Group 3 sigma factor gene, sigJ, a key regulator of desiccation tolerance, regulates the synthesis of extracellular polysaccharide in cyanobacterium Anabaena sp. strain PCC 7120.

The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.     

Isolation and purification of an axenic diazotrophic drought-tolerant cyanobacterium, Nostoc commune, from natural cyanobacterial crusts and its utilization for field research on soils polluted with radioisotopes

Nitrogen fixation and drought tolerance confer the ability to grow on dry land, and some terrestrial cyanobacteria exhibit these properties. These cyanobacteria were isolated in an axenic form from Nostoc commune clusters and other sources by modifying the method used to isolate the nitrogen-fixing and drought-tolerant cyanobacterium Nostoc sp. HK-01. Of these cyanobacteria, N. commune, which is difficult to isolate and purify, uses polysaccharides to maintain water, nitrogen fertilizers for nitrogen fixation, and can live in extreme environments because of desiccation tolerance. In this study, we examined the use of N. commune as biosoil for space agriculture and possible absorption of radioisotopes ((134)Cs, (137)Cs). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

The extracellular-matrix-retaining cyanobacterium Nostoc verrucosum accumulates trehalose, but is sensitive to desiccation

The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies, which consist of both cellular filaments and massive extracellular matrix material. In this study, the physiological features of N. verrucosum were investigated and compared with those of the anhydrobiotic cyanobacterium Nostoc commune. Nostoc verrucosum cells were sensitive to desiccation, but tolerant of freeze-thawing treatment in terms of both cell viability and photosynthetic O(2) evolution. Natural colonies of these cyanobacteria contained similar levels of chlorophyll a, carotenoids, the UV-absorbing pigments scytonemin and mycosporine-like amino acids, and uronic acid [a component of extracellular polysaccharides (EPS)]. EPS from both N. verrucosum and N. commune indicated low acidity and a high affinity for divalent cations, although their sugar compositions differed. The WspA protein, known to be a major component of the extracellular matrix of N. commune, was detected in N. verrucosum. Desiccation caused similarly high levels of trehalose accumulation in both cyanobacteria. Although previously considered relevant to anhydrobiosis in the terrestrial cyanobacterium N. commune, the data presented here suggest that extracellular matrix production and trehalose accumulation are not enough for standing extreme desiccation in N. verrucosum.     

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.     

Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

Identification of ten Anabaena sp. genes that under aerobic conditions are required for growth on dinitrogen but not for growth on fixed nitrogen

Heterocysts are specialized cells required for aerobic fixation of dinitrogen by certain filamentous cyanobacteria. Numerous genes involved in the differentiation and function of heterocysts in Anabaena sp. strain PCC 7120 have been identified by mutagenizing and screening for mutants that require fixed nitrogen for growth in the presence of oxygen. We have verified that 10 Anabaena sp. genes, all1338, all1591, alr1728, all3278, all3520, all3582, all3850, all4019, alr4311, and all4388, identified initially by transposon mutagenesis, are such genes by complementing or reconstructing the original mutation and by determining whether the mutant phenotype might be due to a polar effect of the transposon. Elucidation of the roles of these genes should enhance understanding of heterocyst biology.     

Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120

Photoreduction of dinitrogen by heterocyst-forming cyanobacteria is of great importance ecologically and for subsistence rice agriculture. Their heterocysts must have a glycolipid envelope layer that limits the entry of oxygen if nitrogenase is to remain active to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Genes alr5354 (hglD), alr5355 (hglC) and alr5357 (hglB) of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, and hglE of Nostoc punctiforme are required for synthesis of heterocyst envelope glycolipids. Newly identified Fox- mutants bear transposons in nearby open reading frames (orfs) all5343, all5345-asr5349 and alr5351-alr5358. Complementation and other analysis provide evidence that at least orfs all5343 (or a co-transcribed gene), all5345, all5347, alr5348, asr5350-alr5353 and alr5356, but not asr5349, are also required for a Fox+ phenotype. Lipid and sequence analyses suggest that alr5351-alr5357 encode the enzymes that biosynthesize the glycolipid aglycones. Electron microscopy indicates a role of all5345 through all5347 in the normal deposition of the envelope glycolipids.     

Structural and functional characterization of galactooligosaccharides in Nostoc commune: beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]2-beta-D-1,4-anhydrogalactitol and beta-(1-->6)-galactofuranosylated homologues

A new class of galactooligosaccharides has been identified from the terrestrial cyanobacterium Nostoc commune by MS and NMR techniques. These consist of beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]n-beta-d-1,4-anhydrogalactitols with n ranging from 2 to 8, corresponding to compounds designated 1 through 7. In total these saccharides amounted to approximately 0.35% of the dry thallus of N. commune, while in several other cyanobacteria they were not detected. Possibly they play some role in protection from damage by heat and desiccation as suggested by experiments with heterologous systems. For example, phosphoglucomutase (EC 2.7.5.1) from rabbit muscle was protected against heat inactivation by these oligosaccharides, and alpha-amylase (EC 3.2.1.1) from porcine pancreas by the oligosaccharides 6 and 7. The homologues of lower molecular mass, however, enhanced heat sensitivity of alpha-amylase. The viability of Escherichia coli was completely abolished by desiccation, whereas in the presence of 4 survival rates were approximately 50% of controls not subjected to desiccation. The newly identified saccharides are compared with known galactofuranose-based oligo- and polysaccharides and possible biological functions of them are discussed.     

Effects of periodic desiccation on the synthesis of the UV-screening compound, scytonemin, in cyanobacteria

Scytonemin is an ultraviolet radiation (UVR)-screening compound synthesized by some sheathed cyanobacteria exposed to high solar and sky radiation. It is primarily produced in response to UVA radiation, but certain environmental stresses can enhance synthesis. This study focuses on the effects of periodic desiccation on scytonemin synthesis in three desiccation-tolerant cyanobacterial strains, Nostoc punctiforme PCC 73102, Chroococcidiopsis CCMEE 5056 and Chroococcidiopsis CCMEE 246. Nostoc punctiforme and Chroococcidiopsis CCMEE 5056 exposed to UVA radiation produced more concentrated scytonemin screens when experiencing periodic desiccation (i.e. 1 day desiccated for every 2 days hydrated) than when continuously hydrated. A more concentrated scytonemin screen would reduce the amount of UVR damage accrued when cells are desiccated and metabolically inactive. This might allow the cyanobacteria to allocate more energy to systems other than UVR damage repair during rehydration, which would facilitate recovery. The scytonemin screen is extremely stable, remaining largely intact in the sheaths of desiccated N. punctiforme even when continuously exposed to UVA radiation for about 2 months. In contrast to the above findings, scytonemin synthesis in Chroococcidiopsis CCMEE 246, a strain that produces scytonemin constitutively under low visible light (no UVA), was partially inhibited by periodic desiccation.     

Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.     

Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.     

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold). These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.     

THE ECOLOGY OF NOSTOC

Nostoc is a genus of filamentous cyanobacteria that can form macroscopic or microscopic colonies and is common in both terrestrial and aquatic habitats. Much of the success of Nostoc in terrestrial habitats is related to its ability to remain desiccated for months or years and fully recover metabolic activity within hours to days after re-hydration with liquid water . Nostoc can also withstand repeated cycles of freezing and thawing and, thus, is an important component of extreme terrestrial habitats in the Arctic and Antarctic. The ability to fix atmospheric N ₂ can provide an advantage in nitrogen-poor environments . Nostoc also has the ability to screen damaging ultraviolet light in terrestrial and shallow benthic habitats. The genus potentially could be important in paddy rice culture because it fixes nitrogen that may later be released and used by plants; it also may play a role in soil formation and may increase nitrogen input to natural aquatic and terrestrial ecosystems. The abilities to survive in terrestrial habitats and fix N ₂ are important in symbiotic interactions with fungi (lichens), liverworts, hornworts, mosses, ferns, cycads, and the angiosperm Gunnera. Nostoc is somewhat resistant to predation; this probably is related to production of large amounts of sheath material, synthesis of microcystin-like toxins by some strains, and formation of colonies that are too large for many algivores to consume. Some organisms can subsist on Nostoc, although it may not be a preferred food source. Lytic cyanophages also infect Nostoc, but little is known about population control of Nostoc in its natural environment, Late Precambrian fossils resembling Nostoc have been described, and Nostoc possibly has been an important component of many terrestrial and aquatic communities since that time .     

Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme

The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.     

A pair of iron-responsive genes encoding protein kinases with a Ser/Thr kinase domain and a His kinase domain are regulated by NtcA in the Cyanobacterium Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N(2) when combined nitrogen is not available in the growth medium. It has a family of 13 genes encoding proteins with both a Ser/Thr kinase domain and a His kinase domain. The function of these enzymes is unknown. Two of them are encoded by pkn41 (alr0709) and pkn42 (alr0710). These two genes are separated by only 72 bp on the chromosome, and our results indicate that they are cotranscribed. The expression of pkn41 and pkn42 is induced by iron deprivation irrespective of the nature of the nitrogen source. Mutants inactivating either pkn41, pkn42, or both grow similarly to the wild type under normal conditions, but their growth is impaired either in the presence of an iron chelator or under conditions of nitrogen fixation and iron limitation, two situations where the demand for iron is particularly strong. Consistent with these results, these mutants display lower iron content than the wild type and a higher level of expression for nifJ1 and nifJ2, which encode pyruvate:ferredoxin oxidoreductases. Both nifJ1 and nifJ2 are known to be induced by iron limitation. NtcA, a global regulatory factor for different metabolic pathways, binds to the putative promoter region of pkn41, and the induction of pkn41 in response to iron limitation no longer occurs in an ntcA mutant. Our results suggest that ntcA not only regulates the expression of genes involved in nitrogen and carbon metabolism but also coordinates iron acquisition and nitrogen metabolism by activating the expression of pkn41 and pkn42.     

Characterization of nifH gene expression, modification and rearrangement in Nodularia spumigena strain AV1

The annually reoccurring blooms that characterize the surface waters of the Baltic Sea are dominated by filamentous, heterocystous cyanobacteria such as Nodularia spumigena. In a previous study, we have demonstrated that N. spumigena strain AV1 differentiates heterocysts in the absence of detectable nitrogen fixation activity, an unusual physiological trait that is clearly distinct from other well-studied cyanobacteria. To further analyze the uncoupling between these two processes, we analyzed the gene expression and modification of the nitrogenase enzyme (the enzyme responsible for nitrogen fixation) in N. spumigena AV1, as well as in several other N. spumigena strains. Here, we demonstrate the occurrence of two nifH gene copies in N. spumigena strain AV1, only one of which is located in a complete nifHDK cluster and several NifH protein forms. Furthermore, we demonstrate the occurrence of a DNA rearrangement mechanism acting within the nifH gene copy located in the nifHDK cluster and present only in the strains exhibiting the previously reported uncoupling between heterocyst differentiation and nitrogen fixation processes. These data stress the existence of a distinct and complex regulatory circuit related to nitrogen fixation in this ecologically significant bloom-forming cyanobacterium.