JET‐SUSPENDED,CALCITE‐BALLASTED CYANOBACTERIAL WATERWARTS IN A DESERT SPRING1

We describe a population of colonial cyanobacteria (waterwarts) that develops as the dominant primary producer in a bottom‐fed, O₂‐poor, warm spring in the Cuatro Ciénegas karstic region of the Mexican Chihuahuan Desert. The centimeter‐sized waterwarts were suspended within a central, conically shaped, 6‐m deep well by upwelling waters. Waterwarts were built by an Aphanothece‐like unicellular cyanobacterium and supported a community of epiphytic filamentous cyanobacteria and diatoms but were free of heterotrophic bacteria inside. Sequence analysis of 16S rRNA genes revealed that this cyanobacterium is only distantly related to several strains of other unicellular cyanobacteria (Merismopedia, Cyanothece, Microcystis). Waterwarts contained orderly arrangements of mineral crystallites, made up of microcrystalline low‐magnesium calcite with high levels of strontium and sulfur. Waterwarts were 95.9% (v/v) glycan, 2.8% cells, and 1.3% mineral grains and had a buoyant density of 1.034 kg·L^?1. An analysis of the hydrological properties of the spring well and the waterwarts demonstrated that both large colony size and the presence of controlled amounts of mineral ballast are required to prevent the population from being washed out of the well. The unique hydrological characteristics of the spring have likely selected for both traits. The mechanisms by which controlled nucleation of extracellular calcite is achieved remain to be explored.     

Comparative studies on physiological responses to phosphorus in two phenotypes of bloom-forming <Emphasis Type="Italic">Microcystis</Emphasis>

Toxic Microcystis blooms frequently occur in eutrophic water bodies and exist in the form of colonial and unicellular cells. In order to understand the mechanism of Microcystis dominance in freshwater bodies, the physiological and biochemical responses of unicellular (4 strains) and colonial (4 strains) Microcystis strains to phosphorus (P) were comparatively studied. The two phenotype strains exhibit physiological differences mainly in terms of their response to low P concentrations. The growth of four unicellular and one small colonial Microcystis strain was significantly inhibited at a P concentration of 0.2 mg l⁻¹; however, that of the large colonial Microcystis strains was not inhibited. The results of phosphate uptake experiments conducted using P-starved cells indicated that the colonial strains had a higher affinity for low levels of P. The unicellular strains consumed more P than the colonial strains. Alkaline phosphatase activity in the unicellular strains was significantly induced by low P concentrations. Under P-limited conditions, the oxygen evolution rate, F _v/F _m, and ETR _max were lower in unicellular strains than in colonial strains. These findings may shed light on the mechanism by which colonial Microcystis strains have an advantage with regard to dominance and persistence in fluctuating P conditions. Handling editor: L. Naselli-Flores     

Measuring carbon and N2 fixation in field populations of colonial and free‐living unicellular cyanobacteria using nanometer‐scale secondary ion mass spectrometry1

Unicellular cyanobacteria are now recognized as important to the marine N and C cycles in open ocean gyres, yet there are few direct in situ measurements of their activities. Using a high‐resolution nanometer scale secondary ion mass spectrometer (nanoSIMS), single cell N₂ and C fixation rates were estimated for unicellular cyanobacteria resembling N₂ fixer Crocosphaera watsonii. Crocosphaera watsonii‐like cells were observed in the subtropical North Pacific gyre (22°45′ N, 158°0′ W) as 2 different phenotypes: colonial and free‐living. Colonies containing 3–242 cells per colony were observed and cell density in colonies increased with incubation time. Estimated C fixation rates were similarly high in both phenotypes and unexpectedly for unicellular cyanobacteria 85% of the colonial cells incubated during midday were also enriched in ¹⁵N above natural abundance. Highest ¹⁵N enrichment and N₂ fixation rates were found in cells incubated overnight where up to 64% of the total daily fixed N in the upper surface waters was attributed to both phenotypes. The colonial cells retained newly fixed C in a sulfur‐rich matrix surrounding the cells and often cells of both phenotypes possessed areas (<1 nm) of enriched ¹⁵N and ¹³C resembling storage granules. The nanoSIMS imaging of the colonial cells also showed evidence for a division of N₂ and C fixation activity across the colony where few individual cells (<34%) in a given colony were enriched in both ¹⁵N and ¹³C above the colony average. Our results provide new insights into the ecophysiology of unicellular cyanobacteria.     

Milk Kefir: Ultrastructure,Antimicrobial Activity and Efficacy on Aflatoxin B1 Production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

The association of kefir microbiota was observed by electron microscopic examination. Scanning electron microscopic (SEM) observations revealed that kefir grain surface is very rough and the inner portions had scattered irregular holes on its surface. The interior of the grain comprised fibrillar materials which were interpreted as protein, lipid and a soluble polysaccharide, the kefiran complex that surrounds yeast and bacteria in the grain. Yeast was observed more clearly than bacteria on the outer portion of the grain. Transmission electron microscopic (TEM) observations of kefir revealed that the grain comprised a mixed culture of yeast and bacteria growing in close association with each other. Microbiota is dominated by budded and long-flattened yeast cells growing together with lactobacilli and lactococci bacteria. Bacterial cells with rounded ends were also observed in this mixed culture. Kefir grains, kefir suspensions, and kefiran were tested for antimicrobial activities against several bacterial and fungal species. The highest activity was obtained against Streptococcus faecalis KR6 and Fusarium graminearum CZ1. Growth of Aspergillus flavus AH3 producing for aflatoxin B1 for 10 days in broth medium supplemented with varying concentrations of kefir filtrate (%, v/v) showed that sporulation was completely inhibited at the higher concentrations of kefir filtrate (7–10%, v/v). The average values of both mycelial dry weights and aflatoxin B1 were completely inhibited at 10% (v/v). This is the first in vitro study about the antifungal characteristics of kefir against filamentous fungi which was manifested by applying its inhibitory effect on the productivity of aflatoxin B1 by A. flavus AH3.     

Investigation of saturated and aromatic hydrocarbon resistance mechanisms in Pseudomonas aeruginosa IBBML1

Pseudomonas aeruginosa IBB_ML1, isolated from Poeni petroleum sludge, was able to tolerate and degrade both saturated (n-hexane, n-heptane, n-hexadecane, cyclohexane) and aromatic (benzene, ethylbenzene, propylbenzene, xylene isomers, styrene) hydrocarbons. Molecular studies have revealed that the high hydrocarbon resistance of Pseudomonas aeruginosa IBB_ML1 could be due to the action of members of the HAE1 (hydrophobe/amphiphile efflux 1) family of transporters. It is further possible that additional mechanisms may account for the tolerance of Pseudomonas aeruginosa IBB_ML1 to hydrocarbons, and a combination of short-term and long-term mechanisms may act together in the adaptation of Pseudomonas aeruginosa IBB_ML1 cells to saturated and aromatic hydrocarbons. β-galactosidase activity measurements revealed that there was significant induction of the lacZ gene in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of either 5% and 10% (v/v) saturated or aromatic hydrocarbons, compared with control (cells incubated without hydrocarbons). Rhodamine 6G accumulation in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of 5% and 10% (v/v) saturated hydrocarbons was higher than rhodamine 6G accumulation in cells grown in the presence of 5% and 10% (v/v) aromatic hydrocarbons. The study of cellular and molecular modifications to Pseudomonas aeruginosa IBB_ML1 induced by 5% and 10% (v/v) saturated and aromatic hydrocarbons revealed a complex response of bacterial cells to the presence of different hydrophobic substrates in the culture medium.     

The Establishment of the Hydra-Chlorella Symbiosis: Recognition of Potential Algal Symbionts

The epithelial cells lining the gastric cavity of the freshwater hydra, Hydra viridis, harbor unicellular algal symbionts of the genus Cblorella. It has long been known that these hydra cells can readily phagocytose algal cells and will sequester those algae that have the potential to form a symbiotic association. In this paper the evidence is discussed for when and how recognition of potential symbionts by hydra cells occurs, i.e. during phagocytosis or during the subsequent intracellular events leading to sequestration of algal symbionts.     

Antagonistic association of the chlorellavorus bacterium (“Bdellovibrio” chlorellavorus) withChlorella vulgaris

The chlorellavorus bacterium (Bdellovibrio chlorellavorus Gromov and Mamkaeva 1972) attaches to (but does not enter) cells of the unicellular green alga,Chlorella, which is killed and the cell contents of which are digested. The bacterium is pleomorphic (vibrios 0.3 μm wide; cocci 0.6 μm wide), and it has a Gram-negative cell wall structure pili, and a single, unsheathed, polar flagellum. Division may occur only in bacterial cells attached to algal cells, an attachment mediated by a pad (245×36 nm) of unknown composition. Bacterial growth occurs only in the presence of liveChlorella cells, and not on various bacteriological culture media, killedChlorella cells, 4 strains ofPrototheca, or 24 strains of Gram-negative bacteria. The chlorellavorus bacterium may not require algal protein synthesis, since the bacterium grows on algae in the presence of cycloheximide (30 μg/ml). Although the DNA base composition of the chlorellavorus bacterium (50 mol % G+C) is in the same range asBdellovibrio bacteriovorus, its ultrastructure, developmental cycle, host range, and format of its intermicrobial association all distinguish the chlorellavorus bacterium from members of the genusBdellovibrio.     

Role of Cell Hydrophobicity on Colony Formation in Microcystis (Cyanobacteria)

Colony formation is highly import ant for the competitive advantage of the cyanobacterium Microcystis over other phytoplankton species. The laboratory‐grown colonial Microcystis strains isolated from Lake Taihu (China) maintained colonial forms under the low light condition (10 μE m^–2 s^–1). The cell surface hydrophobicities of the Microcystis colonies were measured by cyanobacterial adherence to xylene in comparison with unicellular Microcystis strains. The cells of the tested colonial strains were all hydrophobic, while the cells of the tested unicellular strains were all hydrophilic. Incubation under the higher light condition (75 μE m^–2 s^–1) leaded to the significant decrease in the cell hydrophobicities of the colonial Microcystis and the transition from colonial forms to unicellular forms. These findings indicated that the cell hydrophobicity of Microcystis may play a role in cell‐cell adherence and colony formation. Phosphate‐limitation, nitrate‐limitation and pH did not affect cell hydrophobicities of colonial Microcystis. Treatment with proteolytic enzymes had no effect on the cell hydrophobicity, indicating that cell surface proteins did not contribute to high cell hydrophobicity. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Some Factors Influencing the in vitro Infectivity and Replication of Encephalitozoon cuniculi*

SYNOPSIS. Rabbit Encephalitozoon cuniculi were propagated in vitro using rabbit choroid plexus (RCP) cells. The organisms reached maximum titer and numbers by 15 days. The source and in vitro passage level of RCP cells moderately influenced the sensitivity of the cells to infection. Cells less than 1 week old were significantly less sensitive than older cells. A moderate increase in infectivity for RCP cells was demonstrated with increasing organism passage level in vitro. Rabbit E. cuniculi were not affected by penicillin-streptomycin or gentamicin in the culture medium. The organism survived more than 9 days in buffer at 37 C and least 24 days at 4 and 20 C. Storage at -70 C or in liquid nitrogen was successful for at least 6 months. Encephalitozoon cuniculi survived 60 but not 120 min at 56 C. They were killed after 10 min of autoclaving and by 2% (v/v) Lysol, 10% (v/v) formalin and 70% (v/v) ethyl alcohol. The organisms survived at least 24 h at pH 9 or pH 4 and were not affected by sonication, freezing and thawing, or distilled water but lost significant infectivity after 24 h in CsCl or 40% (w/v) sucrose.     

Growth inhibition of unicellular and colonial Microcystis strains (Cyanophyceae) by compounds isolated from rice (Oryza sativa) hulls

The algicidal effects of crude and pure rice hull extracts on the growth of Microcystis aeruginosa were investigated using cultured unicellular and colonial strains. Upon treatment with rice hull crude extract (RHE), growth inhibition of unicellular M. aeruginosa was much higher than that of colonial M. aeruginosa. However, purified compounds from the crude extract, β-sitosterol-β-d-glucoside and dicyclohexanyl orizane, powerfully inhibited the growth of colonial M. aeruginosa cells. At the same concentrations, the two compounds were almost equipotent (66% and 80% growth inhibition for colonial M. aeruginosa, respectively; P < 0.05). As rice hulls are readily obtainable, and as extracts show high algicidal activity (targeting colonial algae rather than unicellular organisms) at low concentrations, the results suggest that some pure compounds extracted from rice hulls, such as β-sitosterol-β-d-glucoside and dicyclohexanyl orizane, may serve as environmentally friendly agents for controlling the growth of toxic colonial M. aeruginosa in eutrophic waters.     

Influence of carbon source on growth,biochemical composition and pigmentation of Ankistrodesmus convolutus

The unicellular chlorophyte Ankistrodesmus convolutus Corda was grown in the light using inorganic medium (Bold's Basal Medium, BBM) and BBM enriched with 0.1% w/v of glucose, sodium acetate, sodium citrate or sodium bicarbonate. Glucose supported the highest specific growth rate (μ = 0.93 d^-1) and gave the highest biomass (453 mg dry weight L^-1) at the time of harvest. Of four glucose concentrations (0.05, 0.1, 0.25, 0.5% w/v), best growth was attained at 0.1% w/v. At 0.5% w/v glucose, the cells had high carbohydrates but low lipids and proteins. The relative amounts of 16:0, 18:0, 18:1 and 18:2 increased at the expense of 18:3(n-3) in the carbon-supplemented cultures and at glucose concentrations higher than 0.1% w/v. Cultures grown on glucose had less chlorophyll and carotenoid contents than cultures grown on other carbon sources. Chlorophyll and carotenoid contents decreased with increasing glucose concentrations in the medium.     

Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm^-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm^-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - RuBP carboxylase D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39     

Tumour-expressed tissue factor inhibits cellular cytotoxicity

Aims: The association between tissue factor (TF) expression and increased rate of tumour metastasis is well established. In this study, we have examined the hypothesis that the expression of TF by disseminated tumour cells confers protection against immune recognition and cytotoxicity. Materials and methods: A hybrid EGFP-TF protein was expressed in HT29 colon carcinoma and K562 lymphoblast cell lines. To assess the cytotoxic activity against tumour cells over-expressing TF, a novel method was used, based on the direct measurement of fluorescently labelled HT29 or K562 target cells. Results: Upon challenge with peripheral blood mononuclear cells (PBMC), tumour cells expressing TF partially evaded cellular cytotoxicity (Δ=15–40% reduction in cytotoxicity). Moreover, the influence of TF was not primarily dependent on its procoagulant function, although the inclusion of 20% (v/v) plasma did lower the rate of cytotoxicity against untransfected cells. However, expression of a truncated form of TF, devoid of the cytoplasmic domain, did not mediate any degree of inhibition of cytotoxicity, suggesting that the protective function of TF is principally due to this domain. Conclusions: We conclude that TF can promote immune evasion in tumour cells expressing this protein leading to increased survival and therefore metastatic rate in such cells.     

Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Hair,encoding a single C2H2 zinc‐finger protein,regulates multicellular trichome formation in tomato

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome‐wide association study (GWAS) on type‐I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map‐based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc‐finger protein. Transgenic experiments showed that hair‐absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type‐I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein–protein interaction between them was further detected through pull‐down and yeast two‐hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.     

Differences between adrenomedullary adrenaline and noradrenaline cells: quantitative electron-microscopic evaluation of their differential cellular association with supporting cells

Quantitative differences in cellular association of adrenomedullary chromaffin cells with other types of cells, mainly supporting cells, were studied. Adrenaline (A) and noradrenaline (NA) cells were compared. Electron micrographs (12000 x) of profiles of A and NA cells, bordering against other types of cells, were used for quantitative evaluation. Supporting cells constituted the majority of the non-chromaffin cell types. Occurrence frequencies of chromaffin cells contiguous with other types of cells were: (1) higher for A cells (68.9%, 199/289) than for NA cells (11.0%, 34/309) in case of small contact regions (²-test: P<0.001) and (2) higher for NA cells (68.3%, 211/309) than for A cells (9.7%, 28/289) in case of extended contact regions (P<0.001). In conclusion, the extent of cellular association with supporting cells was remarkably lower in A cells than in NA cells. Such an arrangement is likely to be appropriate for the extensive, homogeneous control and amplified response characteristic of A cells, and for the close range, complex control and more diverse responses characteristic of NA cells.Supported in part by The Karoji Memorial Fund for Medical Research in Hirosaki University, JapanThe authors, TK, TS and GT, wish to dedicate their part of this work to Dr. W.B. Quay on the occasion of his 65th birthday     

DnaK and GroEL chaperones are recruited to the Bacillus subtilis membrane after short-term ethanol stress

Aims: To find out membrane tolerance strategy to ethanol in Bacillus subtilis that possesses a powerful system of protection against environmental stresses. Methods and Results: Cytoplasmic membranes of B. subtilis were severely affected by even short‐term exposure to 3% (v/v) ethanol: the growth rate and membrane protein synthesis were markedly reduced, and no adaptive alterations in phospholipids were detected. Simultaneously, steady‐state DPH fluorescence anisotropy (r_ss) showed that the membrane rigidity increased substantially. Analysis of the membrane phosphoproteome using in vitro labelling with [γ‐³²P]ATP revealed the association of DnaK and GroEL chaperones with membrane, indicating a stress induction process. Upon a long‐term 3% (v/v) ethanol stress, the cell growth accelerated slightly and the composition of polar head groups and fatty acids of membrane phospholipids underwent an extensive reconstruction. Correspondingly, membrane fluidity turned back to the original r_ss values of the control cells. Conclusions: In B. subtilis, the adaptive response to short‐term ethanol stress comprises the recruitment of molecular chaperones on the impaired membrane structure; consequently, the phospholipid synthesis is restored and membrane fluidity adapts properly to the continuing ethanol stress. Significance and Impact of the Study: These findings underline the role of membrane lipids in establishing tolerance towards ethanol and also suggest the contribution of molecular chaperones to the membrane and cell recovery.     

Production of (R)-3-pentyn-2-ol through stereoinversion of racemic 3-pentyn-2-ol by Nocardia fusca AKU 2123

Wet cells of Nocardia fusca AKU 2123 are good catalysts for the production of (R)-3-pentyn-2-ol (PYOH) from (RS)-PYOH through a stereoinversion reaction. Under optimal conditions (350 mM potassium phosphate buffer, pH 8.0, 30% (w/v) wet cells, 0.12% NADPH, 10% glucose, and 30 U/ml glucose dehydrogenase) (R)-PYOH of high optical purity (98.7% e.e.) was produced from 2% (v/v) (RS)-PYOH with a yield of 70.4% by 140 h incubation. Received: 22 January 1999 / Received revision: 23 April 1999 / Accepted: 1 May 1999     

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE1

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form‐genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form‐genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form‐genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form‐genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form‐genus Cyanothece were observed to be in a close relationship with our isolates.