EFFECT OF LYSOZYME (MURAMIDASE) ON MARINE AND FRESHWATER BLUE-GREEN ALGAE1

Marine blue-green algae, Lyngbya Lagerheimii, Microcoleus chthonoplastes, Plectonema terebrans, Agmenellum quadruplicatum, and freshwater blue-green algae, Anacystis nidulans, Anabaena variabilis, Nostoc muscorura, and Oscillatoria sp. treated with lysozyme (muramidase) formed spheroplasts but not protoplasts. The time needed for spheroplast induction varied with the species. Approximate internal osmotic pressures of the blue-green algae were determined. Marine algae generally had a higher osmotic pressure than freshwater algae.     

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF TEN POLAR AND NEAR‐POLAR STRAINS WITHIN THE OSCILLATORIALES (CYANOBACTERIA)1

An approximately 1400‐bp region of the 16S rRNA gene was sequenced for 10 polar or near‐polar strains putatively placed in the Oscillatorialean genera Oscillatoria, Phormidium, and Lyngbya obtained from the University of Toronto Culture Collection to assess phylogenetic relationships. The strains were also examined for thylakoid structure and cell division type with TEM as well as traditional morphology with LM. Phylogenetic trees constructed using parsimony, distance, and maximum likelihood methods were similar in topology. If the original epithets applied to the sequenced strains (both polar and those from GenBank) were used, it was clear that taxa were not monophyletic. However, using the revised taxonomic system of Anagnostidis and Komárek, we were able to reassign these strains to their current correct taxa (species, genus, and family). When these assignments were made, it was determined that the molecular sequence data analyses were congruent with morphology and ultrastructure. Nine of the polar strains were found to be new species, and eight were described as such: Arthronema gygaxiana Casamatta et Johansen sp. nov., Pseudanabaena tremula Johansen et Casamatta sp. nov., Leptolyngbya angustata Casamatta et Johansen sp. nov., Phormidium lumbricale Johansen et Casamatta sp. nov., Microcoleus glaciei Johansen et Casamatta sp. nov., Microcoleus rushforthii Johansen et Casamatta sp. nov., Microcoleus antarcticus Casamatta et Johansen sp. nov., Microcoleus acremannii Casamatta et Johansen sp. nov. Some genera (Leptolyngbya and Microcoleus) were clearly not monophyletic and require future revision.     

Nitrogen fixation by blue-green algal communities in the intertidal zone of the lagoon of Aldabra Atoll

Summary All eight types of blue-green algal community sampled from the intertidal sediments of the lagoon of Aldabra showed detectable rates of acetylene reduction, and six of them showed at least some examples of rates considered by previous authors to be rapid. When the reduction rates are related to the chlorophyll a contents of the communities, the maximum rates recorded come in the following order: Hyella balani, Scytonema sp., Hyella balani-Schizothrix sp., Calothrix crustacea, Rivularia sp., Microcoleus chthonoplastes, Hyella balani-purple sulphur bacteria, Pleurocapsa-Chroococcus. With the exception of the mixed Hyella balani-purple sulphur bacterial community, the rate of acetylene reduction was in all experiments greater in the light than in the dark, the difference being significant (P<0.05) in the majority of cases. The rates were similar in all experiments whether incubation was carried out with sea or brackish water. It seems probable that nitrogen fixation by blue-green algal communities makes an important addition to the lagoon ecosystem, and that not only heterocystous, but also some non-heterocystous, species are involved. Among the latter, the data for Hyella balani and Microcoleus chthonoplastes are especially convincing.     

Isolation and Characterization of Cyanobacteria from Microbial Mats of the Ebro Delta,Spain

Isolation and identification of several strains of cyanobacteria from microbial mats of the Ebro Delta, Spain, are described. A series of tenfold dilutions was the first step of isolation. Self-isolation techniques, which use one or several physiological characteristics of a cyanobacterium, were applied in some cases to obtain enrichment cultures. Twelve filamentous strains were isolated and stored in axenic culture. As only a few cyanobacterial species can be frozen and revived without any cryoprotective agent, preservation of isolated strains was assayed with several cryoprotective solutions. Methanol and glycerol were not suitable as cryoprotective agents for most of the isolates. Dimethyl sulfoxide (DMSO) was apparently the best cryoprotector. A new method, which used a filter paper as a growing substratum that later could be directly stored at −80°C, was successfully used. A morphological study of each strain under light and electron microscopy was made to classify them. All isolated strains belong to phylum BX, Class 1, subsection III of the Bergey's manual of systematic bacteriology, 2nd ed., vol. 1. Most genera are included in the LPP group as Lyngbya aestuarii and Microcoleus chthonoplastes. Received: 30 April 2002 / Accepted: 31 May 2002     

Soil stabilization by a prokaryotic desert crust: Implications for Precambrian land biota

A cyanophyte dominated mat, desert crust, forms the ground cover in areas measuring hundreds of square meters in Utah and smaller patches in Colorado. The algal mat shows stromatolitic features such as sediment trapping and accretion, a convoluted surface, and polygonal cracking. Sand and clay particles are immobilized by a dense network of filaments of the two dominating cyanophyte species,Microcoleus vaginatus andM. chthonoplastes, which secrete sheaths to which particles adhere. These microorganisms can tolerate long periods of desiccation and are capable of instant reactivation and migration following wetting. Migration occurs in two events: 1. immediately following wetting of dry mat, trichomes are mechanically expelled from the sheath as it swells during rehydration, and 2. subsequently, trichomes begin a self-propelled gliding motility which is accompanied by further production of sheath. The maximum distance traveled on solid agar by trichomes ofMicrocoleus vaginatus during a 12 hour period of light was 4.8 cm. This corresponds to approximately 500 times the length of the fastest trichome, and provides a measure of the potential for spreading of the mat in nature via the motility of the trichomes.Dehydration resistence of the sheath modifies the extracellular environment of the trichomes and enables their transition to dormancy. Following prolonged wetting and evaporative drying of the mat in the laboratory, a smooth wafer-like crust is formed by the sheaths ofMicrocleus trichomes that have migrated to the surface. Calcium carbonate precipitates among the algal filaments under experimental conditions, indicating a potential for mat lithification and fossilization in the form of a caliche crust. It is suggested that limestones containing tubular microfossils may, in part, be of such an origin.The formation of mature Precambrian soils may be attributable to soil accretion, stabilization, and biogenic modification by blue-green algal land mats similar to desert crust.     

Algal nitrogen fixation on the lava field of Heimaey,Iceland

Summary Nitrogen fixation (C₂H₂ reduction) by blue-green algae occurring on the juvenile lava field of Heimaey, Iceland was examined both in the laboratory (potential at 20° C and 39° C) and in the field, three and a half years after the volcanic eruption.Already at this early stage of colonization representatives of unicellular and filamentous heterocystous and non-heterocystous blue-green algae were commonly observed. The predominating algae were Nostoc sp. (20° C) and Schizothrix sp. — Microcoleus chthonoplastes, (39° C), the former often in association with the protonemata-rhizoids of moss plants.The potential for nitrogen fixation was recorded at an average rate of 109.2 (20° C) and 138.1 (39° C) ng N g^-1 h^-1 in soil collected from localities randomly distributed over the lava field.Tests for nitrogen fixation performed in situ revealed significant fixation activities in all the eleven localities subject to examination. The activities ranged from 2.8 to 63.4 (mean 21.5) ng N g^-1 h^-1 and 1.9 to 17.7 (mean 7.9) ng N cm^-2 h^-1.All the nitrogen fixation data noted imply that blue-green algae contribute a substantial part of the nitrogen input to the lava. Further, it was found that material incubated under micro-aerophilic conditions exhibited considerably enhanced nitrogenase activity.The role of nitrogen-fixing blue-green algae in general and Nostoc muscorum in particular in being suitable as pioneering organisms preparing the bare lava for ingress of other plants is also discussed.     

Benthic cyanobacterial bloom impacts the reefs of South Florida (Broward County, USA)

Benthic cyanobacteria of the genus Lyngbya can form prominent mats and blooms in tropical and subtropical coral reef and seagrass habitats worldwide. A Lyngbya bloom on the reef tract offshore of Broward County, Florida, was first noted in 2002, and although it is seasonally variable in its distribution and abundance, it has persisted and spread over the past 3 years. In this study, the most abundant species of Lyngbya found in the blooms have been identified and compared to other species of Lyngbya by morphological and molecular methods. The most common species of Lyngbya is consistent with the properties of Lyngbya confervoides C. Agardh. The 16S ribosomal DNA sequence shares 88–92% identity with other known Lyngbya sequences, suggesting that this bloom consists primarily of a new, previously unsequenced species of Lyngbya. The second most common Lyngbya in the bloom is consistent with Lyngbya polychroa. This persistent bloom is a concern because it smothers octocorals and other invertebrates and negatively impacts these southeastern Florida reefs.     

Carbonic Anhydrase Activity of Alkalophilic Cyanobacteria from Soda Lakes

The activity and intracellular partition of carbonic anhydrase (CA) were studied in alkalophilic cyanobacteria, an inhabitant of soda lakes at pH 9–10. In the homogenates of Rhabdoderma lineare, Rhabdoderma sp., and Microcoleus chthonoplastes, high activity of CA was found, similar to that in eukaryotic microalgae. The activity of CA calculated on the basis of chlorophyll and protein was higher for the soluble (sCA) than for membrane (mCA) protein fraction. Intact cells of all cyanobacteria under investigation also showed CA activity that implies the presence of extracellular form(s). The extracellular CA in benthic M. chthonoplastes was localized, at least partly, in a vast glycocalix (gCA) as shown by Western blotting and the measurement of enzyme activity in the isolated glycocalix preparations. Probing gCA from M. chthonoplastes with the antibodies against thylakoid CA from Chlamydomonas reinhardtii (Cah3) demonstrated that gCA belongs to the -type of enzyme and has the structure identical to that of Cah3. The extracellular CA of M. chthonoplastes manifested the maximum activity at pH 7 and 10, but not at pH 6 and 9. An increase in medium pH from 7.2 to 9.6 resulted only in slight alkalization of the cytoplasm in R. lineare, from 7.1 to 7.5. It follows that true alkalophils can maintain the pH inside the cell at the near-neutral level in spite of high pH (10.2) level in the cultural medium.     

pH and Eh on Aldabra atoll 1. comparison of marine and reshwater environments

An account is given of the range of pH and Eh in marine and freshwater aquatic sites on one atoll (Aldabra, Indian Ocean). There is a very wide range in values of pH and Eh for both marine and freshwater environments, the range for pH in general being wider in freshwater ones and Eh in marine ones. Photosynthetic microbial communities on lagoon sediments may be arranged in an approximate order of their association with sediments of particular Eh values. These are, from high to low: Scytonema sp., pennate diatoms, Schizothrix sp., Microcoleus chthonoplastes, Hyella balani, purple phototrophic bacteria.     

Grazer interactions with four species of Lyngbya in southeast Florida

Blooms of the toxic cyanobacteria Lyngbya spp. have been increasing in frequency and severity in southeast Florida in recent years. Lyngbya produces many active secondary metabolites which often act as feeding deterrents to generalist herbivores, possibly increasing the longevity of these nuisance blooms. Whilst diverse arrays of small invertebrate consumers are often found in association with Lyngbya, little is known of their grazing selectivity among species of Lyngbya. This study examines the feeding preference of grazers for four local Lyngbya species (Lyngbya majuscula, Lyngbya confervoides, Lyngbya polychroa and Lyngbya spp.). Stylocheilus striatus and Haminoea antillarum showed no dietary selectivity between L. polychroa, L. majuscula and Lyngbya spp. in multiple choice feeding assays, whereas Bulla striata showed a distinct preference for L. polychroa (P < 0.001). To determine whether preference might be related to species-specific secondary metabolites, L. majuscula, L. confervoides and L. polychroa non-polar and polar extracts were incorporated into artificial diets and offered to a range of mesograzers. No significant difference was noted in feeding stimulation or deterrence amongst extracts and the controls for any of the grazers. When fed a monospecific diet of L. polychroa, S. striatus consumed more (P < 0.001) and attained a higher daily biomass (P = 0.004) than S. striatus fed L. confervoides. As L. polychroa and L. confervoides often co-exist on local coral reefs and yield dense numbers of S. striatus, host switching to a more palatable species of Lyngbya may have important implications regarding top-down control of local blooms leading to proliferation of one species and decimation of another. S. striatus fed a diet of L. polychroa consumed more (P = 0.003), had a greater increase in body mass (P = 0.020) and higher conversion efficiency (P = 0.005) than those fed L. confervoides regardless of host origin. Possible explanations for host switching between species of Lyngbya related to morphology, toxicity and nutrient requirements for growth are discussed.     

Significance of the biomineralizing microniche in a lyngbya (cyanobacterium) travertine

Observations of the spatial arrangement of Lyngbya (Phormidium) incrustatum tri‐chomes in a travertine crust allowed prediction of the frequency of four types of microniche favorable for photosynthetically induced calcification. The most frequent type of microniche occurred at “cross‐over”; points where two trichomes made contact at a single point. Such points occurred very frequently (～6000/mm² travertine). Microniches more favorable to photocalcification consist of groups of three or more trichomes lying side by side, enclosing narrow tubes of interstitial water. These configurations were far less frequent and their effects would be negligible in Lyngbya travertines. In the calcified cyanobacteria Rivularia and Schizothrix such configurations may be much more common. The existence of such microniches demonstrated that regions favorable for photocalcification occur in travertine‐encrusted cyanobac‐terial mats and may initiate carbonate precipitation. However, the bulk of the carbonate precipitated in such travertines is known to be formed through inorganic processes.     

Sacrificial cell death and trichome breakage in an oscillatoriacean blue-green alga: the role of murein

Summary Trichomes of Microcoleus vaginatus, a motile blue-green alga of the family Oscillatoriaceae, were studied by light and electron microscopy in an effort to determine the sites of trichome breakage during production of hormogonia.According to the evidence presented herein, transcellular breakage of trichomes is the only mechanism of hormogonium production in M. vaginatus. Tearing of the murein sacculus appears to be necessary and sufficient for transcellular breakage to ensue. As Fuhs and earlier investigators have correctly claimed, this process always involves the death of the cell whose wall is torn.When trichomes of M. vaginatus break across cells to produce hormogonia, the murein sacculus usually tears along a circumferential set of junctional pores. This particular mechanism of trichome breakage is not universal among members of the family Oscillatoriaceae.This report is based on a thesis submitted in partial fulfillment of the requirements for a Ph. D. degree in Biology at Harvard University.     

Morphological, Chemical, and Genetic Diversity of Tropical Marine Cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales)

Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium Lyngbya majuscula, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections. We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of L. majuscula Gomont, Lyngbya spp., and Symploca spp. from Guam and the Republic of Palau. The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra. Secondary metabolites were analyzed by two-dimensional thin-layer chromatography. Each collection possessed a distinct cellular morphology that readily distinguished Lyngbya spp. from Symploca spp. Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of L. majuscula. A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of Lyngbya and Symploca but yielded a basal polytomy for Lyngbya. Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of L. majuscula showed 0 to 1.3% divergence. Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of Lyngbya and Symploca as well as the monophyly of Lyngbya bouillonii and L. majuscula, genetic divergence was not correlated with chemical and morphological differences. These data suggest that 16S rDNA sequence analyses do not predict chemical variability among Lyngbya species. Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among Lyngbya species.     

Carbonic Anhydrase of the Alkaliphilic Cyanobacterium Microcoleus chthonoplastes

The activity of carbonic anhydrase (CA) was studied in different cell fractions of the alkaliphilic cyanobacterium Microcoleus chthonoplastes. The activity of this enzyme was found in the soluble and membrane protein fractions, as well as in intact cells and in a thick glycocalyx layer enclosing the cyanobacterium cells. The localization of CA in glycocalyx of M. chthonoplastes was shown by western blot analysis and by immunoelectron microscopy studies with antibodies to the thylakoid CA from Chlamydomonas reinhardtii (Cah3). At least one of the CA forms occurring in M. chthonoplastes CA was shown to be an -type enzyme. A possible mechanism of the involvement of the glycocalyx CA in calcification of cyanobacteria is discussed.     

PHYLOGENY AND GENETIC VARIANCE IN TERRESTRIAL MICROCOLEUS (CYANOPHYCEAE) SPECIES BASED ON SEQUENCE ANALYSIS OF THE 16S rRNA GENE AND ASSOCIATED 16S–23S ITS REGION1

Thirty‐one strains of Microcoleus were isolated from desert soils in the United States. Although all these taxa fit the broad definition of Microcoleus vaginatus (Vaucher) Gomont in common usage by soil algal researchers, sequence data for the 16S rRNA gene and 16S–23S internal transcribed spacer (ITS) region indicated that more than one species was represented. Combined sequence and morphological data revealed the presence of two morphologically similar taxa, M. vaginatus and Microcoleus steenstrupii Boye‐Petersen. The rRNA operons of these taxa were sufficiently dissimilar that we suspect the two taxa belong in separate genera. The M. vaginatus clade was most similar to published sequences from Trichodesmium and Arthrospira. When 16S sequences from the isolates we identified as M. steenstrupii were compared with published sequences, our strains grouped with M. chthonoplastes (Mertens) Zanardini ex Gomont and may have closest relatives among several genera in the Phormidiaceae. Organization within the 16S–23S ITS regions was variable between the two taxa. Microcoleus vaginatus had either two tRNA genes (tRNA^Ile and tRNA^Ala) or a fragment of the tRNA^Ile gene in its ITS regions, whereas M. steenstrupii had rRNA operons with either the tRNA^Ile gene or no tRNA genes in its ITS regions. Microcoleus vaginatus showed no subspecific variation within the combined morphological and molecular characterizations, with 16S similarities ranging from 97.1% to 99.9%. Microcoleus steenstrupii showed considerable genetic variability, with 16S similarities ranging from 91.5% to 99.4%. In phylogenetic analyses, we found that this variability was not congruent with geography, and we suspect that our M. steenstrupii strains represent several cryptic species.     

EPIPHYTES ON CLADOPHORA GLOMERATA IN THE GREAT LAKES AND ST. LAWRENCE SEAWAY WITH PARTICULAR REFERENCE TO THE RED ALGA CHROODACTYLON RAMOSUM (= ASTEROCYTIS SMARGDINA)1

The range of Cladophora glomerata² along the east and north shorelines of the Great Lakes and St. Lawrence Seaway extends form just east of Montreal to Thunder Bay on Lake Superior. However, it does not occur at sites sampled in Georgian Bay, the North Channel or eastern Lake Superior. The dominant epiphytes on Cladophora throughout this range are the blue-green algae, particularly Lyngbya diguetii, L. epiphytica and Chamaesiphon incrustans, which account for 53 to 90% of the cell density. The diatoms Cocconeis pediculus and Rhoicosphenia curvata contribute to much of the remaining density. The red alga Chroodactylon ramosum is a minor component of Cladophora epiphyton but is widespread in Lakes Ontario, Erie and Huron. Filament morphology, cell diameters and lengths of Chroodactylon are quite similar to those of marine forms. In addition, this alga has been reported to be quite tolerant of a wide range of salinities. Chroodactylon has been found in 33 freshwater sites throughout North America and 30 of these are from the Great Lakes or its drainage basin. Therefore, it appears possible that Chroodactylon, like Bangia atropurpurea, has originated in the Great Lakes by a migration from the Atlantic Ocean.     

COLEOFASCICULUS GEN. NOV. (CYANOBACTERIA): MORPHOLOGICAL AND MOLECULAR CRITERIA FOR REVISION OF THE GENUS MICROCOLEUS GOMONT1

Species currently classified within the cyanobacterial genus Microcoleus were determined to fall into two distinct clades in a 16S rDNA phylogeny, one containing taxa within the Oscillatoriaceae, the other containing taxa within the Phormidiaceae. The two lineages were confirmed in an analysis of the 16S–23S internal transcribed spacer (ITS) region sequences and secondary structures. The type species for Microcoleus is M. vaginatus Gomont, and this taxon belongs in the Oscillatoriaceae. Consequently, Microcoleus taxa in the Phormidiaceae must be placed in separate genera, and we propose the new genus Coleofasciculus to contain marine taxa currently placed in Microcoleus. The type species for Coleofasciculus is the well‐studied and widespread marine mat‐forming species Microcoleus chthonoplastes (Mert.) Zanardini ex Gomont. Other characters separating the two families include type of cell division and thylakoid structure.