Ferredoxin Gene Research Articles

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus.

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.IMPORTANCENitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10mM) resulted in the production of 4.4g/L isopropanol and 19.8g/L Isopropanol-Butanol-Ethanol mixture.

Metal additives for boosting hydrogen production in anaerobic fermentation: Focus on the change of gene expression and cost analysis

Dark fermentation has emerged as a promising strategy for sustainable hydrogen production. However, the low hydrogen yield produced by anaerobic microbes continues to be a challenge. Although metal ion additives have been proposed to enhance hydrogen production, their entire impacts and underlying mechanisms are poorly understood. The present study aimed to examine the effects of Fe2+, Ni2+, Mg2+, and Mo6+ on dark fermentative hydrogen production, as well as their influences on microbial community analysis and gene expression in a batch system. The experiments were conducted under anaerobic conditions at 37 °C, utilizing 100 mM glucose as a substrate and an initial pH of 6.8. The results showed that Mo6+ at 0.015 mg-Mo6+/L significantly enhanced the hydrogen yield (2.21-fold increment) by upregulating nitrogenase gene expression (2.19-fold increment). Fe2+ at 50 mg-Fe2+/L was the second most effective metal ion, with a 1.81-fold increase in hydrogen yield and upregulation of ferredoxin (1.83-fold increment) and pyruvate formate-lyase gene (3.36-fold increment) expression. Mg2+ at 70 mg-Mg2+/L and Ni2+ at 25 mg-Ni2+/L also increased hydrogen yield by 1.51-fold and 1.31-fold, respectively, with upregulation of glyceraldehyde-3-phosphate dehydrogenase (1.02-fold and 1.81-fold increments, respectively) and ATP synthase (1.46-fold and 1.10-fold increments, respectively) expressions for both metal additives, pyruvate formate-lyase (2.77-fold increment) and pyruvate kinase (1.44-fold increment) gene expression for Mg2+. Regarding the mixed metal approach, it was observed that although certain combinations of mixed metals, such as Mg2++Mo6+, resulted in an enhancement of hydrogen yield by a factor of 1.37-fold, the increment was found to be lower than that of an individual metal. Therefore, the results of this study suggest that using a single metal would be more effective. Economic potential calculations further revealed that Mo6+ exhibited the highest economic feasibility, with the lowest expense of 2.02x10−5 $-metal/m3-H2, accounting for 0.002% of the average total production cost. This was followed by Mg2+, Fe2+, and Ni2+. Overall, our findings provide mechanistic insights into the use of metal ion additives for clean hydrogen production and offer a foundation for optimising their economic feasibility in large-scale hydrogen production.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 wasdetermined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

The Novel Monooxygenase Gene dipD in the dip Gene Cluster of Alcaligenes faecalis JQ135 Is Essential for the Initial Catabolism of Dipicolinic Acid.

Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.

Genome-wide identification and characterization of the Prunus persica ferredoxin gene family and its role in improving heat tolerance

Ferredoxin is involved in many biological processes, such as carbon fixation, nitrogen assimilation, chlorophyll metabolism, and fatty acid synthesis, and it plays a role in plant resistance to stress. However, the functions of Fds in peach during stress are unclear. In this study, 11 members of the peach Fd gene family were identified and divided into six groups (I– VI). We carried out bioinformatics analysis on these sequences, analyzed the physical and chemical properties of PpFd protein and the cis-elements in its promoter region, and predicted and compared the differences in gene structure and conserved protein motifs among groups. The results showed that the PpFd protein was highly conserved in plant species. In addition, overexpression of PpFd08 significantly increased the tolerance of transgenic tomato to high-temperature stress. The transcriptome analysis and qRT-PCR results of PpFd08 transgenic apple calli showed that PpFd08 might enhance heat resistance by modulating the expression of heat tolerance related genes. The results of this study provide a new understanding for the further study of the function of PpFd protein in peach and a candidate gene for improving the heat resistance of peach.

Electro-fermentation enhances H2 and ethanol co-production by regulating electron transfer and substrate transmembrane transport

Electro-fermentation systems (EFSs) are an emerging technology capable of regulating microbial fermentation pathways by tuning oxidation-reduction potentials and electron flow. However, there is limited understanding of the bioenergy conversion and metabolic regulation of fermentative bacteria in EFSs. In this study, we investigated how electrode potentials in EFSs affect the metabolic products and global transcriptome expression of Ethanoligenens harbinense. The E. harbinense-inoculated anodic EFS (AEFS) with a poised potential of 0 V or the cathodic EFS (CEFS) with a poised potential of 0 V (vs. Ag/AgCl reference electrode) obtained the maximum H2 production of 1888–1986 mL/L-medium, which increased by 23–26% compared with open-circuit fermentation (OC-EFSs). The highest H2 yield of 1.190 ± 0.009–1.197 ± 0.001 mol-H2/mol-glucose was obtained by the AEFS0 and the OC-EFS. Ethanol production of AEFS-0.4 increased by 30.7 ± 13.3% compared with OC-EFSs. In addition, glucose uptake and cell growth in the EFS were enhanced with an increase in cellular energy supply. Transcriptome analysis revealed that overexpression of the [FeFe]-hydrogenase, ferredoxin, and rubredoxin genes in the AEFS with a poised potential of 0 V promoted the H2 production rate. Genes involved in electron transfer and reduced nicotinamide adenine dinucleotide (NADH) regeneration were upregulated in the AEFS, leading to more ethanol production. In addition, substrate transmembrane transport was suppressed by underexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporter system-related genes at lower or higher potentials. These results confirm that an EFS effectively regulates the metabolite spectrum of H2-producing bacteria by coordinating electron transfer, NADH regeneration, and substrate transmembrane transport to provide a flexible approach for improving bioenergy production by fermentative bacteria.

Toxic and protective mechanisms of cyanobacterium Synechocystis sp. in response to titanium dioxide nanoparticles.

An increasing production and use of titanium dioxide nanoparticles (TiO2 NPs) pose a huge threat to phytoplankton since they are largely released into aquatic environments, which represent a sink for TiO2 NPs. However, toxicity and protective mechanisms of cyanobacteria in response to TiO2 NPs remain elusive. Here we investigated toxic effects of two sizes of TiO2 NPs (50 and 10nm) and one bulk TiO2 (200nm) on a cyanobacterium, Synechocystis sp. and their possible protective mechanisms. We found that 10nm TiO2 NPs caused significant growth and photosynthesis inhibition in Synechocystis sp. cells, largely reflected in decreased growth rate (38%), operational PSII quantum yields (40%), phycocyanin (51%) and allophycocyanin (63%), and increased reactive oxygen species content (245%), superoxide dismutase activity (46%). Also, transcriptomic analysis of Synechocystis sp. exposure to 10nm TiO2 NPs showed the up-regulation of D1 and D2 protein genes (psbA and psbD), ferredoxin gene (petF) and F-type ATPase genes (e.g., atpB), and the down-regulation of psbM and psb28-2 in PS II. We further proposed a conceptual model to explore possible toxic and protective mechanisms for Synechocystis sp. under TiO2 nanoparticle exposure. This study provides mechanistic insights into our understanding of Synechocystis sp. responses to TiO2 NPs. This is essential for more accurate environmental risk assessment approaches of nanoparticles in aquatic ecosystems by governmental environmental agencies worldwide.

Simultaneous Genome Sequencing of Prosthecochloris ethylica and Desulfuromonas acetoxidans within a Syntrophic Mixture Reveals Unique Pili and Protein Interactions.

Strains of Chloropseudomonas ethylica, 2-K, N2, and N3 are known to be composed of a syntrophic mixture of a green sulfur bacterium and a sulfur-reducing colorless component. Upon sequence analysis, the green sulfur photosynthetic bacterial component of strain N3 was dominant and was readily sequenced, but the less abundant sulfur-reducing bacterial component was apparent only when analyzed by metagenomic binning. Whole-genome comparison showed that the green bacterium belonged to the genus Prosthecochloris and apparently was a species for which there was no genome sequence on file. For comparison, we also sequenced the genome of Prosthecochloris sp. DSM 1685, which had previously been isolated from the 2-K mixture in pure culture and have shown that all three Prosthecochloris genomes belong to a new species, which we propose to be named Prosthecochloris ethylica comb. nov. Whole genomes were also sequenced for the isolated Desulfuromonas strains DSM 1675 (from strain 2-K) and DSM 1676 (from strain N2) and shown to be nearly identical to the genome found in the N3 mixture. The genome of the green sulfur bacterium contains large genes for agglutination proteins, similar to the ones proposed to be involved in larger photosynthetic consortia of Chlorochromatium aggregatum. In addition, we also identified several unique “tight adhesion (tad)” pili genes that are presumably involved in the formation of cell–cell interactions. The colorless component, on the other hand, contained a unique large multiheme cytochrome C and unique genes for e-pili (geopilin) formation, genetically clustered with a conserved ferredoxin gene, which are all expected to play an electron transfer role in the closed sulfur cycle in the syntrophic mixture. The findings from the simultaneous genome sequencing of the components of Cp. ethylica have implications for the phenomenon of direct interspecies interactions and coupled electron transfer in photosynthetic symbionts. The mechanisms for such interactions appear to be more common in the environment than originally anticipated.

The Transfer of the Ferredoxin Gene From the Chloroplast to the Nuclear Genome Is Ancient Within the Paraphyletic Genus Thalassiosira.

Ferredoxins are iron–sulfur proteins essential for a wide range of organisms because they are an electron transfer mediator involved in multiple metabolic pathways. In phytoplankton, these proteins are active in the mature chloroplasts, but the petF gene, encoding for ferredoxin, has been found either to be in the chloroplast genome or transferred to the nuclear genome as observed in the green algae and higher plant lineage. We experimentally determined the location of the petF gene in 12 strains of Thalassiosira covering three species using DNA sequencing and qPCR assays. The results showed that petF gene is located in the nuclear genome of all confirmed Thalassiosira oceanica strains (CCMP0999, 1001, 1005, and 1006) tested. In contrast, all Thalassiosira pseudonana (CCMP1012, 1013, 1014, and 1335) and Thalassiosira weissflogii (CCMP1010, 1049, and 1052) strains studied retained the gene in the chloroplast genome, as generally observed for Bacillariophyceae. Our evolutionary analyses further extend the dataset on the localization of the petF gene in the Thalassiosirales. The realization that the petF gene is nuclear-encoded in the Skeletonema genus allowed us to trace the petF gene transfer back to a single event that occurred within the paraphyletic genus Thalassiosira. Phylogenetic analyses revealed the need to reassess the taxonomic assignment of the Thalassiosira strain CCMP1616, since the genes used in our study did not cluster within the T. oceanica lineage. Our results suggest that this strains’ diversification occurred prior to the ferredoxin gene transfer event. The functional transfer of petF genes provides insight into the evolutionary processes leading to chloroplast genome reduction and suggests ecological adaptation as a driving force for such chloroplast to nuclear gene transfer.

Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea

BackgroundEpothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts.ResultsSchlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029.ConclusionGene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives.

Metabolic regulation of ethanol-type fermentation of anaerobic acidogenesis at different pH based on transcriptome analysis of Ethanoligenens harbinense

BackgroundEthanol-type fermentation, one of the fermentation types in mixed cultures of acidogenesis with obvious advantages such as low pH tolerance and high efficiency of H2 production, has attracted widespread attentions. pH level greatly influences the establishment of the fermentation of carbohydrate acidogenesis by shaping community assembly and the metabolic activity of keystone populations. To explore the adaptation mechanisms of ethanol-type fermentation to low pH, we report the effects of initial pH on the physiological metabolism and transcriptomes of Ethanoligenens harbinense—a representative species of ethanol-type fermentation.ResultsDifferent initial pH levels significantly changed the cell growth and fermentation products of E. harbinense. Using transcriptomic analysis, we identified and functionally categorized 1753 differentially expressed genes (DEGs). By mining information on metabolic pathways, we probed the transcriptional regulation of ethanol–H2 metabolism relating to pH responses. Multiple pathways of E. harbinense were co-regulated by changing gene expression patterns. Low initial pH down-regulated the expression of cell growth- and acidogenesis-related genes but did not affect the expression of H2 evolution-related hydrogenase and ferredoxin genes. High pH down-regulated the expression of H2 evolution- and acidogenesis-related genes. Multiple resistance mechanisms, including chemotaxis, the phosphotransferase system (PTS), and the antioxidant system, were regulated at the transcriptional level under pH stress.ConclusionsEthanoligenens adapted to low pH by regulating the gene expression networks of cell growth, basic metabolism, chemotaxis and resistance but not H2 evolution-related genes. Regulation based on pH shifts can represent an important approach to establish and enhance ethanol-type fermentation. The complete gene expression network of ethanol fermentative bacteria for pH response provides valuable insights into the acidogenic fermentation, and offers an effective regulation strategy for the sustainable energy recovery from wastewater and solid waste.

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification.IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

Improved GFP Variants to Study Gene Expression in Haloarchaea.

The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten amino acid substitutions, resulting in mGFP6. Using mGFP6 as reporter, we studied six haloarchaeal promoters exhibiting different promoter strengths. The strongest activity was observed with the housekeeping promoters Pfdx of the ferredoxin gene and P2 of the ribosomal 16S rRNA gene. Much lower activities were determined for the promoters of the p-vac region driving the expression of gas vesicle protein (gvp) genes in Halobacterium salinarum PHH1. The basal promoter strength dropped in the order PpA, PpO > PpF, PpD. All promoters showed a growth-dependent activity pattern. The GvpE-induced activities of PpA and PpD were high, but lower compared to the Pfdx or P2 promoter activities. The mGFP6 reporter was also used to investigate the regulatory effects of 5′-untranslated regions (5′-UTRs) of three different gvp mRNAs. A deletion of the 5′-UTR always resulted in an increased expression, implying a negative effect of the 5′-UTRs on translation. Our experiments confirmed mGFP6 as simple, fast and sensitive reporter to study gene expression in haloarchaea.

Effects of pH value on the expression of key iron/sulfur oxidation genes during bioleaching of chalcopyrite on thermophilic condition

Physicochemical factors such pH value would affect the microbial metabolism during chalcopyrite bioleaching. To this end, the effects of pH on the expression of critical functional genes during bioleaching were evaluated. A mixed culture of moderate thermophiles was used for chalcopyrite bioleaching at initial pH values of 1.0, 2.0, and 3.0, and bioleaching processes were monitored via measuring the physicochemical parameters. Quantitative real-time PCR assay was used to monitor the dynamics of microbial community structures and the expression of critical iron/sulfur oxidation genes (4Fe-4S ferredoxin and sulfate adenylyltransferase genes, respectively). Redundancy analysis and calculation of correlation coefficients were used to reveal linkages between gene expression and various physicochemical factors. The leaching processes at initial pH of 1.0 and 3.0 were prolonged compared with that at initial pH of 2.0. It was shown that Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus were the dominant species during the early stage in free and attached cells, respectively, while Ferroplasma thermophilum became predominant in the later phase. The gene expression in Sulfobacillus thermosulfidooxidans and Ferroplasma thermophilum was greatly affected by pH values. On the other hand, the relationship between pH and gene expression in Acidithiobacillus caldus was not significant. The study unraveled the importance of pH value on chalcopyrite bioleaching, and pH selectively influenced the expression of key functional genes of some specific species.

Comparative transcriptome analysis of a long-time span two-step culture process reveals a potential mechanism for astaxanthin and biomass hyper-accumulation in Haematococcus pluvialis JNU35

BackgroundAmong all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process.ResultsIn the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine–urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass.ConclusionsTo the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, β-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.

Adaption of microarray primers for iron transport and homeostasis gene expression in Pseudomonas fluorescens exposed to nano iron

Modified protocols were adapted for PCR and culture based methods for the analysis of Pseudomonas fluorescens cells exposed to nanoscale zero-valent iron (NZVI) and iron (Fe) in bacterial growth nutrient media was determined by a modified atomic absorption spectrometric (AAS) analysis method. We adapted sets of microarray primers used to quantify gene expression of pvdS and a bacterioferritin-associated ferredoxin gene for use in real-time quantitative reverse transcription (qRT-PCR) analysis. pvdS is one of a cluster of genes regulating the synthesis of the siderophore pyoverdine that was also measured using chrome azrul S (CAS) plates.•The current protocol provides a detailed qRT-PCR method for quantifying genes involved in the acquisition and utilization of Fe in P. fluorescens cells exposed to NZVI.•The qRT-PCR results were independently corroborated with 2 culture based methods, growth curves and chrome azurol S (CAS) plate.•The modified AAS method was used to measure Fe in Tryptic Soy Broth (TSB) medium where sodium (Na) causes inference in iron measurement.

Reduced expression of iron transport and homeostasis genes in Pseudomonas fluorescens during iron uptake from nanoscale iron

Plant growth-promoting rhizobacteria (PGPR) have beneficial effects on the host plant that include acquisition of nutrients such as iron (Fe). In this study, the impact of varying concentrations of nanoscale zero-valent iron (NZVI, 1, 2 and 5 g L−1) on a PGPR, Pseudomonas fluorescens' viability, growth and production of siderophores was investigated. Microscale zero-valent iron (MZVI) particles were also included in this study to determine and compare the effects of particle size on Fe acquisition and use by Pseudomonas fluorescens. Siderophores chelate insoluble forms of Fe that are then rendered bioavailable to P. fluorescens as well as some plants. This research indicates that the Fe species derived from lower concentrations of NZVI additions (1 g L−1) are bioavailable and can be utilized as a nutrient by P. fluorescens without the bacteria producing siderophores, an energy intensive process. Also, NZVI application as a fertilizer has promise for better utilization of Fe by plants. P. fluorescens colony forming units (log10 CFU mL−1) growth in cultures containing 1 g L−1 NZVI were significantly higher exceeded (14 units) than those grown without NZVI additions (13 units). The majority of P. fluorescens cells treated with 2 and 5 g L−1 were nonviable and produced limited colony forming units (4 units). The effects of NZVI on siderophore production by P. fluorescens were assessed using chrome azurol S (CAS) plates and real time quantitative reverse transcription PCR (qRT-PCR). Further, expression of genes involved in Fe utilization [putative pvdS (PFL 4190) and a bacterioferritin-associated ferredoxin gene (PFL 4858)] were targeted using real time quantitative reverse transcription PCR (qRT-PCR). Expression of both genes was below detection limits in cultures exposed to NZVI but measurable in MZVI and control treatments, again indicating potential ease in uptake of Fe by P. fluorescens from NZVI. Transmission electron microscopy (TEM) revealed the presence of NZVI particles on the exterior and interior of cells; energy dispersive X-ray spectroscopy (EDS) verified that the material was derived from NZVI and that Fe concentrations were higher in the interior of cells exposed to 1 g L−1 NZVI compared to the control. The research findings from this study indicate that NZVI has the potential to accumulate in plant growth-promoting rhizobacteria and affect the growth of organisms in environments like the rhizobiome which may affect plant growth positively. This research opens up opportunities for future studies to determine the effects of Fe and other nutrients and micronutrients derived from nanomaterials, particularly in oligotrophic systems.

Characterization of photosynthetic ferredoxin from the Antarctic alga Chlamydomonas sp. UWO241 reveals novel features of cold adaptation

The objective of this work was to characterize photosynthetic ferredoxin from the Antarctic green alga Chlamydomonas sp. UWO241, a key enzyme involved in distributing photosynthetic reducing power. We hypothesize that ferredoxin possesses characteristics typical of cold-adapted enzymes, namely increased structural flexibility and high activity at low temperatures, accompanied by low stability at moderate temperatures. To address this objective, we purified ferredoxin from UWO241 and characterized the temperature dependence of its enzymatic activity and protein conformation. The UWO241 ferredoxin protein, RNA, and DNA sequences were compared with homologous sequences from related organisms. We provide evidence for the duplication of the main ferredoxin gene in the UWO241 nuclear genome and the presence of two highly similar proteins. Ferredoxin from UWO241 has both high activity at low temperatures and high stability at moderate temperatures, representing a novel class of cold-adapted enzymes. Our study reveals novel insights into how photosynthesis functions in the cold. The presence of two distinct ferredoxin proteins in UWO241 could provide an adaptive advantage for survival at cold temperatures. The primary amino acid sequence of ferredoxin is highly conserved among photosynthetic species, and we suggest that subtle differences in sequence can lead to significant changes in activity at low temperatures.

The major leaf ferredoxin Fd2 regulates plant innate immunity in Arabidopsis.

Ferredoxins, the major distributors for electrons to various acceptor systems in plastids, contribute to redox regulation and antioxidant defence in plants. However, their function in plant immunity is not fully understood. In this study, we show that the expression of the major leaf ferredoxin gene Fd2 is suppressed by Pseudomonas syringae pv. tomato (Pst) DC3000 infection, and that knockout of Fd2 (Fd2-KO) in Arabidopsis increases the plant's susceptibility to both Pst DC3000 and Golovinomyces cichoracearum. On Pst DC3000 infection, the Fd2-KO mutant accumulates increased levels of jasmonic acid and displays compromised salicylic acid-related immune responses. Fd2-KO also shows defects in the accumulation of reactive oxygen species induced by pathogen-associated molecular pattern-triggered immunity. However, Fd2-KO shows enhanced R-protein-mediated resistance to Pst DC3000/AvrRpt2 infection, suggesting that Fd2 plays a negative role in effector-triggered immunity. Furthermore, Fd2 interacts with FIBRILLIN4 (FIB4), a harpin-binding protein localized in chloroplasts. Interestingly, Fd2, but not FIB4, localizes to stromules that extend from chloroplasts. Taken together, our results demonstrate that Fd2 plays an important role in plant immunity.