Nostoc Punctiforme Research Articles

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Wind erosion control using inoculation of aeolian sand with cyanobacteria

AbstractWind erosion is a natural phenomenon that has been increasing due to anthropogenic activities. Wind erosion has detrimental consequences on the environment, and its prevention is a critical issue in many arid regions of the world. The topic of this study was an artificial formation of biocrusts to diminish wind erosion by inoculation of the aeolian sand by two filamentous cyanobacteria of Nostoc punctiforme and Microcoleus vaginatus. The erodibility of the sand was examined in a wind–tunnel equipped with a sand particle counter (SPC). The erosion rate, number of emitted particles, and flux of eroded mass from the surface of the sand were measured as indicators of the erodibility at five wind velocities of 6, 10, 15, 20, and 25 m/s. Results suggested that biocrust of M. vaginatus had thicker interparticle bonding and more resistance than biocrust of N. punctiforme against wind erosion; however, both of cyanobacterial crust reduced the erodibility indicators by two to three orders of magnitude.

Laboratory study on biophysicochemical improvement of desert sand

Losses of soil biodiversity, stability, and fertility are primary factors causing degradation in drylands; therefore, finding a sustainable conservation practice is a critical issue for reversing desertification in these areas. By inoculation of two filamentous cyanobacteria, i.e., Nostoc punctiforme and Microcoleus vaginatus, on infertile desert sand, this study examined the component, structure, permeability, and strength of the created biocrusts. Moreover, wind erodibility of the biocrusts was tested by a wind tunnel, equipped with a sand particle counter (SPC). With having higher amounts of polysaccharide and Chl-a contents after 21 days of cultivation, the M. vaginatus-formed biocrust showed comparatively higher values of nutrients and strength and lower values of permeability and erodibility. Concerning wind erosion of the sand samples, both biocrusts reduced surface erosion of the sand several thousand times, in conditions without saltating particles. Nevertheless, in the presence of saltating particles, the erosion resistance of the treated samples decreased significantly.

The Hybrid Histidine Kinase HrmK Is an Early-Acting Factor in the Hormogonium Gene Regulatory Network.

Filamentous, heterocyst-forming cyanobacteria belonging to taxonomic subsections IV and V are developmentally complex multicellular organisms capable of differentiating an array of cell and filament types, including motile hormogonia. Hormogonia exhibit gliding motility that facilitates dispersal, phototaxis, and the establishment of nitrogen-fixing symbioses. The gene regulatory network (GRN) governing hormogonium development involves a hierarchical sigma factor cascade, but the factors governing the activation of this cascade are currently undefined. Here, using a forward genetic approach, we identified hrmK, a gene encoding a putative hybrid histidine kinase that functions upstream of the sigma factor cascade. The deletion of hrmK produced nonmotile filaments that failed to display hormogonium morphology or accumulate hormogonium-specific proteins or polysaccharide. Targeted transcriptional analyses using reverse transcription-quantitative PCR (RT-qPCR) demonstrated that hormogonium-specific genes both within and outside the sigma factor cascade are drastically downregulated in the absence of hrmK and that hrmK may be subject to indirect, positive autoregulation via sigJ and sigC Orthologs of HrmK are ubiquitous among, and exclusive to, heterocyst-forming cyanobacteria. Collectively, these results indicate that hrmK functions upstream of the sigma factor cascade to initiate hormogonium development, likely by modulating the phosphorylation state of an unknown protein that may serve as the master regulator of hormogonium development in heterocyst-forming cyanobacteria.IMPORTANCE Filamentous cyanobacteria are morphologically complex, with several representative species amenable to routine genetic manipulation, making them excellent model organisms for the study of development. Furthermore, two of the developmental alternatives, nitrogen-fixing heterocysts and motile hormogonia, are essential to establish nitrogen-fixing symbioses with plant partners. These symbioses are integral to global nitrogen cycles and could be artificially recreated with crop plants to serve as biofertilizers, but to achieve this goal, detailed understanding and manipulation of the hormogonium and heterocyst gene regulatory networks may be necessary. Here, using the model organism Nostoc punctiforme, we identify a previously uncharacterized hybrid histidine kinase that is confined to heterocyst-forming cyanobacteria as the earliest known participant in hormogonium development.

Effects of cyanobacterial suspensions as bio-fertilizers on growth factors and the essential oil composition of chamomile, Matricaria chamomilla L.

The cyanobacterial species used in this study were collected from medicinal plant beds and were cultured using nitrate free BG-11 medium. As heterocystous cyanobacteria fix atmospheric nitrogen and some of them additionally produce phytohormones, we hypothesized that using a suspension of beneficial cyanobacterial species in the soil of the plant might enhance its growth and would probably also affect the essential oil of this plant. Based on a seedling growth test, the cyanobacterial species Nostoc carneum, Wollea vaginicola and Nostoc punctiforme were selected for a pot trial. A glasshouse experiment with seven replicates was designed to evaluate the effects of the cyanobacterial suspensions on Matricaria chamomilla L. growth and essential oil production. The cyanobacterial suspensions were applied 15 days after planting. Fifty days after flowering the plants were harvested and the plant growth parameters were measured. Additionally, the chemical composition of soil samples from both the treatments and control pots were analyzed. All of the studied cyanobacterial suspensions had a significant effect on the weight of essential oil as well as on root growth. The chamazulene content of the essential oil in plants treated with N. carneum was 26.9% more than in the control. HPLC analysis revealed that the cyanobacterial species used in this experiment were able to produce plant growth–promoting hormones such as indole 3-acetic acid, indole 3-propionic acid and indole 3-butyric acid. There was a significant positive correlation between cyanobacterial IAA and all of the growth factors, as well as some constituents of the essential oil.

An in silico Study of Two Transcription Factors Controlling Diazotrophic Fates of the Azolla Major Cyanobiont Trichormus azollae.

The cyanobiont Trichormus azollae lives symbiotically within fronds of the genus Azolla, and assimilates atmospheric nitrogen upon N-limitation, which earmarks this symbiosis to be a valuable biofertilizer in rice cultivation, among many other benefits that also include carbon sequestration. Therefore, studying the regulation of nitrogen fixation in Trichormus azollae is of great importance and benefit, especially the two topmost rungs of regulation, the NtcA and HetR transcription factors that are able to regulate the expression of myriads of downstream genes. Bioinformatics tools were used to zoom in on the NtcA and HetR transcription factors from Trichormus azollae to elaborate on what makes this particular cyanobiont different from other symbiotic as well as more distinct counterparts, in their commitment to nitrogen fixation. The utility of Azolla plants in tropical agriculture in particular merits the “top down N-regulation” by cyanobiont as a significant niche area of study, to make sense of superior N-fixing capabilities. The Trichormus azollae NtcA sequence was found as a phylogenetic outlier to horizontally infecting cyanobionts, which points to a distinct identity compared to symbiotic counterparts. There were borderline (60%-70%) levels of acceptable bootstrap support for the phylogenetic position of the Azolla cyanobiont’s NtcA protein compared to other cyanobionts. Furthermore, the NtcA global nitrogen regulator in the Azolla cyanobiont has an extra cysteine at position 128, in addition to two other more conspicuous cysteines (positions, 157 and 164). A simulated homology model of the NtcA protein from Trichormus azollae, points to a single unique cysteine (Cysteine-128) as a key residue at the center of a lengthy C-helix, which forms a coiled-coil interface, through likely disulfide bond formation. Three cysteine (Cysteines: 128, 157, 164) architecture is exclusively found in Trichormus azollae and is absent in other cyanobacteria. A separate proline to alanine mutation in position 97—again exclusive to Trichormus azollae—appears to influence the flexibility of effector binding domain (EBD) to 2-oxoglutarate. The Trichormus azollae HetR sequence was found outside of horizontally-infecting cyanobiont sequences that formed a common clade, with the exception of the cyanobiont from the genus Cycas that formed one line of descent with the Trichormus azollae counterpart. Five (out of 6) serines predicted to be phosphorylated in the Trichormus azollae HetR sequence, are conserved in the Nostoc punctiforme counterpart, showcasing that phosphorylation is likley conserved in both vertically-transmitted and horizontally-acquired cyanobionts. A key Serine-127, within a conserved motif TSLTS, although conserved in heterocystous subsection IV and V cyanobacteria, are mutated in subsection III cyanobacteria that form trichomes but are unable to form heterocysts. I conclude that the NtcA protein from Trichormus azollae to be strategically divergent at specific amino acids that gives it an advantage in function as a 2-oxoglutarate-mediated transcription factor. The Trichormus azollae HetR transcription factor appears to possess parallel functionality to horizontally acquired counterparts. Especially Cysteine-128 in the NtcA transcription factor of the Azolla cyanobiont is an interesting proposition for future structure-function studies.

Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P).

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

Antiviral activity of five filamentous cyanobacteria against coxsackievirus B3 and rotavirus.

Coxsackievirus B3 (CVB3) and rotavirus (RV) are pathogens of some chronic human diseases. The aim of this study was to determine in vitro antiviral activity of some cyanobacteria against RV and CVB3 infections. Five cyanobacteria were collected from Egypt, identified, and analyzed biochemically. Then, the inhibition of the cytopathic effect of RV and CVB3 viruses by cyanobacterial extracts was examined. Methanol extract of the cyanobacterial isolates showed high antiviral activity against CVB3 with Therapeutic index (TI) of 50.0, 30.0, 27.6, 16.6, and 20.0 for Leptolyngbya boryana, Arthrospira platensis, Nostoc punctiforme, Oscillatoria sp., and Leptolyngbya sp., respectively. The extracts reduced CVB3 titers comparing to 50% tissue culture infectious doses (TCID50) with values 3.25-5.75 log10 of TCID50. Moreover, extracts of A. platensis, and Oscillatoria sp. exhibited high antiviral activity against RV with TI values of 45 and 42.5, respectively, and a reduction in virus titers by 5.75 log10 and 5.5 log10 of TCID50, respectively. Extracts of L. boryana, Leptolyngbya sp., and N. punctiforme had a moderate to low antiviral activity against RV with TI ranging between 2.8 and 7, respectively, and a reduction in virus titers between 0.5 log10 and 1.5 log10 of TCID50, respectively. This study concluded that extracts of five cyanobacterial isolates possess a potent antiviral effect against CVB3 and RV, making them promising sources of new safe antiviral drugs.

Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA

Ferritin-like proteins, Dps (DNA-binding protein from starved cells), store iron and play a key role in the iron homeostasis in bacteria, yet their iron releasing machinery remains largely unexplored. The electron donor proteins that may interact with Dps and promote the mobilization of the stored iron have hitherto not been identified. Here, we investigate the binding capacity of the two atypical Dps proteins NpDps4 and NpDps5 from Nostoc punctiforme to isolated ferredoxins. We report NpDps-ferredoxin interactions by fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET) methods. Dynamic light scattering, size exclusion chromatography and native gel electrophoresis results show that NpDps4 forms a dodecamer at both pH 6.0 and pH 8.0, while NpDps5 forms a dodecamer only at pH 6.0. In addition, FCS data clearly reveal that the non-canonical NpDps5 interacts with DNA at pH 6.0. Our spectroscopic analysis shows that [FeS] centers of the three recombinantly expressed and isolated ferredoxins are properly incorporated and are consistent with their respective native states. The results support our hypothesis that ferredoxins could be involved in cellular iron homeostasis by interacting with Dps and assisting the release of stored iron.

The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps protein class that can be broadly found among cyanobacteria.

Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.

Generation of Active Protease Depending on Peptide-Protein Interactions Using Interaction-Dependent Native Chemical Ligation and Protein Trans-Splicing

Abstract An artificial signal transduction system has been constructed by employing engineered human immunodeficiency type-1 (HIV-1) protease and Nostoc punctiforme PCC73102 (Npu) DnaE intein. While the truncation of four amino acid residues at the N-terminus of HIV-1 protease diminished its activity, the attachment of the PQIT sequence into the truncated protease by protein trans-splicing (PTS) reconstituted the enzymatic activity. By combining interaction-dependent native chemical ligation (IDNCL) with the PTS reaction, the peptide-protein interaction was clearly detected by measuring HIV-1 protease activity. Src homology domain 2 (SH2) of c-Src (SrcSH2) and phosphopeptides were used as model binding pairs. HIV-1 protease activities were dose-dependently increased after the IDNCL-PTS reaction when the peptides containing pYEEI (pY = phosohotyrosine) and pYEE sequences were used as the input peptides. HIV-1 protease activity generated by IDNCL-PTS might activate several enzymes, and therefore, the artificial signal transduction system might be available in synthetic biology.

Unlocking the Spatial Control of Secondary Metabolism Uncovers Hidden Natural Product Diversity in Nostoc punctiforme.

Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.

Creation of thermostable l-tryptophan dehydrogenase by protein engineering and its application for l-tryptophan quantification

l-Tryptophan dehydrogenase is a new NAD+-dependent amino acid dehydrogenase discovered in Nostoc punctiforme. The enzyme is involved in scytonemin biosynthesis and is highly selective toward l-tryptophan. By a growth-dependent molecular evolution technique, a thermostable mutant enzyme was selected successfully. l-Tryptophan concentration in human plasma was successfully determined using the thermostable mutant of l-tryptophan dehydrogenase.

A Tripartite, Hierarchical Sigma Factor Cascade Promotes Hormogonium Development in the Filamentous Cyanobacterium Nostoc punctiforme.

Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and frequently, nitrogen fixation as well. As a result, they contribute substantially to global primary production and nitrogen cycles. Furthermore, the multicellular filamentous cyanobacteria in taxonomic subsections IV and V are developmentally complex, exhibiting an array of differentiated cell types and filaments, including motile hormogonia, making them valuable model organisms for studying development. To investigate the role of sigma factors in the gene regulatory network (GRN) controlling hormogonium development, a combination of genetic, immunological, and time-resolved transcriptomic analyses were conducted in the model filamentous cyanobacterium Nostoc punctiforme, which, unlike other common model cyanobacteria, retains the developmental complexity of field isolates. The results support a model where the hormogonium GRN is driven by a hierarchal sigma factor cascade, with sigJ activating the expression of both sigC and sigF, as well as a substantial portion of additional hormogonium-specific genes, including those driving changes to cellular architecture. In turn, sigC regulates smaller subsets of genes for several processes, plays a dominant role in promoting reductive cell division, and may also both positively and negatively regulate sigJ to reinforce the developmental program and coordinate the timing of gene expression, respectively. In contrast, the sigF regulon is extremely limited. Among genes with characterized roles in hormogonium development, only pilA shows stringent sigF dependence. For sigJ-dependent genes, a putative consensus promoter was also identified, consisting primarily of a highly conserved extended -10 region, here designated a J-Box, which is widely distributed among diverse members of the cyanobacterial lineage.IMPORTANCE Cyanobacteria are integral to global carbon and nitrogen cycles, and their metabolic capacity coupled with their ease of genetic manipulation make them attractive platforms for applications such as biomaterial and biofertilizer production. Achieving these goals will likely require a detailed understanding and precise rewiring of these organisms' GRNs. The complex phenotypic plasticity of filamentous cyanobacteria has also made them valuable models of prokaryotic development. However, current research has been limited by focusing primarily on a handful of model strains which fail to reflect the phenotypes of field counterparts, potentially limiting biotechnological advances and a more comprehensive understanding of developmental complexity. Here, using Nostoc punctiforme, a model filamentous cyanobacterium that retains the developmental range of wild isolates, we define previously unknown definitive roles for a trio of sigma factors during hormogonium development. These findings substantially advance our understanding of cyanobacterial development and gene regulation and could be leveraged for future applications.

Expression and characterization of a lipid peroxidase from Nitrosomonas europeae structurally related to prostaglandin H2 synthase

Prostaglandin‐endoperoxide synthase (PGHS; EC 1.14.99.1) catalyzes the two step synthesis of prostaglandin H2, an important precursor in the synthesis of prostaglandins and eicosanoids. The first step of the reaction involves the addition of two molecules of oxygen to arachidonic acid to form prostaglandin G2. In the second step, the C15 hydroperoxide in prostaglandin G2 is reduced to an alcohol, generating prostaglandin H2. PGHS has been isolated and characterized from many different eukaryotes (both vertebrates and invertebrates) and has recently been expressed from an open reading frame in the red algae Gracilaria vermiculophylla. A number of putative PGHS orthologs have been identified in bacterial genomes. In this study, a putative PGHS protein from Nitrosomosas europeae has been cloned, expressed, and characterized. Soluble Nitrosomonas protein was isolated to greater than 90% purity exploiting an N‐terminal (his)6 tag. The expressed protein exists in solution as an oligomer, likely a tetramer, and binds heme at a ratio of approximately 0.40 heme molecules per protein monomer. The expressed protein is incapable of adding oxygen to unsaturated lipid substrates, but is a capable peroxidase using both hydrogen peroxide and 15(S)‐hydroperoxy eicosapentaenoic acid as substrates. This result contrasts experiments using a similar protein previously expressed from the genome of Nostoc punctiforme, which exhibits lipoxygenase, but not peroxidase, activity. Structural modeling of the Nostoc and Nitrosomonas proteins yields superimposable structures. Mutagenesis studies of the Nostoc protein are in progress to generate peroxidase activity in the protein product.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Widely Conserved ebo Cluster Is Involved in Precursor Transport to the Periplasm during Scytonemin Synthesis in Nostoc punctiforme.

Scytonemin is a dimeric indole-phenol sunscreen synthesized by some cyanobacteria under conditions of exposure to UVA radiation. While its biosynthetic pathway has been elucidated only partially, comparative genomics reveals that the scytonemin operon often contains a cluster of five highly conserved genes (ebo cluster) of unknown function that is widespread and conserved among several bacterial and algal phyla. We sought to elucidate the function of the ebo cluster in the cyanobacterium Nostoc punctiforme by constructing and analyzing in-frame deletion mutants (one for each ebo gene and one for the entire cluster). Under conditions of UVA induction, all ebo mutants were scytoneminless, and all accumulated a single compound, the scytonemin monomer, clearly implicating all ebo genes in scytonemin production. We showed that the scytonemin monomer also accumulated in an induced deletion mutant of scyE, a non-ebo scytonemin gene whose product is demonstrably targeted to the periplasm. Confocal autofluorescence microscopy revealed that the accumulation was confined to the cytoplasm in all ebo mutants but that that was not the case in the scyE deletion, with an intact ebo cluster, where the scytonemin monomer was also excreted to the periplasm. The results implicate the ebo cluster in the export of the scytonemin monomer to the periplasm for final oxidative dimerization by ScyE. By extension, the ebo gene cluster may play similar roles in metabolite translocation across many bacterial phyla. We discuss potential mechanisms for such a role on the basis of structural and phylogenetic considerations of the ebo proteins.IMPORTANCE Elucidating the biochemical and genetic basis of scytonemin constitutes an interesting challenge because of its unique structure and the unusual fact that it is partially synthesized in the periplasmic space. Our work points to the ebo gene cluster, associated with the scytonemin operon of cyanobacteria, as being responsible for the excretion of scytonemin intermediates from the cytoplasm into the periplasm during biosynthesis. Few conserved systems have been described that facilitate the membrane translocation of small molecules. Because the ebo cluster is well conserved among a large diversity of bacteria and algae and yet insights into its potential function are lacking, our findings suggest that translocation of small molecules across the plasma membrane may be its generic role across microbes.

Magnesium Stable Isotope Fractionation on a Cellular Level Explored by Cyanobacteria and Black Fungi with Implications for Higher Plants.

In a controlled growth experiment we found that the cyanobacterium Nostoc punctiforme has a bulk cell 26Mg/24Mg ratio (expressed as δ26Mg) that is -0.27‰ lower than the growth solution at a pH of ca. 5.9. This contrasts with a recently published δ26Mg value that was 0.65‰ higher than growth solution for the black fungus Knufia petricola at similar laboratory conditions, interpreted to reflect loss of 24Mg during cell growth. By a mass balance model constrained by δ26Mg in chlorophyll extract we inferred the δ26 Mg value of the main Mg compartments in a cyanobacteria cell: free cytosolic Mg (-2.64‰), chlorophyll (1.85‰), and the nonchlorophyll-bonded Mg compartments like ATP and ribosomes (-0.64‰). The lower δ26Mg found in Nostoc punctiforme would thus result from the absence of significant Mg efflux during cell growth in combination with either (a) discrimination against 26Mg during uptake by desolvation of Mg or transport across protein channels or (b) discrimination against 24Mg in the membrane transporter during efflux. The model predicts the preferential incorporation of 26Mg in cells and plant organs low in Mg and the absence of isotope fractionation in those high in Mg, corroborated by a compilation of Mg isotope ratios from fungi, bacteria, and higher plants.

Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H2O2 detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1-5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H2O2 However, no iron oxidation with O2 as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

Design and characterization of a synthetic minimal promoter for heterocyst-specific expression in filamentous cyanobacteria.

Short and well defined promoters are essential for advancing cyanobacterial biotechnology. The heterocyst of Nostoc sp. is suggested as a microbial cell factory for oxygen sensitive catalysts, such as hydrogenases for hydrogen production, due to its microoxic environment. We identified and predicted promoter elements of possible significance through a consensus strategy using a pool of heterocyst-induced DIF+ promoters known from Anabaena sp. PCC 7120. To test if these conserved promoter elements were crucial for heterocyst-specific expression, promoter-yfp reporter constructs were designed. The characterization was accomplished by replacing, -35 and -10 regions and the upstream element, with well described elements from the trc promoter of Escherichia coli, which is also functional in Nostoc sp. From the in vivo spatial fluorescence of the different promoter-yfp reporters in Nostoc punctiforme ATCC 29133, we concluded that both the consensus -35 and extended -10 regions were important for heterocyst-specific expression. Further that the promoter strength could be improved by the addition of an upstream element. We designed a short synthetic promoter of 48 nucleotides, PsynDIF, including a consensus DIF1 sequence, a 17 base pair stretch of random nucleotides and an extended consensus -10 region, and thus generated the shortest promoter for heterocyst-specific expression to date.

Production of alkanes from CO2 by engineered bacteria

BackgroundMicrobial biosynthesis of alkanes is considered a promising method for the sustainable production of drop-in fuels and chemicals. Carbon dioxide would be an ideal carbon source for these production systems, but efficient production of long carbon chains from CO2 is difficult to achieve in a single organism. A potential solution is to employ acetogenic bacteria for the reduction of CO2 to acetate, and engineer a second organism to convert the acetate into long-chain hydrocarbons.ResultsIn this study, we demonstrate alkane production from CO2 by a system combining the acetogen Acetobacterium woodii and a non-native alkane producer Acinetobacter baylyi ADP1 engineered for alkane production. Nine synthetic two-step alkane biosynthesis pathways consisting of different aldehyde- and alkane-producing enzymes were combinatorically constructed and expressed in A. baylyi. The aldehyde-producing enzymes studied were AAR from Synechococcus elongatus, Acr1 from A. baylyi, and a putative dehydrogenase from Nevskia ramosa. The alkane-producing enzymes were ADOs from S. elongatus and Nostoc punctiforme, and CER1 from Arabidopsis thaliana. The performance of the pathways was evaluated with a twin-layer biosensor, which allowed the monitoring of both the intermediate (fatty aldehyde), and end product (alkane) formation. The highest alkane production, as indicated by the biosensor, was achieved with a pathway consisting of AAR and ADO from S. elongatus. The performance of this pathway was further improved by balancing the relative expression levels of the enzymes to limit the accumulation of the intermediate fatty aldehyde. Finally, the acetogen A. woodii was used to produce acetate from CO2 and H2, and the acetate was used for alkane production by the engineered A. baylyi, thereby leading to the net production of long-chain alkanes from CO2.ConclusionsA modular system for the production of drop-in liquid fuels from CO2 was demonstrated. Among the studied synthetic pathways, the combination of ADO and AAR from S. elongatus was found to be the most efficient in heterologous alkane production in A. baylyi. Furthermore, limiting the accumulation of the fatty aldehyde intermediate was found to be beneficial for the alkane production. Nevertheless, the alkane productivity of the system remained low, representing a major challenge for future research.