Heterocystous Cyanobacteria Research Articles

In vitro antimicrobial, antioxidant and molecular study of Nostoc punctiforme isolated from the rice field

Cyanobacteria are photosynthetic prokaryotes and are reservoir of bioactive compounds having substantial antimicrobial and antioxidant activity. They serve as a rich source of structurally novel and biologically active metabolites, holding great potential for various pharmaceutical applications. The aim of this study is to investigate in vitro antimicrobial and antioxidant activities of the extracts of Nostoc punctiforme, a filamentous heterocystous cyanobacterium predominant in the rice fields of Lakhanpur (Bargarh District), Odisha. The heterocystous cyanobacteria was collected from rice field and identified by means of morphological and molecular approach (Nostoc punctiforme Jbr02). Subsequently, in vitro antimicrobial and antioxidant activity was assayed using acetone, methanol, and benzene extract. The cyanobacterial extracts established important antimicrobial actions against bacterial and fungal pathogens with significant MIC values from 62.5 to 500 μg/ml. The methanolic extract displayed better inhibitory activities against both V. Cholerae and P. verrucosun with the MIC value 62.5μg/ml while no such activity was recorded in benzene extract against B. subtilis, A. niger and F. oxysporum pathogens. The antioxidant study, depicted that the methanolic extract showed relatively higher antiradical activity of IC50 542±0.012 μg/ml among the three solvents. The Ferric reducing assay result was also maximum in methanolic extract (0.478±0.78) as compared to benzene and acetone extract. These findings infer that Nostoc punctiforme has potential antimicrobial and antioxidant activity, unveiling its potential as a valuable source of bioactive compounds. Thus, N. punctiforme in specific and cyanobacteria in general are one of the most promising candidates for commercial applications.

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Genome Analysis Coupled With Transcriptomics Reveals the Reduced Fitness of a Hot Spring Cyanobacterium Mastigocladus laminosus UU774 Under Exogenous Nitrogen Supplement.

The present study focuses on the stress response of a filamentous, AT-rich, heterocystous cyanobacterium Mastigocladus laminosus UU774, isolated from a hot spring, Taptapani, located in the eastern part of India. The genome of UU774 contains an indispensable fragment, scaffold_38, of unknown origin that is implicated during severe nitrogen and nutrition stress. Prolonged exposure to nitrogen compounds during starvation has profound adverse effects on UU774, leading to loss of mobility, loss of ability to fight pathogens, reduced cell division, decreased nitrogen-fixing ability, reduced ability to form biofilms, reduced photosynthetic and light-sensing ability, and reduced production of secreted effectors and chromosomal toxin genes, among others. Among genes showing extreme downregulation when grown in a medium supplemented with nitrogen with the fold change > 5 are transcriptional regulator gene WalR, carbonic anhydrases, RNA Polymerase Sigma F factor, fimbrial protein, and twitching mobility protein. The reduced expression of key enzymes involved in the uptake of phosphate and enzymes protecting oxygen-sensitive nitrogenases is significant during the presence of nitrogen. UU774 is presumed to withstand heat by overexpressing peptidases that may be degrading abnormally folded proteins produced during heat. The absence of a key gene responsible for heterocyst pattern formation, patS, and an aberrant hetN without a functional motif probably lead to the formation of a chaotic heterocyst pattern in UU774. We suggest that UU774 has diverged from Fischerella sp. PCC 9339, another hot spring species isolated in the United States.

Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT

Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H2O2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H2O2-treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H2O2. The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H2O2-consumption assays. Furthermore, an entrance channel for H2O2 was identified and key residues implicated in H2O2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide “redox switch” is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.

Salinity as a key control on the diazotrophic community composition in the southern Baltic Sea

Abstract. Over the next decade, the Baltic Sea is predicted to undergo severe changes including decreased salinity due to altered precipitation related to climate changes. This will likely impact the distribution and community composition of Baltic Sea dinitrogen-fixing (N2-fixing) microbes, among which heterocystous cyanobacteria are especially adapted to low salinities and may expand to waters with currently higher salinity, including the Danish Strait and Kattegat, while other high-salinity-adapted N2 fixers might decrease in abundance. In order to explore the impact of salinity on the distribution and activity of different diazotrophic clades, we followed the natural salinity gradient from the eastern Gotland and Bornholm basins through the Arkona Basin to the Kiel Bight and combined N2 fixation rate measurements with a molecular analysis of the diazotrophic community using the key functional marker gene for N2 fixation nifH, as well as the key functional marker genes anfD and vnfD, encoding for the two alternative nitrogenases. We detected N2 fixation rates between 0.7 and 6 nmol N L−1 d−1, and the diazotrophic community was dominated by the cyanobacterium related to Nodularia spumigena and the small unicellular, cosmopolitan cyanobacterium UCYN-A. Nodularia was present in gene abundances between 8.07 × 105 and 1.6 × 107 copies L−1 in waters with salinities of 10 and below, while UCYN-A reached gene abundances of up to 4.5 × 107 copies L−1 in waters with salinity above 10. Besides those two cyanobacterial diazotrophs, we found several clades of proteobacterial N2 fixers and alternative nitrogenase genes associated with Rhodopseudomonas palustris, a purple non-sulfur bacterium. Based on principal component analysis (PCA), salinity was identified as the primary parameter describing the diazotrophic distribution, while pH and temperature did not have a significant influence on the diazotrophic distribution. While this statistical analysis will need to be explored in direct experiments, it gives an indication for the future development of diazotrophy in a freshening Baltic Sea with UCYN-A retracting to more saline North Sea waters and heterocystous cyanobacteria expanding as salinity decreases.

Antenna Modification Leads to Enhanced Nitrogenase Activity in a High Light-Tolerant Cyanobacterium.

ABSTRACTBiological nitrogen fixation is an energy-intensive process that contributes significantly toward supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria, light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems relies on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remain inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors larger amounts of PBS in its heterocysts, and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains large amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities, the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities.

ANTIMICROBIAL POTENTIALS OF TWO FRESHWATER HETEROCYSTOUS CYANOBACTERIA WESTIELLOPSIS PROLIFICA AND NOSTOCHOPSIS LOBATUS COLLECTED FROM SIMILIPAL BIOSPHERE RESERVE, ODISHA, INDIA

The Similipal Biosphere Reserve, an unique ecosystem in the eastern part of India, occupy the northern boundary of Odisha state, lies between 21°28´ to 22°08´ North latitude and 86°04´ to 86°37´ East longitude, covering a vast area of 5578 sq. km in the district of Mayurbhanj. Westiellopsis prolifica and Nostochopsis lobatus, are a group of heterocystous, true branched, filamentous cyanobacteria belonging to the order Stigonematales, rarely found in the aquatic bodies of this region in the state of Odisha. In the present investigation, an attempt has been made to evaluate the antimicrobial potentials of Westiellopsis prolifica and Nostochopsis lobatus collected from flowing streams of Similipal Biosphere Reserve. The organisms were cultured in nitrogen free BG-11 medium and raised to axenic state. Crude metabolites were extracted from cultured test organisms in late log phase using organic solvents of different polarity viz. methanol and chloroform. The metabolites were tested against some clinically significant microorganisms including bacteria and fungi using agar cup diffusion method and the results of antimicrobial activity of cyanobacteria are summarized. The findings revealed that pattern of inhibition varied with respect to the cyanobacterial strains used, nature of solvent extracts and the pathogenic microorganisms tested. Westiellopsis prolifica was found to be more potent that inhibit all the pathogenic bacteria and fungi both in methanol and chloroform extract. This study illustrates that heterocystous cyanobacteria from freshwater bodies could be a potent source of antimicrobial agents and further characterization of active metabolites and evaluation of their pharmacological potentials are also needed.

Growth and photosynthesis acclimated response of the cyanobacterium Fischerella sp. FS 18 exposed to extreme conditions: alkaline pH, limited irradiance, and carbon dioxide concentration.

The true-branching heterocystous cyanobacterium Fischerella sp. FS 18 is widely distributed in paddy fields (North) and petroleum polluted soils (South) in Iran. This investigation tested the hypothesis that the cyanobacterium can acclimatize under the combined effect of extreme environmental conditions. Here, we analysed the physiological response of the cyanobacterium under extremely limited irradiance (2μmol photon m-2s-1); limited carbon dioxide concentration (no aeration) at alkaline pHs (9 and 11) for up to 96h. When the cyanobacterium was exposed to these extreme conditions at pH 11, we observed a decline in growth, oxygen liberation, photosystems ratio, chlorophyll a, and phycobilisomes activity compared to pH 9 after 24h. Besides, we registered a significant decrease in maximum photochemical efficiency and activity of photosystem II at pH 11. The comparative single-cell study revealed that pH 9 caused higher efficiency of photosystem II and I, while increasing alkalinity pH 11 led to disturbed phycobilisomes activity after 24h. This strain was able to recover its structures after 96h. In addition, spectroscopy analyses revealed the presence of the Mycosporine-like amino acid at pH 9.

Trichome Lengths of the Heterocystous N2-Fixing Cyanobacteria in the Tropical Marginal Seas of the Western North Pacific

Calothrix rhizosoleniae and Richelia intracellularis are heterocystous cyanobacteria found in the tropical oceans. C. rhizosoleniae commonly live epiphytically on diatom genera Chaetoceros (C-C) and Bacteriastrum (B-C) while R. intracellularis live endosymbiotically within Rhizosolenia (R-R), Guinardia (G-R), and Hemiaulus (H-R); although, they occasionally live freely (FL-C and FL-R). Both species have much shorter trichomes than the other marine filamentous cyanobacteria such as Trichodesmium spp. and Anabaena gerdii. We investigated the trichome lengths of C. rhizosoleniae and R. intracellularis in the South China Sea (SCS) and the Philippine Sea (PS) between 2006 and 2014. On average, H-R had the shortest trichome lengths (3.5 cells/trichome), followed by B-C and C-C (4.9–5.2 cells/trichome) and FL-C (5.9 cells/trichome), and R-R, G-R, and FL-R had the longest trichome lengths (7.4–8.3 cells/trichome). Field results showed the trichome lengths of C-C and B-C did not vary seasonally or regionally. However, FL-C and H-R from the SCS and during the cool season had longer trichomes, where/when the ambient nutrient concentrations were higher. R-R, G-R, and FL-R also showed regional and seasonal variations in trichome length. Ultrastructural analysis found no gas vesicles within the C. rhizosoleniae cells to assist in buoyancy regulation. Results suggest that the trichome lengths of C. rhizosoleniae and R. intracellularis might be regulated by their diatom hosts’ symbiotic styles and by ambient nutrients. Short trichome length might help C. rhizosoleniae and R. intracellularis to stay in the euphotic zone regardless as to whether they are free-living or symbiotic.

Response of paddy field cyanobacterium, Westiellopsis prolifica Janet to the inorganic nitrogenous fertilizers

Cyanobacteria are prokaryotic nitrogen fixing microorganism which played a significant role in the nitrogen fixation and contributed much to the submerged rice cropping system of the world. It can luxuriantly flourish in paddy fields and providing nitrogen as well as various growth promoting substances that influence the growth of paddy plants. India, being an important rice producing country of the world, depends upon various inorganic fertilizers for rice cultivation. The heterocystous cyanobacterium, Westiellopsis prolifca is a common inhabitant in most of the paddy fields of Kerala. The growth and other biochemical parameters upon the growth of cyanobacterium were assayed. The nitrogenous fertilizer ammonium sulphate influences the chlorophyll-a synthesis, protein content and growth rate. But the ammonia excretion was drastically reduced in cyanobacterial cultures upon the treatment with inorganic fertilizers.

Atmospheric CO2 availability induces varying responses in net photosynthesis, toxin production and N2 fixation rates in heterocystous filamentous Cyanobacteria (Nostoc and Nodularia)

Heterocystous Cyanobacteria of the genus Nodularia form major blooms in brackish waters, while terrestrial Nostoc species occur worldwide, often associated in biological soil crusts. Both genera, by virtue of their ability to fix N2 and conduct oxygenic photosynthesis, contribute significantly to global primary productivity. Select Nostoc and Nodularia species produce the hepatotoxin nodularin and whether its production will change under climate change conditions needs to be assessed. In light of this, the effects of elevated atmospheric CO2 availability on growth, carbon and N2 fixation as well as nodularin production were investigated in toxin and non-toxin producing species of both genera. Results highlighted the following:Biomass and volume specific biological nitrogen fixation (BNF) rates were respectively almost six and 17 fold higher in the aquatic Nodularia species compared to the terrestrial Nostoc species tested, under elevated CO2 conditions.There was a direct correlation between elevated CO2 and decreased dry weight specific cellular nodularin content in a diazotrophically grown terrestrial Nostoc species, and the aquatic Nodularia species, regardless of nitrogen availability.Elevated atmospheric CO2 levels were correlated to a reduction in biomass specific BNF rates in non-toxic Nodularia species.Nodularin producers exhibited stronger stimulation of net photosynthesis rates (NP) and growth (more positive Cohen’s d) and less stimulation of dark respiration and BNF per volume compared to non-nodularin producers under elevated CO2 levels.This study is the first to provide information on NP and nodularin production under elevated atmospheric CO2 levels for Nodularia and Nostoc species under nitrogen replete and diazotrophic conditions.

Adverse effects of nitrogenous compounds on nitrogen-fixing cyanobacterium Anabaena solitaria Klebahn

The nitrogen-fixing heterocystous cyanobacterium Anabaena solitaria was tested for its response to different concentrations of nitrogen sources, namely nitrate and ammonia. To compare both the effect of concentration and type of nitrogen source, gradual concentrations were prepared for both ammonia and nitrate. The results showed concentration was significantly important on the frequency of heterocyst formation where highest nitrogen compounds concentrations were inhibitory for heterocyst formation. On the other hand, the effect of the type of nitrogenous compounds tested was insignificant which only proves that they are equally usable by cyanobacteria which will use them instead of transforming one of its cells to heterocyst thus not performing nitrogen fixation. Results also revealed the massive growth of heterotrophic bacteria in presence of nitrogenous compounds. This indicates that the presence of nitrogenous compounds in fields in which nitrogen-fixing cyanobacteria are to be used as biofertilizers can be inhibitory for nitrogen fixation and stimulatory for heterotrophic bacterial growth which could be pathogenic and harmful to plants. The use of more nitrogen-fixing cyanobacteria with the minimum amount of nitrogenous compounds is recommended.

Effect of Heat Stress and the Recovery Potential of Heterocystous Cyanobacterium, Anabaena iyengarii Bharadwaja 1935

Cyanobacteria, the major photosynthetic organisms, cover a large surface area of this planet. These organisms, being photosynthetic, have the capacity for sequestration of atmospheric carbon dioxide, a significant greenhouse gas that causes global warming. In this work, we have collected, developed pure culture, and identified 25 cyanobacterial species from semi arid agricultural rice fields of western Odisha with the high-temperature environmental setting. The purpose was to screen the cyanobacteria that can survive and grow at high temperatures with high photosynthetic efficiency. Cyanobacteria belong to genera Nostoc, Anabaena, Calothrix, and Hapalosiphon are observed to survive at 45°C. Among the cyanobacterial species, Anabaena iyengarii 17-SKD-2014 was found to exhibit higher growth, protein content, photosynthetic pigments, and photosynthetic O2 evolution at 45°C in comparison to other cyanobacterial isolates. Further, this cyanobacterium was grown at 50°C to analyze the cellular viability, and only up to ninth day incubated culture could recover from high-temperature stress after transferring to 25°C. Even though this indigenous cyanobacterial species failed to survive at 50°C in the laboratory conditions beyond a time limit, but this could be biotechnologically manipulated for effective carbon dioxide sequestration contributing to minimization of global warming.

Cyanobacteria-Derived Proline Increases Stress Tolerance in Arabidopsis thaliana Root Hairs by Suppressing Programmed Cell Death.

Nitrogen-fixing heterocystous cyanobacteria are used as biofertilizer inoculants for stimulating plant growth but can also alleviate plant stress by exometabolite secretion. However, only a small number of studies have focused on elucidating the identity of said bioactives because of the wide array of exuded compounds. Here, we used the root hair assay (RHA) as a rapid programmed cell death (PCD) screening tool for characterizing the bioactivity of cyanobacteria Nostoc muscorum conditioned medium (CM) on Arabidopsis thaliana root hair stress tolerance. We found that heat-stressed A. thaliana pre-treated with N. muscorum CM fractions exhibited significantly lower root hair PCD levels compared to untreated seedlings. Treatment with CM increased stress tolerance by suppressing PCD in root hairs but not necrosis, indicating the bioactive compound was specifically modulating the PCD pathway and not a general stress response. Based on documented N. muscorum exometabolites, we identified the stress-responsive proline as a compound of interest and strong evidence from the ninhydrin assay and HPLC indicate that proline is present in N. muscorum CM. To establish whether proline was capable of suppressing PCD, we conducted proline supplementation experiments. Our results showed that exogenous proline had a similar effect on root hairs as N. muscorum CM treatment, with comparable PCD suppression levels and insignificant necrosis changes. To verify proline as one of the biologically active compounds in N. muscorum CM, we used three mutant A. thaliana lines with proline transporter mutations (lht1, aap1 and atprot1-1::atprot2-3::atprot3-2). Compared with the wild-type seedlings, PCD-suppression in lht1and aap1 mutants was significantly reduced when supplied with low proline (1–5 μM) levels. Similarly, pre-treatment with N. muscorum CM resulted in elevated PCD levels in all three mutant lines compared to wild-type seedlings. Our results show that plant uptake of cyanobacteria-derived proline alters their root hair PCD sensitivity threshold. This offers evidence of a novel biofertilizer mechanism for reducing stress-induced PCD levels, independent of the existing mechanisms documented in the literature.

Carbamate, Organophosphate and Organochlorine Pesticidal Effect on the Qualitative Occurrence and Survivability of Blue-green Algae

Blue-green algae make a valuable contribution to the nitrogenous soil fertility by fixing atmospheric nitrogen and are of great agricultural importance as biofertilizer. Four commercial grade pesticides, Carbamate, Furadan and Sevin and Organophosphate, Rogor and Organochlorine, Endotaf were used to study their effect on the survivability and qualitative occurrence of blue-green algae by taking survival on the control plate as 100%. Stock solutions of these pesticides were prepared freshly for experiments in the sterilized BG-11 media and added to the culture media to obtain the desired concentrations of 100, 250, 500 and 1000 ppm. After 30 days of incubation, the blue-green algal forms appeared in the culture flask were identified using standard monographs. The pragmatic results indicated that, soil blue-green algae show variable resistance to pesticide treatments. The survival percentage of the tested blue-green algae was reduced upto 50 percent at 500 ppm of carbamate pesticides, Furadan and Sevin. While with Rogor only 10.52% and Endotaf 5.26% survivability was noticed at 500 ppm dose level. Furthermore, the organochlorine pesticide Endotaf was more toxic than the organophosphate, Rogor and the carbamates, Sevin and Furadan in sequence to the blue-green algae. It was cocluded that higher doses of pesticide application in the crop fields i.e. more than 100 ppm of Furadan, Sevin and Rogor and even at 100 ppm of Endotaf, qualitative and quantitative occurrence of heterocystous and non- heterocystous blue-green algae was decreased considerably. This suggests that, indiscriminate use of these pesticides may cause adverse effects on the nitrogen fixing blue-green algae of various crop fields, which has a direct influence on total productivity. In general, the sensitiveness of different blue-green algae to pesticide application was found to be more in sheathless heterocystous and unicellular forms than the heterocystous ensheathed and non- heterocystous ensheathed forms.

Land degradation effects on composition of pioneering soil communities: An alternative successional sequence for dryland cyanobacterial biocrusts

Abstract In drylands, soil surfaces in interplant spaces are usually covered by biocrusts, which consist of communities of heterotrophic and chemoautotrophic bacteria, cyanobacteria, mosses, lichens, microalgae, fungi and other organisms. Cyanobacteria are of special interest because of their capacity to promote biocrust succession or increase soil fertility and stability. Therefore, some studies have analyzed their communities in different ecosystems, focusing on how different factors, such as temperature or altitude, influence their composition. Nevertheless, to our knowledge, the relationship between ecosystem degradation and cyanobacterial community composition has not yet been studied in depth. This could be determinant for the successful development of tools for restoring degraded biocrusts by cyanobacterial inoculation. Thus, the objective of this study was to analyze the effect of the ecosystem degradation level on cyanobacteria composition from topsoil communities, where they are keystone pioneering organisms. To do this, we analyzed the cyanobacterial diversity by molecular sequencing (16S rRNA gene) of the DNA extracted from biocrusts at different developmental stages, which were collected from three ecosystems in southeastern Spain. The selected ecosystems represent different “land-condition” states as a result of degradation processes. In one of them soil was removed by mining (the Gádor quarry), the second is a natural badland area (El Cautivo) where water erosion is intense, and the third ecosystem is a well-preserved area (Balsa Blanca). Our findings show that cyanobacterial richness decreases (up to 28 OTUs) as degradation increases and biocrust developmental stage decreases. Also, the relative abundances of most of the species were significantly correlated with the degradation state of the sampling site, either showing a positive or negative trend. Two of the species which increased in abundance with site degradation, and were especially abundant in incipient biocrusts, were Leptolyngbya frigida and Trichocoleus desertorum, while other species, also showing an increase in abundance with degradation, but having a higher relative abundance in most developed biocrusts, were Nostoc commune, Tolypothrix distorta and Scytonema sp. The significant correlation of these species with degradation at different biocrusts developmental stages, suggests an alternative developmental sequence for drylands, at least in more degraded ecosystems. In less degraded ones, the composition of the major cyanobacterial groups followed the common pattern of bundle-forming cyanobacteria (54.7%) pertaining to the Microcoleus genus followed by other non-heterocystous filamentous (17.4%), unicellular/colonial (7.5%) and heterocystous cyanobacteria (1.1%). In comparison, the cyanobacterial groups dominating the most incipient biocrusts, colonizing the most degraded soil, were the filamentous non-heterocystous (50.7%) and the bundle-forming cyanobacteria (48.9%). Therefore, our results show that some cyanobacterial species, which do not belong to traditional pioneer genera, are frequent colonizers of degraded soils, and then, they could be potentially used for producing a more efficient inoculum for inducing biocrust formation and restoring degraded soils. Finally, it is also remarkable that L. frigida appears as dominant in some biocrusts from drylands (up to 74.9% of abundance) being therefore demonstrated its wide distribution in nonpolar biomes and its capacity to also inhabit degraded arid soils.

A novel septal protein of multicellular heterocystous cyanobacteria is associated with the divisome.

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.

A polyketide synthase HglEA, but not HglE2, synthesizes heterocyst specific glycolipids in Anabaena sp. PCC 7120.

Heterocysts are the specialized cells for nitrogen fixation in some filamentous cyanobacteria. To protect the oxygen labile nitrogen fixing enzyme, nitrogenase, heterocysts keep their inner environment microoxic by developing layers of barrier on the outside of their outer membranes. Heterocyst specific glycolipids (Hgls) are constituents of the layer of barrier and amphipathic compounds, synthesized from a very long chain fatty alcohol as a hydrophobic tail and a sugar as a polar head. In the model heterocystous cyanobacterium Anabaena sp. PCC 7120, Hgls are made of fatty alcohol with 26 carbons and a glucose, linked by an ether bond in alpha configuration. The fatty alcohol is synthesized via reactions of a polyketide synthase, HglEA. In Anabaena sp. PCC 7120, another polyketide synthase HglE2 shared more than 50% identity in an amino acid sequence with HglEA and is expected to be involved in Hgls synthesis. However, no direct evidence has been reported. Here, we experimentally show that HglEA is the contributor of Hgls synthesis, and that HglE2 is not involved in the development of the heterocyst specific glycolipid layer.

Elements of the heterocyst-specific transcriptome unravelled by co-expression analysis in Nostoc sp. PCC 7120.

Nitrogen is frequently limiting microbial growth in the environment. As a response, many filamentous cyanobacteria differentiate heterocysts, cells devoted to N2 fixation. Heterocyst differentiation is under the control of the master regulator HetR. Through the characterization of the HetR-dependent transcriptome in Nostoc sp. PCC 7120, we identified the new candidate genes likely involved in heterocyst differentiation. According to their maximum induction, we defined E-DIF (early in differentiation) and L-DIF (late in differentiation) genes. Most of the genes known to be involved in the critical aspects of heterocyst differentiation or function were also classified into these groups, showing the validity of the approach. Using fusions to gfp, we verified the heterocyst-specific transcription of several of the found genes, antisense transcripts and potentially trans-acting sRNAs. Through comparative sequence analysis of promoter regions, we noticed the prevalence of the previously described DIF1 motif and identified a second motif, called DIF2, in other promoters of the E-DIF cluster. Both motifs are widely conserved in heterocystous cyanobacteria. We assigned alr2522 as a third member, besides nifB and nifP, to the CnfR regulon. The elements identified here are of interest for understanding cell differentiation, engineering of biological nitrogen fixation or production of O2 -sensitive molecules in cyanobacteria.

Assessment of sedimentary heterocyst glycolipids as tracers of freshwater input to the Changjiang Estuary and East China Sea

Heterocyst glycolipids (HGs) are biomarkers of important freshwater and marine diazotrophs known as heterocystous cyanobacteria. Recent research has found that HGs with hexose moieties (C6) are derived from free-living, mostly freshwater species while those with pentose moieties (C5) are derived from diatom-symbiont, mostly marine species. Here, we analyzed 39 surface sediments from a study area ranging from the Changjiang Estuary to the offshore East China Sea for HGs to investigate distribution patterns at this freshwater-seawater interface. We detected both C5 and C6 HGs in these samples; however, they had different spatial distributions: the C6 HG abundance was highest at the freshwater end of the estuary and decease offshore, whereas the abundance of C5 HGs displayed almost inverse pattern. We hypothesized that such distributions were likely caused by the mixing of freshwater and seawater, leading to a mixture of HGs derived from inland waters and/or soils and the marine environment. The proportion of C6 HGs to total HGs, defined as the FHG index, decreased with distance offshore, possibly reflecting the delivery of freshwater into the ocean. The FHG index changed curvilinearly with salinity, likely resulting from concentration-based weighting of end-member contributions. The FHG index correlated linearly (r2 = 0.76) with δ13Corg, suggesting that FHG could be used to indicate relative contributions of freshwater-delivered terrestrial organic matter in this estuarine and coastal region. However, the FHG index did not linearly correlate with the branched and isoprenoid tetraether (BIT) index, another proxy used for terrestrial input, likely because the BIT index is predominantly controlled by variations in marine crenarchaeol production.