Nostoc cyanobacterial inoculation in South African agricultural soils enhances soil structure, fertility, and maize growth

Many soils in South Africa have low nutrient supply, poor structural stability and are prone to soil erosion due to susceptibility to surface sealing and crusting. Two crusting soils from the Eastern Cape Province, South Africa were used to evaluate the effects of inoculation with a strain of Nostoc on soil structure, fertility and maize growth. The Nostoc suspension was uniformly applied over potted soils at a rate of 6g (dry weight) per square meter soon after maize germination. Nostoc inoculation increased soil N by 17% and 40% in Hertzog and Guquka soils, respectively. Soil C was also increased significantly and this increase was strongly associated with that of soil N (R ² = 0.838). The highest contents of soil C, soil N and mineral N, however, were found in non-cropped Nostoc inoculated soils. Nostoc inoculation increased maize dry matter yields by 49% and 40% in Hertzog and Guquka soils, respectively. Corresponding increases in maize tissue N were 23% and 14%, respectively. Scanning electron microscopy (SEM) revealed that soil particles and fragments of non-cropped inoculated soils had coatings of extracellular polymeric substances (EPS) with other particles enmeshed in networks of filaments, whilst by contrast little or no EPS and/or filaments were observed on cropped and/or non-inoculated soils. This was consistent with chemical analysis which showed that Nostoc caused significant increases in the EPS and soil C contents of non-cropped soils. The proportion of very stable aggregates was increased by inoculation with Nostoc possibly due to the greater quantities of soil C and EPS observed in inoculated soils. Inoculated soils cropped with maize had a lower proportion of stable aggregates presumably due to their low soil C and EPS contents compared to non-cropped soils. The results suggested that Nostoc could improve the fertility and structural stability of the studied degraded soils.     

Pure culture and reconstitution of the Anthoceros-Nostoc symbiotic association

The partners of the symbiotic association between Anthoceros punctatus L. and Nostoc spp. have been cultured separately in a pure state. The symbiotic association was reconstituted following dual culture in liquid Anthoceros growth medium with a variety of axenic Nostoc isolates and mutant strains. The heterocyst frequency of competent Nostoc strains increased four- to fivefold when in symbiotic association relative to free-living N₂-grown cultures. Dinitrogen fixation by symbiotic Nostoc supported the growth of Anthoceros tissue, although this growth was nitrogen-limited relative to that supported by exogenous ammonium. When the association was reconstituted in the presence of two or three wild-type and mutant Nostoc strains some of these strains were found to compete in infection of Anthoceros tissue and a fraction of the symbiotic Nostoc colonies contained more than one strain. Exogenous ammonium did not affect infection, but repressed development of the symbiotic Nostoc colonies in Anthoceros tissue, and symbiotic Nostoc in N₂-grown Anthoceros tissue appeared to regress from the symbiotic state in the presence of exogenous ammonium. The results show that the Anthoceros-Nostoc symbiotic association is amenable to specific experimental manipulations; their implications are discussed with respect to infection of Anthoceros tissue and control of the development of symbiotic Nostoc.     

Occurrence and Localization of Phycoerythrin in Symbiotic Nostoc of Cycas revoluta and in the Free-Living Isolated Nostoc 7422

The phycobiliprotein phycoerythrin was localized in symbiotic and free-living Nostoc of the cycad Cycas using immunocytochemistry. In symbiotic Nostoc, phycoerythrin was associated with the thylakoid membranes of vegetative cells and absent from heterocysts. Similar cellular/subcellular localization was observed between symbiotic Nostoc and the free-living Cycas isolate Nostoc 7422.     

Sequence Variation of the tRNALeu Intron as a Marker for Genetic Diversity and Specificity of Symbiotic Cyanobacteria in Some Lichens

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA^Leu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA^Leu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA^Leu (UAA) intron can be of great value when examining cyanobacterial diversity.     

Molecular identification of Ostreopsis cf. ovata in filter feeders and putative predators

Ostreopsis ovata Fukuyo is a benthic dinoflagellate widespread from tropical to subtropical and warm temperate coastal areas world-wide. Since the species produces palytoxin-like substances that can accumulate in seafood, the apparent expansion of its range in recent years represents an increasing risk for human health. This leads to the necessity of monitoring protocols that enable the rapid detection of the presence of this microalga in environmental samples and sea-food. We developed an identification protocol based on real-time PCR (qPCR) to detect O. cf. ovata presence in different matrices. The protocol was proved to be able to reveal microalgal traces in both soft tissues and intervalvar liquid of mussels exposed to O. cf. ovata in natural and experimental conditions as well as in seawater samples. O. cf. ovata could also be detected in mussel tissues after the end of the bloom, when it was no longer detectable in sea water. We were able to detect O. cf. ovata in copepods fed on unialgal cultures as well. Cell density estimates based on standard curves resulted to be comparable to direct microscopical counts. The method is therefore suitable to ascertain the origin of palytoxin-like substances in toxic seafood. In addition, our results confirm that mussels and other predators can actually ingest O. cf. ovata cells and act as a vector for toxin transfer through both benthic and planktonic food webs.     

Nodularin,a cyanobacterial toxin,is synthesized in planta by symbiotic Nostoc sp.

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. ‘Macrozamia riedlei 65.1'' and Nostoc sp. ‘Macrozamia serpentina 73.1'' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng μg−1 chlorophyll a in Nostoc sp. ‘Macrozamia riedlei 65.1'' and 12.5±8.4 ng μg−1 Chl a in Nostoc sp. ‘Macrozamia serpentina 73.1'' extracts. Further scans indicated the presence of the rare isoform [L-Har²] nodularin, which contains ℒ-homoarginine instead of ℒ-arginine. Nodularin was also present at 1.34±0.74 ng ml⁻¹ (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har²] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har²] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.     

Genetic Diversity of Nostoc Symbionts Endophytically Associated with Two Bryophyte Species

The diversity of the endophytic Nostoc symbionts of two thalloid bryophytes, the hornwort Anthoceros fusiformis and the liverwort Blasia pusilla, was examined using the tRNA^Leu (UAA) intron sequence as a marker. The results confirmed that many different Nostoc strains are involved in both associations under natural conditions in the field. The level of Nostoc diversity within individual bryophyte thalli varied, but single DNA fragments were consistently amplified from individual symbiotic colonies. Some Nostoc strains were widespread and were detected from thalli collected from different field sites and different years. These findings indicate a moderate level of spatial and temporal continuity in bryophyte-Nostoc symbioses.     

N-terminus conservation in the terminal pigment of phycobilisomes from a prokaryotic and eukaryotic alga

High molecular weight polypeptides from phycobilisomes, believed to be involved in facilitating the energy flow from phycobilisomes to thylakoids, are conserved in the prokaryote Nostoc sp. and the eukaryote Porphyridium cruentum. Partial N-terminal sequence analysis of the phycobilisome-polypeptides of Nostoc (94 kilodalton) and Porphyridium (92 kilodalton) revealed 55% identity in the first 20 residues, but no significant homology with sequences of other phycobiliproteins or phycobilisome-linkers. Polypeptides (94 and 92 kilodalton) from Nostoc thylakoids free of phycobilisomes, previously presumed to be involved in the phycobilisome-thylakoid linkage (M Mimuro, CA Lipschultz, E Gantt 1986 Biochim Biophys Acta 852: 126) exhibit the same immunocrossreactivity but are different from the 94 kilodalton-phycobilisome polypeptide by having blocked N-termini and a different amino acid composition.     

Chlorophyll-Protein Complexes of the Cyanophyte, Nostoc sp

Four chlorophyll-protein complexes have been resolved from the cyanophyte, Nostoc sp., by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis at 4 C. Complexes solubilized by SDS from Spinacia oleracea were run for comparison. As has been well documented, the P700-chlorophyll a-protein complex from the higher plant and blue-green algal samples are similar, and the light-harvesting pigment protein complex is present only in the former. Most noteworthy are two closely migrating chlorophyll proteins in Nostoc sp. which have approximately the same mobility as a single chlorophyll-protein band resolvable from spinach. The absorption maximum of the complex from spinach is at 667 nanometers, and those of the two complexes from Nostoc sp. are at 667 and 669 nanometers; the fluorescence emission maximum at −196 C is at 685 nanometers, and the 735 nanometer fluorescence peak, characteristic of the P700-chlorophyll a-protein complex, is absent. The apoproteins of these new complexes from Nostoc sp. and spinach are in the kilodalton range. It appears that at least one of these two chlorophyll-protein complexes from Nostoc sp. compares with those recently described by others from higher plants and green algae as likely photosystem II complexes, perhaps containing P680, although no photochemical data are yet available.     

DIFFERENCES BETWEEN CULTURED AND FRESHLY ISOLATED CYANOBIONT FROMPELTIGERA—IS THERE SYMBIOSIS-SPECIFIC REGULATION OF A GLUCOSE CARRIER?

The cyanobiont (Nostoc) of the lichenPeltigera horizontaliswas isolated using a procedure of repeated grinding, differential centrifugation and partitioning in an aqueous two-phase system of dextran/polyethylene glycol. A yield of 22% was calculated on the basis of chlorophyll recovery from the thallus. Freshly isolatedNostocdiffered little from that kept in prolonged culture with respect to the relative activities of a number of enzymes of carbohydrate metabolism in the soluble fraction. However, much higher specific activities of the disproportionating transglycosylase and starch phosphorylase, higher intracellular concentrations of glucose, maltooligosaccharides and glycogen and a lower rate of glucose uptake were evident in freshly isolatedNostoc. These activities and pool sizes, as well as CO₂-fixation capability, stayed roughly constant for at least 5 h after isolation; glucose release, which is characteristic for the symbioticNostoc, dropped sharply within 1 h. These observations suggest the transport of glucose out of the cell, rather than that the activities of cytoplasmic enzymes may be a point of early regulation during transition ofNostocfrom the lichenized to the free-living state.     

In-Situ Determination of the Mechanical Properties of Gliding or Non-Motile Bacteria by Atomic Force Microscopy under Physiological Conditions without Immobilization

We present a study about AFM imaging of living, moving or self-immobilized bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900 µm/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5 MPa and 40±5 to 310±30 kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.     

The application of two-step linear temperature program to thermal analysis for monitoring the lipid induction of Nostoc sp. KNUA003 in large scale cultivation

Recently, microalgae was considered as a renewable energy for fuel production because its production is nonseasonal and may take place on nonarable land. Despite all of these advantages, microalgal oil production is significantly affected by environmental factors. Furthermore, the large variability remains an important problem in measurement of algae productivity and compositional analysis, especially, the total lipid content. Thus, there is considerable interest in accurate determination of total lipid content during the biotechnological process. For these reason, various high-throughput technologies were suggested for accurate measurement of total lipids contained in the microorganisms, especially oleaginous microalgae. In addition, more advanced technologies were employed to quantify the total lipids of the microalgae without a pretreatment. However, these methods are difficult to measure total lipid content in wet form microalgae obtained from large-scale production. In present study, the thermal analysis performed with two-step linear temeperature program was applied to measure heat evolved in temperature range from 310 to 351 °C of Nostoc sp. KNUA003 obtained from large-scale cultivation. And then, we examined the relationship between the heat evolved in 310–351 °C (H_E) and total lipid content of the wet Nostoc cell cultivated in raceway. As a result, the linear relationship was determined between H_E value and total lipid content of Nostoc sp. KNUA003. Particularly, there was a linear relationship of 98% between the H_E value and the total lipid content of the tested microorganism. Based on this relationship, the total lipid content converted from the heat evolved of wet Nostoc sp. KNUA003 could be used for monitoring its lipid induction in large-scale cultivation.     

Root Colonization and Phytostimulation by Phytohormones Producing Entophytic Nostoc sp. AH-12

Nostoc, a nitrogen-fixing cyanobacterium, has great potential to make symbiotic associations with a wide range of plants and benefit its hosts with nitrogen in the form of nitrates. It may also use phytohormones as a tool to promote plant growth. Phytohormones [cytokinin (Ck) and IAA] were determined in the culture of an endophytic Nostoc isolated from rice roots. The strain was able to accumulate as well as release phytohormones to the culture media. Optimum growth conditions for the production of zeatin and IAA were a temperature of 25 °C and a pH of 8.0. Time-dependent increase in the accumulation and release of phytohormones was recorded. To evaluate the impact of cytokinins, an ipt knockout mutant in the background of Nostoc was generated by homologous recombination method. A sharp decline (up to 80 %) in the zeatin content was observed in the culture of mutant strain Nostoc AHM-12. Association of the mutant and wild type strain with rice and wheat roots was studied under axenic conditions. The efficacy of Nostoc to colonize plant root was significantly reduced (P < 0.05) as a result of ipt inactivation as evident by low chlorophyll a concentration in the roots. In contrast to the mutant strain, wild type strain showed good association with the roots and enhanced several growth parameters, such as fresh weight, dry weight, shoot length, and root length of the crop plants. The study clearly demonstrated that Ck is a tool of endophytic Nostoc to colonize plant root and promote its growth.     

Sub-Cellular Element Analysis of a Cyanobacterium (Nostoc sp.) in Symbiosis with Gunnera manicata by ESI and EELS

Element analysis using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) was performed in a symbiotic Nostoc sp. strain found in the upper stem tissue of Gunnera manicata, and in Nostoc PCC 9229, a free-living heterocyst-forming cyanobacterium able to enter into symbiosis with the angiosperm Gunnera in reconstitution experiments. ESI and EELS unequivocally identified the four elements nitrogen (N), sulphur (S), phosphorus (P) and oxygen (O) in different inclusion bodies of these biological specimens. High amounts of nitrogen were solely detected in huge cyanophycin granules in vegetative cells of the symbiotic Nostoc strain, whereas large polyphosphate bodies, containing high amounts of phosphorus, sulphur and oxygen, could be seen in the free-living Nostoc PCC 9229. The latter were usually not present or, when found, very small in vegetative cells of the cyanobiont.     

Bioactivity-guided isolation of antimicrobial metabolite from Xylaria sp.

Symbiotic plant-microbe metabolic interactions not only have beneficial effects on plants but also contribute to rich, unmatched and complex chemical biodiversity with biological potential. Systematic delineated bioprospecting of fungal diversity associated with Ficus pumila Linn (Moraceae) for antimicrobial metabolite revealed Xylaria sp. FPL-25(M). The present study describes bioactivity guided fractionation prioritized for antimicrobial potential. Thus, chemical investigation of culture broth of Xylaria sp. FPL-25(M) by bioactivity guided fractionation with spectroscopic techniques revealed bioactive metabolite xylobovide-9-methyl ester. The xylobovide-9-methyl ester exhibited broad-spectrum antimicrobial activity. However, Gram-positive bacteria and fungi were more susceptible than Gram-negative bacteria. The present study results represent bioassay-based screening strategy which facilitates rapid, efficient and reliable approach for endophytic strain prioritization for novel bioactive molecules.     

Diversity of Endosymbiotic Nostoc in Gunnera magellanica (L) from Tierra del Fuego, Chile

Global warming is causing ice retreat in glaciers worldwide, most visibly over the last few decades in some areas of the planet. One of the most affected areas is the region of Tierra del Fuego (southern South America). Vascular plant recolonisation of recently deglaciated areas in this region is initiated by Gunnera magellanica, which forms symbiotic associations with the cyanobacterial genus Nostoc, a trait that likely confers advantages in this colonisation process. This symbiotic association in the genus Gunnera is notable as it represents the only known symbiotic relationship between angiosperms and cyanobacteria. The aim of this work was to study the genetic diversity of the Nostoc symbionts in Gunnera at three different, nested scale levels: specimen, population and region. Three different genomic regions were examined in the study: a fragment of the small subunit ribosomal RNA gene (16S), the RuBisCO large subunit gene coupled with its promoter sequence and a chaperon-like protein (rbcLX) and the ribosomal internal transcribed spacer (ITS) region. The identity of Nostoc as the symbiont was confirmed in all the infected rhizome tissue analysed. Strains isolated in the present study were closely related to strains known to form symbioses with other organisms, such as lichen-forming fungi or bryophytes. We found 12 unique haplotypes in the 16S rRNA (small subunit) region analysis, 19 unique haplotypes in the ITS region analysis and 57 in the RuBisCO proteins region (rbcLX). No genetic variability was found among Nostoc symbionts within a single host plant while Nostoc populations among different host plants within a given sampling site revealed major differences. Noteworthy, interpopulation variation was also shown between recently deglaciated soils and more ancient ones, between eastern and western sites and between northern and southern slopes of Cordillera Darwin. The cell structure of the symbiotic relationship was observed with low-temperature scanning electron microscopy, showing changes in morphology of both cyanobiont cells (differentiate more heterocysts) and plant cells (increased size). Developmental stages of the symbiosis, including cell walls and membranes and EPS matrix states, were also observed.