Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina

Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO₂ evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM α-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.Key words: arginase, lectin, Peltigera canina, recognition, urease     

A cyanobacterial β-actin-like protein,responsible for lichenized <Emphasis Type="Italic">Nostoc</Emphasis> sp. motility towards a fungal lectin

Lichenized Nostoc cells isolated from the lichen Peltigera canina develop chemotactic movement towards a lectin purified from the lichen thallus. Inhibitors of cytoskeleton dynamics, such as phalloidin, latrunculin A and blebbistatin, impede cell displacement. The inhibition of chemotaxis produced by the combined action of phalloidin and blebbistatin is largely reversed by GTP and its analogs, GTP(γ)S and GDP(β)S, as well as by cyclic AMP. Movement implies a rearrangement of the cytoskeleton causing cell polarity, which is, in turn, inhibited by phalloidin and latrunculin A, as revealed by confocal microscopy. F-actin fibers composing Nostoc cytoskeleton have been visualized by immunocytochemical techniques associated with transmission electron microscopy.     

The occurrence of fimbriae on a N 2 2 -fixing cyanobacterium which occurs in lichen symbiosis

The Nostoc cyanobiont of the lichen Peltigera canina when grown on N₂ possesses, in the motile stage, discrete unbranched non-flagellar appendages (fimbriae or pili). These arise from the host cell surface in a peritrichous manner, have an axial hole, are 7.0 ±0.3 nm in diameter and are up to 3 m long. They do not haemagglutinate guinea pig red blood corpuscles and differ from the major fimbrial types reported for Gram-negative heterotrophic bacteria and from sex pili. They may be involved in motility and specificity in symbiotic cyanobacteria.     

Sarcosine: a possible regulatory compound in the Peltigera praetextata-Nostoc symbiosis

Abstract In analyses of free amino acids in the lichen Peltigera praetextata high levels of sarcosine ( N -methylglycine) were found.
Sarcosine was found to stimulate nitrogenase activity of the cyanobiont Nostoc , isolated from Peltigera canina . This stimulation was optimal at 1 mM sarcosine, a concentration which is close to that calculated as being present in a water-saturated lichen thallus. Sarcosine also was found to inhibit the activity of glutamine synthetase in Nostoc . This inhibitory effect may account for the stimulation of nitrogenase activity and may be important in the symbiotic regulation of cyanophilic lichens.     

Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism

The CO₂ dependence of net CO₂ assimilation was examined in a number of green algal and cyanobacterial lichens with the aim of screening for the algal/cyanobacterial CO₂-concentrating mechanism (CCM) in these symbiotic organisms. For the lichens Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and P. neopolydactyla (Gyeln.) Gyeln., the photosynthetic performance was also compared between intact thalli and their respective photobionts, the green alga Coccomyxa PA, isolated from Peltigera aphthosa and the cyanobacterium Nostoc PC, isolated from Peltigera canina. More direct evidence for the operation of a CCM was obtained by monitoring the effects of the carbonic-anhydrase inhibitors acetazolamide and ethoxyzolamide on the photosynthetic CO₂use efficiency of the photobionts. The results strongly indicate the operation of a CCM in all cyanobacterial lichens investigated and in cultured cells of Nostoc PC, similar to that described for free-living species of cyanobacteria. The green algal lichens were divided into two groups, one with a low and the other with a higher CO₂-use efficiency, indicative of the absence of a CCM in the former. The absence of a CCM in the low-affinity lichens was related to the photobiont, because free-living cells of Coccomyxa PA also apparently lacked a CCM. As a result of the postulated CCM, cyanobacterial Peltigera lichens have higher rates of net photosynthesis at normal CO₂ compared with Peltigera aphthosa. It is proposed that this increased photosynthetic capacity may result in a higher production potential, provided that photosynthesis is limited by CO₂ under natural conditions.     

Cyanobiont specificity in some Nostoc-containing lichens and in a Peltigera aphthosa photosymbiodeme

The cyanobacterial symbionts in some Nostoc -containing lichens were investigated using the nucleotide sequence of the highly variable cyanobacterial tRNA^Leu (UAA) intron. When comparing different Nostoc -containing lichens, identical intron sequences were found in different samples of the same lichen species collected from two remote areas. This was true for all species where this comparison was made ( Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and Nephroma arcticum (L.) Torss.). With one exception, a specific intron sequence was never found in more than one lichen species. However, for two of the species, Peltigera aphthosa and Nephroma arcticum , two different cyanobionts were found in different samples. By examining a P. aphthosa photosymbiodeme it could be shown that the same Nostoc is present in both bipartite and tripartite lobes of this lichen. It is thus possible for one cyanobiont/ Nostoc to form the physiologically different symbioses that are found in bipartite and tripartite lichens. The connection between photobiont identity and secondary chemistry is discussed, as a correlation between differences in secondary chemistry and different cyanobionts/ Nostoc s in the species Peltigera neopolydactyla (Gyeln.) Gyeln. was observed. It is concluded that more knowledge concerning the photobiont will give us valuable information on many aspects of lichen biology.     

Spatial patterns of photobiont diversity in some Nostoc-containing lichens

Patterns of photobiont diversity were examined in some Nostoc -containing lichens using the nucleotide sequence of the cyanobacterial tRNA^Leu (UAA) intron. Lichen specimens collected in northwestern USA were analysed and the sequence data were compared with tRNA^Leu(UAA) intron sequences previously obtained from lichens in northern Europe. Generally, it is the species identity of a lichen rather than the geographical origin of the specimen that determines the identity of the cyanobiont. Identical intron sequences were found in Peltigera membranacea specimens collected in Oregon (USA) and in Sweden, and very similar sequences were also found in Nephroma resupinatum thalli collected in Oregon and Finland. Furthermore, in mixed assemblages where two Peltigera species grew in physical contact with each other, the different lichen species housed different photobiont strains. There is however not a one-to-one relation between mycobiont and photobiont as some intron sequences were found in more than one lichen species, and different intron sequences were found in different samples of some lichen taxa. Peltigera venosa exhibited a higher level of photobiont diversity than any other lichen species studied, and several intron sequences could for the first time be obtained from a single thallus. It is not clear whether this is evidence of lower cyanobiont specificity, or reflects an ability to exhibit different degrees of lichenization with different Nostoc strains. In one specimen of P. venosa , which contained bipartite cyanosymbiodemes and tripartite, cephalodiate thalli, both thallus types contained the same intron sequence.     

Fasciclin domain proteins are present in nostoc symbionts of lichens

Differences in the soluble protein fraction between the freshly isolated cyanobiont of lichen Peltigera membranacea, the corresponding free-living strain, and Nostoc punctiforme were analyzed. One protein, which was among the most prominent proteins of the freshly isolated cyanobiont, was expressed at a lower level in the corresponding free-living strain and was not detected at all on the two-dimensional gels of N. punctiforme. This protein was partially sequenced, and the corresponding open reading frame (ORF) in the N. punctiforme genome was identified. This ORF contains a fasciclin domain typical of a class of surface-associated proteins involved in cell adhesion. Similar fasciclin motif-containing genes have previously been shown to be symbiotically induced in other symbiotic systems.     

The Nostoc-Nephroma symbiosis: localization, distribution pattern and levels of key proteins involved in nitrogen and carbon metabolism of the cyanobiont

The qualitative distribution and quantitative estimates of nitrogenase (EC 1.7.99.2), glutamine synthetase (EC 6.3.1.2), phycoerythrin and ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were studied in the cyanobacterium Nostoc residing in internal cephalodia of the tripartite lichen Nephroma arcticum L. Polyclonal antisera, raised in rabbit against the proteins, and goat anti-rabbit IgG conjugated to 10 nm gold were used as probes to detect the antigens by transmission electron microscopy. Western blot analyses demonstrated the monospecificity of the antisera. Nitrogenase was localized in heterocysts, with vegetative cells showing a label intensity comparable to the background. Distribution of the antigen within the heterocysts was uniform. Glutamine synthetase labelling was very low, but appeared to be distributed in both cell types. An intense phycoerythrin labelling was associated with the thylakoid region of the vegetative cells, whereas a much lower labelling was observed in the heterocyst. No significant differences were found between cyanobionts in younger and older cephalodia except for the nitrogenase labelling, which was higher in heterocysts of the cyanobiont in younger cephalodia. Most of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) label was present in vegetative cells. The Rubisco label was pronounced in the carboxysomes, whereas the label in the cytoplasm, on a unit area basis, was much lower. Heterocysts showed a label intensity similar to that of the vegetative cell cytoplasm. In Nostoc of the bipartite lichen Peltigera canina L., the Rubisco protein showed a comparable distribution pattern, but the average number of carboxysomes per vegetative cell was about 4 times higher.     

The α-amylase inhibitor of bean seed: two-step proteolytic maturation in the protein storage vacuoles of the developing cotyledon

In the nitrogen fixing symbiosis between Nostoc and the angiosperm Gunnera , the cyanobiont is found in stem glands and is thought to have a heterotrophic mode of nutrition. To investigate whether the photosynthetic machinery in the cyanobiont is down-regulated in the symbiosis, the presence of the phycobiliproteins, phycoerythrin and phycocyanin, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) in cyanobionts of Gunnera magellanica Lam. and in a free-living (cultured) isolate of the cyanobacterium was studied by immunoelectron microscopy. Carboxysomes were numerous in all vegetative cells (ca 3.5 per cell section), and on an area basis they showed a high Rubisco label compared to the cytoplasm; but recalculation on a volume basis demonstrated that the carboxysomal fraction of Rubisco decreased in the cyanobiont along the plant stem. Along the whole Gunnera stem both types of phycobiliproteins were present in the symbiotic Nostoc and in amounts equivalent to or above those detected in the free-living isolate. As the symbiotic Nostoc is located intracellularly, out of reach of light in the plant stem, the findings indicate a lack of regulation of the photosynthetic protein synthesis in the symbiotic state.     

PRELIMINARY OBSERVATIONS ON THE GROWTH OF TRANSPLANTEDPELTIGERA CANINAUNDER SEMI-NATURAL CONDITIONS

Peltigera caninathalli have been successfully transplanted onto soil in a garden and in flowerpots. Garden samples showed marked seasonality and achieved growth rates of 6·4 cm per year. Pot-grown samples showed variation in the growth of individual thallus lobes and established that, under different soil hydration regimes, permanently hydrated thalli could sustain considerable linear growth rates for at least 140 days.     

Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria

Lectins were extracted from whole fern grindings ofAzolla pinnata (AP) andAzolla filiculoides (AF) by precipitation with ammonium sulfate to 20% of saturation. At high pH both lectins dissociate into inactive subunits (5000 mol wt) which reassociate into active aggregates (>500,000 mol wt) following concentration by ammonium sulfate precipitation or freezing and thawing. Although amino sugars inhibited hemagglutinating activity of both AP and AF lectins,d-fructose was inhibitory only to the AP lectin hemagglutinating activity, andd-galactose was slightly inhibitory to the AP lectin but not to the AF lectin. Both lectins exhibited specificity for freshly extracted cyanobionts from homologous fern species: AP lectin agglutinated cyanobiont filaments from AP, but not from AF; AF lectin agglutinated cyanobiont filaments from AF, but not AP. Neither lectin reacted with cultured cyanobionts from either fern species. Hemagglutinating titers were likewise reduced by adsorption of these lectins to homologous cyanobiont cells. This report provides strong suggestive evidence for specificity in this N-fixing symbiosis between aquatic fern and cyanobacterium.     

Drought-induced structural alterations at the mycobiont-photobiont interface in a range of foliose macrolichens

Summary Cryotechniques, such as low temperature scanning electron microscopy (LTSEM) and freeze-substitution for transmission electron microscopy (TEM), were applied to two cyanobacterial and three green algal macrolichens in order to locate free water and to visualize drought-induced structural alterations at the mycobiont—photobiont interface. The following species were examined:Peltigera canina/Nostoc punctiforme, Sticta sylvatica/Nostoc sp. (both Peltigerales),Parmelia sulcata/Trebouxia impressa, Hypogymnia physodes/Trebouxia sp. (both Lecanorales), andXanthoria parietina/Trebouxia arboricola (Teloschistales). In all species free water was confined to the symplast and the apoplast. No intercellular water reservoirs were found in the gas-filled thallus interior. Thalline fluctuations in water content reflect fluctuations in apoplastic and symplastic water. All the taxonomically diverse lichen photobionts have access to water and dissolved nutrients via the fungal apoplast only. Drought stress (i.e., water content 20%/dw and below) caused dramatic shrinkage and deformation in all cell types. At any level of hydration the fungal and algal protoplast maintained close contact with the cell wall. This applied to the cyanobacterial photobionts and their murein sacculus and gelatinous sheath too. Although the cytoplasm of both partners was strongly condensed in desiccated lichens the cellular membrane systems, usually negatively contrasted, were very well preserved. The significance of these data is discussed with regard to the functioning of the symbiotic relationship.     

Preservation of secondary fungal metabolites in herbarium lichen specimens

Fresh picked and herbarium thalli of Cladonia stellaris, C. rangiferina, Allocetraria nivalis, A. cucullata, Cetraria islandica, Peltigera canina, and Nephroma articum epigene lichens were studied using the immune-enzyme analysis. No big difference was observed in the contents of mycotoxin secondary metabolites, i.e., deoxynivalenol, diacetoxyscirpenol, zearalenone, alternariol, citrinin, sterigmatocystin, cyclopiazonic acid, mycophenolic acid, emodin, and PR-toxin. The discovery of these substances in the specimens preserved for several decades shows that lichens have an effective system of conservation of metabolic exchange products.     

Does the Reproductive Strategy Affect the Transmission and Genetic Diversity of Bionts in Cyanolichens? A Case Study Using Two Closely Related Species

Observed levels of population genetic diversity are often associated with differences in species dispersal and reproductive strategies. In symbiotic organisms, the genetic diversity level of each biont should also be highly influenced by biont transmission. In this study, we evaluated the influence of the reproductive strategies of cyanolichen species on the current levels of population genetic diversity of bionts. To eliminate any phylogenetic noise, we selected two closely related species within the genus Degelia, which only differ in their reproductive systems. We sampled all known populations of both species in central Spain and genotyped the fungal and cyanobacterial components of lichen samples using DNA sequences as molecular markers. We applied population genetics approaches to evaluate the genetic diversity and population genetic structure of the symbiotic components of both lichen species. Our results indicate that fungal and cyanobiont genetic diversity is highly influenced by the reproductive systems of lichen fungus. We detected higher bionts genetic diversity values in the sexual species Degelia plumbea. By contrast, the levels of fungal and cyanobiont genetic diversity in the asexual species Degelia atlantica were extremely low (almost clonal), and the species shows a high specificity towards its cyanobiont. Our results indicate that reproduction by vegetative propagules, in species of the genus Degelia, favors vertical transmission and clonality, which affects the species’ capacity for resources and competition, thereby limiting the species to restricted niches.     

Cytoplasmic contractions in growing fungal hyphae and their morphogenetic consequences

Video-enhanced light microscopy of the apical and subapical regions of growing hyphae of several fungal species revealed the existence of momentary synchronized motions of subcellular organelles. First discovered in a temperature-sensitive morphological mutant (ramosa-1) of Aspergillus niger, these seemingly spontaneous cytoplasmic contractions were also detected in wild-type hyphae of A. niger, Neurospora crassa, and Trichoderma atroviride. Cytoplasmic contractions in all fungi lasted about 1 s. Although the cytoplasm recovered its motility and appearance, the contraction usually led to drastic changes in Spitzenkörper (apical body) behavior and hyphal morphology, often both. Within 10 s after the contraction, the Spitzenkörper commonly became dislodged from its polar position; sometimes it disassembled into phase-dark and phase-light components; more commonly, it disappeared completely. Whether partial or complete, the dislocation of the Spitzenkörper was always accompanied by a sharp reduction or cessation of growth, and was usually followed by marked morphological changes that included bulbous hyphal tips, bulges in the hyphal profile, and formation of subapical and apical branches. The cytoplasmic contractions are vivid evidence that the most conspicuous cell organelles (membrane-bound) in living hyphae are interconnected via a contractile cytoskeletal network.     

Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan

Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.     

NITROGEN FIXATION RATE AND CHLOROPHYLL CONTENT OF THE LICHEN PELTIGERA CANINA EXPOSED TO SULFUR DIOXIDE

In general, the rate of nitrogen fixation decreased when the lichen Peltigera canina (L.) Willd. was exposed to sulfur dioxide gas at levels from 0.1 to 500 ppm; at 30 ppm, however, nitrogen fixation was stimulated. Chlorophyll content decreased as level of sulfur dioxide increased.     

Regulation of Dictyostelium morphogenesis by RapGAP3

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3⁻ cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3⁻ cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3⁻ cells may result in reduced directional movement of the mutant cells to the apex of the mound.     

Nitrogenase in free-living and symbiotic cyanobacteria: Immunoelectron microscopic localization

Abstract Nitrogenase (Fe-protein) was localized in the free-living cyanobacterium Anabaena cylindrica and in the cyanobionts of Cycas revoluta and Peltigera aphthosa , using colloidal gold as an immunocytochemical marker. The Fe-protein was found to be evenly distributed throughout the heterocyst cytoplasma in A. cylindrica and in both the cyanobionts, including multiple heterocysts of the C. revoluta cyanobiont. No label was observed in the vegetative cells of free-living A. cylindrica or of the cyanobionts, although the cyanobionts apparently live under microaerobic conditions.