of Ferredoxin 9 reveal a more neutral to basic protein surface. Using a combination of electron paramagnetic resonance spectroscopy and square-wave voltammetry on heterologously produced Ferredoxin 9, this study demonstrates that the protein coordinates 2×[4Fe4S]2+/1+ redox-active and magnetically interacting clusters, with measured redox potentials of -420 ± 9 mV and - 516 ± 10 mV vs SHE. A more in-depth analysis of Fdx9's unique structure and protein sequence suggests that this type of Nil-2[4Fe4S] multi-domain ferredoxin is well conserved in cyanobacteria, bearing structural similarities to proteins involved in homocysteine synthesis in methanogens.

The influence of electron utilization pathways on photosystem I photochemistry in Synechocystis sp. PCC 6803.

The capacity of cyanobacteria to adapt to highly dynamic photon flux and nutrient availability conditions results from controlled management and use of reducing power, and is a major contributing factor to the efficiency of photosynthesis in aquatic environments. The response to changing conditions includes modulating gene expression and protein-protein interactions that serve to adjust the use of electron flux and mechanisms that control photosynthetic electron transport (PET). In this regard, the photochemical activity of photosystem I (PSI) reaction centers can support balancing of cyclic (CEF) and linear electron flow (LEF), and the coupling of redox carriers for use by electron utilization pathways. Therefore, changes in the utilization of reducing power might be expected to result in compensating changes at PSI as a means to support balance of electron flux. To understand this functional relationship, we investigated the properties of PSI and its photochemical activity in cells that lack flavodiiron 1 catalyzed oxygen reduction activity (ORR1). In the absence of ORR1, the oxygen evolution and consumption rates declined together with a shift in the oligomeric form of PSI towards monomers. The effect of these changes on PSI energy and electron transfer properties was examined in isolated trimer and monomer fractions of PSI reaction centers. Collectively, the results demonstrate that PSI photochemistry is modulated through coordination with the depletion of electron demand in the absence of ORR1.

Correction: The influence of electron utilization pathways on photosystem I photochemistry in Synechocystis sp. PCC 6803.

[This corrects the article DOI: 10.1039/D2RA01295B.].

Gene Editing Technologies for Biofuel Production in Thermophilic Microbes.

Thermophilic microbes are an attractive bioproduction platform due to their inherently lower contamination risk and their ability to perform thermostable enzymatic processes which may be required for biomass processing and other industrial applications. The engineering of microbes for industrial scale processes requires a suite of genetic engineering tools to optimize existing biological systems as well as to design and incorporate new metabolic pathways within strains. Yet, such tools are often lacking and/or inadequate for novel microbes, especially thermophiles. This chapter focuses on genetic tool development and engineering strategies, in addition to challenges, for thermophilic microbes. We provide detailed instructions and techniques for tool development for an anaerobic thermophile, Caldanaerobacter subterraneus subsp. tengcongensis, including culturing, plasmid construction, transformation, and selection. This establishes a foundation for advanced genetic tool development necessary for the metabolic engineering of this microbe and potentially other thermophilic organisms.

Inactivation of the uptake hydrogenase in the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS enables a biological water-gas shift platform for H2 production.

Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.

Building a genome engineering toolbox in nonmodel prokaryotic microbes.

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO2 , and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop "design rules" for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.

Mountain pine beetle infestation of lodgepole pine in areas of water diversion.

The Rocky Mountains have experienced extensive infestations from the mountain pine beetle (Dendroctonus ponderosae Hopkins), affecting numerous pine tree species including lodgepole pine (Pinus contorta Dougl. var. latifolia). Water diversions throughout the Rocky Mountains transport large volumes of water out of the basins of origin, resulting in hydrologic modifications to downstream areas. This study examines the hypothesis that lodgepole pine located below water diversions exhibit an increased incidence of mountain pine beetle infestation and mortality. A ground survey verified diversion structures in a portion of Grand County, Colorado, and sampling plots were established around two types of diversion structures, canals and dams. Field studies assessed mountain pine beetle infestation. Lodgepole pines below diversions show 45.1% higher attack and 38.5% higher mortality than lodgepole pines above diversions. These findings suggest that water diversions are associated with increased infestation and mortality of lodgepole pines in the basins of extraction, with implications for forest and water allocation management.

De novo transcriptomic analysis of hydrogen production in the green alga Chlamydomonas moewusii through RNA-Seq.

BACKGROUND: Microalgae can make a significant contribution towards meeting global renewable energy needs in both carbon-based and hydrogen (H2) biofuel. The development of energy-related products from algae could be accelerated with improvements in systems biology tools, and recent advances in sequencing technology provide a platform for enhanced transcriptomic analyses. However, these techniques are still heavily reliant upon available genomic sequence data. Chlamydomonas moewusii is a unicellular green alga capable of evolving molecular H2 under both dark and light anaerobic conditions, and has high hydrogenase activity that can be rapidly induced. However, to date, there is no systematic investigation of transcriptomic profiling during induction of H2 photoproduction in this organism. RESULTS: In this work, RNA-Seq was applied to investigate transcriptomic profiles during the dark anaerobic induction of H2 photoproduction. 156 million reads generated from 7 samples were then used for de novo assembly after data trimming. BlastX results against NCBI database and Blast2GO results were used to interpret the functions of the assembled 34,136 contigs, which were then used as the reference contigs for RNA-Seq analysis. Our results indicated that more contigs were differentially expressed during the period of early and higher H2 photoproduction, and fewer contigs were differentially expressed when H2-photoproduction rates decreased. In addition, C. moewusii and C. reinhardtii share core functional pathways, and transcripts for H2 photoproduction and anaerobic metabolite production were identified in both organisms. C. moewusii also possesses similar metabolic flexibility as C. reinhardtii, and the difference between C. moewusii and C. reinhardtii on hydrogenase expression and anaerobic fermentative pathways involved in redox balancing may explain their different profiles of hydrogenase activity and secreted anaerobic metabolites. CONCLUSIONS: Herein, we have described a workflow using commercial software to analyze RNA-Seq data without reference genome sequence information, which can be applied to other unsequenced microorganisms. This study provided biological insights into the anaerobic fermentation and H2 photoproduction of C. moewusii, and the first transcriptomic RNA-Seq dataset of C. moewusii generated in this study also offer baseline data for further investigation (e.g. regulatory proteins related to fermentative pathway discussed in this study) of this organism as a H2-photoproduction strain.

New method for discovery of starch phenotypes in growing microalgal colonies.

To identify algal strains with altered starch metabolism from a large pool of candidates of growing algal colonies, we have developed a novel, high-throughput screening tool by combining gaseous bleaching of replica transferred colonies and subsequent iodine staining to visualize starch. Screening of healthy growing colonies of microalgae has not been possible previously because high levels of chlorophyll make the detection of starch with an iodine stain impossible. We demonstrated that chlorine dioxide (ClO(2)) removes essentially all chlorophyll from the colonies and enables high-throughput screening of, for example, a population of mutagenized cells or a culture collection isolated in a bioprospecting project.

Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga.

Characterization of genes responsible for the CO-linked hydrogen production pathway in Rubrivivax gelatinosus.

Upon exposure to carbon monoxide, the purple nonsulfur photosynthetic bacterium Rubrivivax gelatinosus produces hydrogen concomitantly with the oxidation of CO according to the equation CO + H(2)O <--> CO(2) + H(2). Yet little is known about the genetic elements encoding this reaction in this organism. In the present study, we use transposon mutagenesis and functional complementation to uncover three clustered genes, cooL, cooX, and cooH, in Rubrivivax gelatinosus putatively encoding part of a membrane-bound, multisubunit NiFe-hydrogenase. We present the complete amino acid sequences for the large catalytic subunit and its electron-relaying small subunit, encoded by cooH and cooL, respectively. Sequence alignment reveals a conserved region in the large subunit coordinating a binuclear [NiFe] center and a conserved region in the small subunit coordinating a [4Fe-4S] cluster. Protein purification experiments show that a protein fraction of 58 kDa molecular mass could function in H(2) evolution mediated by reduced methyl viologen. Western blotting experiments show that the two hydrogenase subunits are detectable and accumulate only when cells are exposed to CO. The cooX gene encodes a putative Fe-S protein mediating electron transfer to the hydrogenase small subunit. We conclude that these three Rubrivivax proteins encompass part of a membrane-bound, multisubunit NiFe-hydrogenase belonging to the energy-converting hydrogenase (Ech) type, which has been found among diverse microbes with a common feature in coupling H(2) production with proton pumping for energy generation.

Energy generation from the CO oxidation-hydrogen production pathway in Rubrivivax gelatinosus.

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H2 production pathway according to the stoichiometry CO + H2O --> CO2 + H2. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H2 activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H2 production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H2 production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H2 reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H2 production from gas streams containing CO.

Hydrogen photoproduction is attenuated by disruption of an isoamylase gene in Chlamydomonas reinhardtii.

DNA insertional transformants of Chlamydomonas reinhardtii were screened chemochromically for attenuated H(2) production. One mutant, displaying low H(2) gas photoproduction, has a nonfunctional copy of a gene that shows high homology to the family of isoamylase genes found in several photosynthetic organisms. DNA gel blotting and gene complementation were used to link this isoamylase gene to previously characterized nontagged sta7 mutants. This mutant is therefore denoted sta7-10. In C. reinhardtii, the STA7 isoamylase gene is important for the accumulation of crystalline starch, and the sta7-10 mutant reported here contains <3% of the glucose found in insoluble starch when compared with wild-type control cells. Hydrogen photoproduction rates, induced after several hours of dark, anaerobic treatment, are attenuated in sta7 mutants. RNA gel blot analysis indicates that the mRNA transcripts for both the HydA1 and HydA2 [Fe]-hydrogenase genes are expressed in the sta7-10 mutant at greater than wild-type levels 0.5 h after anaerobic induction. However, after 1.5 h, transcript levels of both HydA1 and HydA2 begin to decline rapidly and reach nearly undetectable levels after 7 h. In wild-type cells, the hydrogenase transcripts accumulate more slowly, reach a plateau after 4 h of anaerobic treatment, and maintain the same level of expression for >7 h under anaerobic incubation. Complementation of mutant cells with genomic DNA corresponding to the STA7 gene restores both the starch accumulation and H(2) production phenotypes. The results indicate that STA7 and starch metabolism play an important role in C. reinhardtii H(2) photoproduction. Moreover, the results indicate that mere anaerobiosis is not sufficient to maintain hydrogenase gene expression without the underlying physiology, an important aspect of which is starch metabolism.

Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase.

To identify genes necessary for the photoproduction of H(2) in Chlamydomonas reinhardtii, random insertional mutants were screened for clones unable to produce H(2). One of the identified mutants, denoted hydEF-1, is incapable of assembling an active [Fe] hydrogenase. Although the hydEF-1 mutant transcribes both hydrogenase genes and accumulates full-length hydrogenase protein, H(2) production activity is not observed. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase. In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. All organisms with [Fe] hydrogenase and sequenced genomes contain homologues of HydE, HydF, and HydG, which, prior to this study, were of unknown function. Within several prokaryotic genomes HydE, HydF, and HydG are found in putative operons with [Fe] hydrogenase structural genes. Both HydE and HydG belong to the emerging radical S-adenosylmethionine (commonly designated "Radical SAM") superfamily of proteins. We demonstrate here that HydEF and HydG function in the assembly of [Fe] hydrogenase. Northern blot analysis indicates that mRNA transcripts for both the HydEF gene and the HydG gene are anaerobically induced concomitantly with the two C. reinhardtii [Fe] hydrogenase genes, HydA1 and HydA2. Complementation of the bx;1C. reinhardtii hydEF-1 mutant with genomic DNA corresponding to a functional copy of the HydEF gene restores hydrogenase activity. Moreover, co-expression of the C. reinhardtii HydEF, HydG, and HydA1 genes in Escherichia coli results in the formation of an active HydA1 enzyme. This represents the first report on the nature of the accessory genes required for the maturation of an active [Fe] hydrogenase.

Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus.

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.