Increased heterocyst frequency by patN disruption in Anabaena leads to enhanced photobiological hydrogen production at high light intensity and high cell density

The effects of increasing the heterocyst-to-vegetative cell ratio on the nitrogenase-based photobiological hydrogen production by the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were studied. Using the uptake hydrogenase-disrupted mutant (ΔHup) as the parent, a deletion-insertion mutant (PN1) was created in patN, known to be involved in heterocyst pattern formation and leading to multiple singular heterocysts (MSH) in Nostoc punctiforme strain ATCC 29133. The PN1 strain showed heterocyst differentiation but failed to grow in medium free of combined-nitrogen; however, a spontaneous mutant (PN22) was obtained on prolonged incubation of PN1 liquid cultures and was able to grow robustly on N₂. The disruption of patN was confirmed in both PN1 and PN22 by PCR and whole genome resequencing. Under combined-nitrogen limitation, the percentage of heterocysts to total cells in the PN22 filaments was 13–15 and 16–18% under air and 1% CO₂-enriched air, respectively, in contrast to the parent ΔHup which formed 6.5–11 and 9.7–13% heterocysts in these conditions. The PN22 strain exhibited a MSH phenotype, normal diazotrophic growth, and higher H₂ productivity at high cell concentrations, and was less susceptible to photoinhibition by strong light than the parent ΔHup strain, resulting in greater light energy utilization efficiency in H₂ production on a per unit area basis under high light conditions. The increase in MSH frequency shown here appears to be a viable strategy for enhancing H₂ productivity by outdoor cultures of cyanobacteria in high-light environments.

     

Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

Background  

The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Cyanobacteria <Emphasis Type="Italic">Nostoc Punctiforme</Emphasis> from Abyssal Benthos of Lake Baikal: Unique Ecology and Metabolic Potential

A strain of Nostoc punctiforme was isolated from the bottom sediments of the oil seep at Gorevoy Utes (Central Baikal) at a depth of 890 m. The Baikal strain is highly similar (98–99%) to the N. punctiforme CCAP 1453/9 strain and the typical N. punctiforme PCC 73103 strain isolated from soil ecotopes. Based on the analysis of functional genes and mass spectrometry data, we determined that the strain can produce bioactive peptides and polyketides, but does not produce known cyanobacterial toxins, saxitoxin or its analogs, or microcystins. The peptides aeruginosinamide, aeruginosin 606, aeruginosin 98-A, kasumigamide C, and microginin 91-D were recorded in the metabolic profile of the strain. The major ion found in the MALDI mass spectrum is most likely to be an ion of a polyketide substance with unknown function.     

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments

Background

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N₂ fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N₂-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts.

Results

Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N₂-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme.

Conclusions

The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1064) contains supplementary material, which is available to authorized users.     

Iron deficiency influences NtcA‐dependent regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120

In Anabaena sp. PCC 7120, iron is an essential trace element and its availability determines proper functioning of several kinds of metabolisms. Iron deficiency leads to several unavoidable consequences including membrane damage. In the present study, we dealt with the impact of iron deficiency on NtcA (global nitrogen regulator)‐dependent regulation of two important processes, i.e. fatty acid desaturation and heterocyte envelop formation in cyanobacterium Anabaena sp. PCC 7120. In Anabaena sp. PCC 7120, NtcA regulates fatty acid desaturation by regulating enzyme fatty acid desaturases. The NtcA‐based regulation of fatty acid desaturation may be direct or indirect. Furthermore, the expression of genes involved in the heterocyte envelope polysaccharide (HEP) layer formation (hepABCK) and heterocyte‐specific glycolipids (HGLs) synthesis (devH, hglE_A, prpJ and devB) were also under the control of NtcA and reduced under iron deficiency background. The enhanced expression of furA and early downregulation of ntcA under iron deficiency is responsible for reduction in fatty acid desaturation as well as decrease in the expression of genes involved in HEP layer formation and HGL synthesis. Overall results confirmed that iron deficiency influences the NtcA‐based regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120.     

Cloning of Lipoxygenase Genes from a Cyanobacterium, Nostoc punctiforme, and Its Expression in Eschelichia coli

Oxylipin metabolism represents one of the important hormonal and defensive mechanisms employed by plants, algae, or animals. It begins mostly with the reaction of lipoxygenases (LOXs), which catalyze the oxygenation of polyunsaturated fatty acids to form the corresponding hydroperoxides. At present, little information about LOXs in cyanobacteria has been reported. Herein, we report the first isolation of two LOX genes (NpLOX1 and NpLOX2) from a cyanobacterium, Nostoc punctiforme ATCC29133. Incubations of recombinant NpLOX1 and NpLOX2 proteins expressed in Eschelichia coli with linoleic acid resulted in the predominant formation of linoleic acid 13-S-hydroperoxide. Other C18 and C20 fatty acids could also be substrates for NpLOX enzymes. Phylogenetic analysis of NpLOX sequences showed that the NpLOX enzymes shared a high homology with LOX sequence of a bacterial pathogen, Pseudomonas aeruginosa, and these bacterial LOXs formed a subfamily distinct from those of plants, algae, and mammals.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Symbiosis between the cyanobacterium Nostoc and the liverwort Blasia requires a CheR-type MCP methyltransferase

In response to environmental change, the cyanobacterium Nostoc punctiforme ATCC 29133 produces highly adapted filaments known as hormogonia that have gliding motility and serve as the agents of infection in symbioses with plants. Hormogonia sense and respond to unidentified plant-derived chemical signals that attract and guide them towards the symbiotic tissues of the host. There is increasing evidence to suggest that their interaction with host plants is regulated by chemotaxis-related signal transduction systems. The genome of N. punctiforme contains multiple sets of chemotaxis (che)-like genes. In this study we characterize the large che5 locus of N. punctiforme. Disruption of NpR0248, which encodes a putative CheR methyltransferase, results in loss of motility and significantly impairs symbiotic competency with the liverwort Blasia pusilla when compared with the parent strain. Our results suggest that chemotaxis-like elements regulate hormogonia function and hence symbiotic competency in this system.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133

Following exposure to long‐wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole‐alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 cotranscribed genes. A two‐component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator (RR), Npun_F1278, was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain Δ1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, Δ1278 was incapable of producing scytonemin. A phenotypic characterization of Δ1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid‐soluble pigment complement of Δ1278 compared to the wildtype. The mutant, however, had a slower specific growth rate under white light and produced ~2.5‐fold more phycocyanin per cell under UVA than the wildtype. Since Δ1278 does not produce scytonemin, this study demonstrates that the RR gene, Npun_F1278, is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production.     

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P_kasO* or P_neo. The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.     

Isolation and Characterization of Genes Encoding Thermoactive and Thermostable Dextranases from Two Thermotolerant Soil Bacteria

Thermotolerant Paenibacillus strain Dex70-1B and unidentified strain Dex70-34 produce thermoactive dextran-degrading enzymes. Plasmid-based genomic DNA libraries constructed from mixed bacterial cultures containing Dex70-1B or Dex70-34 were screened for the ability to confer dextranolytic activity at 70°C onto Escherichia coli. One gene, designated dex1, was isolated from each strain. The Dex70-1B and Dex70-34 dex1 gene sequences were non-identical, and encoded proteins containing 597 (M_r 68.6 kDa) and 600 amino acids (M_r 69.2 kDa), respectively. The Dex1 amino acid sequences were most similar to one another, and formed a new clade among the family 66 glycosyl hydrolase sequences. Expression of the Dex1 proteins in E. coli produced dextranolytic activity that converted ethanol-insoluble blue dextran into an ethanol-soluble form, suggestive of endodextranases (EC 3.2.1.11). Both enzymes were most active at about 60°C and pH 5.5, and retained more than 70% maximal activity after incubation at 57°C for 9.5 h in the absence of substrate.     

MORPHOLOGICAL AND 16S rRNA GENE EVIDENCE FOR RECLASSIFICATION OF THE PARALYTIC SHELLFISH TOXIN PRODUCING APHANIZOMENON FLOS‐AQUAE LMECYA 31 AS APHANIZOMENON ISSATSCHENKOI (CYANOPHYCEAE)1

A taxonomic reevaluation of the paralytic shellfish toxin (saxitoxins) producing cyanobacterium Aphanizomenon flos‐aquae Ralfs ex Born. & Flah. LMECYA31 was done using morphology and 16S rRNA gene sequences. We found that strain LMECYA31 was incorrectly identified as Aph. flos‐aquae based on (a) lack of bundle formation in trichomes, (b) shape of terminal cells in the trichomes, (c) lower similarity (<97.5%) in the 16S rRNA gene sequences relative to those of Aph. flos‐aquae, and (d) comparison within a phylogenetic tree of 16S rRNA gene sequences. The shape of the terminal trichome cells and the shape and size of the vegetative cell, heterocyst, and akinete in strain LMECYA31 match characters of Aph. issatschenkoi (Ussachew) Proschkina‐Larvernko. 16S rRNA gene sequences and phylogenetic clusters constructed from 16S rRNA gene sequences support our conclusion that strain LMECYA31 should be Aph. issatschenkoi.     

Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli

The docosahexaenoic acid (DHA) biosynthesis gene cluster (pDHA3) from the DHA-producing Moritella marina strain MP-1 includes the genes pfaA, pfaB, pfaC, and pfaD, which are similar to the genes of polyketide biosynthesis. When this cluster was co-expressed in Escherichia coli with M. marina MP-1 pfaE, which encodes phosphopantetheinyl transferase, DHA was biosynthesized. The maximum production of DHA (5% of total fatty acids) was observed at 15°C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.     

PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120

Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and heterocysts. Under diazotrophic conditions, the mutant DeltaprpJ (S20) did not grow, lacked only one of the two heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglE(A) and nifH, two genes necessary for the formation of the glycolipid layer of heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for heterocyst maturation because it regulates the synthesis of only one of the two heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both heterocyst glycolipids.