SIMPLE METHOD FOR RNA PREPARATION FROM CYANOBACTERIA1

A simple and rapid method is presented for the preparation of RNA from various cyanobacteria. Unlike other methods that require a lysis solution, lysozymes, or proteinase K, the proposed method, called the bead–phenol–chloroform (BPC) method, uses silica/zirconia beads, phenol, and chloroform to break the cells and extract RNA more efficiently. Experiments confirm that the BPC method can successfully isolate total RNA from various cyanobacterial strains without DNA contamination, and the extracted RNA samples have a relatively high purity, concentration, and yield. Furthermore, the BPC method is more rapid, simple, and economical when compared with previously reported methods.     

ISOLATION AND CHARACTERIZATION OF CYANOBACTERIA POSSESSING ANTIMICROBIAL ACTIVITY FROM THE SUNDARBANS,THE WORLD’S LARGEST TIDAL MANGROVE FOREST1

Eight obligately halophilic, euryhaline cyanobacteria from intertidal soil were isolated in artificial seawater nutrients III (ASN‐III) medium. Antimicrobial activity, 16S rRNA gene sequences, phenotypic characters as well as growth and antibiosis in response to variable salinity, temperature, phosphate concentration, and pH were studied. Minimum inhibitory concentrations (MIC) of the extracts against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and multiple drug‐resistant clinical isolates ranged between 0.25 and 0.5 mg · mL⁻¹. Cytotoxicity tests showed 73%–84% human colon adenocarcinoma (HT‐29/C1) cell survival at MIC values, indicating that the extracts were nontoxic. Morphologically, six cyanobacteria were assigned to the Lyngbya‐Phormidium‐Plectonema (LPP) group B, and one each was assigned to Oscillatoria and Synechocystis genera. Glycerol, mannitol, and starch supported better photoheterotrophic growth than simpler mono‐ and disaccharides. No heterocyst formation was observed when grown under nitrogen‐starved conditions. All isolates survived 7‰ salinity, grew at minimum 32‰ salinity, and showed sustained growth throughout 32‰–82‰ salinity but matured poorly in freshwater medium supplemented with 30.0 g · L⁻¹ NaCl. Antimicrobial production occurred only at 32‰ salinity. While four of the eight isolates demonstrated sustained growth at 37°C, maximum antimicrobial activity was obtained at 25°C. All strains showed maximum growth and antimicrobial elaboration at 0.04 g · L⁻¹ phosphate. All isolates thrived at pH 9.5; six grew at pH 4.5, though antimicrobial production occurred only at pH 7.5. Molecular phylogenetic analysis based on 16S rRNA gene sequences of the filamentous isolates validated the previous taxonomic affiliations established on morphological characteristics. This is the first study of antimicrobial‐producing halophilic cyanobacteria from the mangroves.     

A SIMPLE AND RAPID TECHNIQUE FOR THE ISOLATION OF DNA FROM MICROALGAE1

A simple method for the purification of PCR‐ quality DNA from microalgae is presented. This method uses the detergent dodecyltrimethylammonium bromide coupled with cell breakage by agitation in the presence of glass beads and chloroform. A final purification step involves a commercial cartridge system. The procedure requires only about 1–2 mL of algal culture and can be completed in about 20 min. DNA suitable for PCR has been obtained from several algal lineages using this method, including numerous green algae and stramenopiles.     

INDUCTION OF VACUOLATED SPHEROPLASTS AND ISOLATION OF VACUOLES IN CYANOBACTERIA1

There is still a great deal of debate about whether cyanobacteria contain vacuoles. This might in part reflect our limited ability to isolate vacuoles. We found and isolated vacuoles from different cyanobacteria during spheroplast preparation. Lysozyme treatment induced two kinds of spheroplasts: vacuolated spheroplasts and nonvacuolated spheroplasts. Upon breakage in distilled water, vacuolated spheroplasts released transparent, spherical, and colorless vacuoles with diameters ranging from 2.3 to 16 μm. Large vacuoles could be generated by fusion of two or three small vacuoles. Additionally, large vacuoles also could engulf small ones or other cellular bodies. The isolated vacuoles could tolerate hypotonic condition, and some could be drawn into a thread. Nonvacuolated spheroplasts released few vacuoles after breaking apart. This successful confirmation and isolation of vacuoles will allow studies of the origin and function of cyanobacterial vacuoles.     

IN-SITU MORPHOLOGY AND OCCURRENCE OF EUCARYOTIC PHOTOTROPHS OF BACTERIAL SIZE IN THE PICOPLANKTON OF ESTUARINE AND OCEANIC WATERS1

Concentrates of the picoplankton (0.2–2.0 μm) sized fraction from the euphotic zone of estuarine and oceanic waters were examined by transmission electron microscopy. In addition to the numerous phototrophic procaryotes (chroococcoid cyanobacteria) previously reported, small phototrophic eucaryotes were observed in 20 of 25 samples examined. Micromonas pusilla (Butcher) Manton and Parks, a 1 × 1.5 μm flagellate, was abundant in estuarine samples in summer. Similar sized cells of non-flagellated chlorophytes, either Nannochloris Naumann or Chlorella Beijerinck, were observed sporadically in many samples. The most ubiquitous microalga was a scaled, non-flagellated prasinophyte that occurred at 9 of 15 different locations on 15 of 20 sampling dates in water samples from Iceland to the Caribbean Sea, This tiny alga (0.5 to 1.0 μm in diam.) is probably the smallest known photo-trophic eucaryote and has not heretofore been described. Enrichment cultures using conventional techniques on several cruises yielded only the Chlorella-type of green alga, as well as numerous isolates of unicellular chroococcoid cyanobacteria.     

蜜环菌遗传测定的单孢分离和培养方法

从平板中直接分离担子菌萌发孢子的单孢分离方法,主要利用自制的毛细管和显徽玻璃针等简单工具操作。该法简便快速,抗污染,获得单孢率高。同时报道一种改进型的担子菌互交不育性测定用培养基MEJA,与国内外目前常用的SR和MEA培养基相比,增加一项判断标准,使互交不育性和互交可育性的反应更加清晰可靠。     

PRESENCE OF PHYCOERYTHRIN IN TWO STRAINS OF PROCHLOROCOCCUS (CYANOBACTERIA) ISOLATED FROM THE SUBTROPICAL NORTH PACIFIC OCEAN

Prochlorococcus is a ubiquitous marine oxyphotobacterium characterized by the presence of DV-chl a and b . In addition, the type strain Prochlorococcus marinus Chisholm et al. CCMP 1375 (or SS120), an isolate from the Sargasso Sea, contains low levels of an unusual phycoerythrin. Until now, it has been unclear if phycoerythrin occurs randomly within this systematic group and if the molecular characteristics of this phycoerythrin are restricted to this single strain. Here, we show that two additional Prochlorococcus strains from the Pacific Ocean also contain similar low levels of phycoerythrin. DNA sequence and phylogenetic analyses demonstrated that this phycoerythrin is very similar to the phycoerythrin of P. marinus SS120 and differs from the classic cyanobacterial phycoerythrins. In contrast, a third isolate from the Arabian Sea lacks phycoerythrin. Based on the DV-chl b:a ratio and 16S rRNA sequence data, we classify the two Pacific phycoerythrin-containing isolates as low-light-adapted strains and the Arabian Sea isolate as a high-light-adapted strain. Thus, we provide further evidence to link the physiology of an individual genotype and the presence or absence of functional phycoerythrin genes within the genus Prochlorococcus .     

RELATIONSHIP BETWEEN THE NUMBER OF DIVIDING AND NONDIVIDING CELLS OF CYANOBACTERIA IN NORTH ATLANTIC PICOPLANKTON1

In the North Atlantic over a wide geographic region that includes various oceanic regimes and a temperature range from 10 to 22° C, an increase in the number of nondividing Synechococcus cells (X) was generally accompanied by a greater-than-proportional increase in the number of dividing cells (Y). As a result, the fraction of dividing cells (FDC = Y · (Y + X)^?1) was positively related to population size (Y + X). Recognizing that FDC is generally greater in a rapidly growing population than in a slowly growing one, our empirical finding implies a positive correlation between specific growth rate and standing stock for Synechococcus. One notable exception occurred during winter (T < 5°C) in a eutrophic coastal embayment when a decrease in cell abundance was not matched by a decrease in FDC.     

DISTRIBUTION OF DIATOMS IN THE PLANKTON OF YAQUINA ESTUARY,OREGON1

The distributional patterns of diatoms in the plankton of the Yaqnina Estuary, Oregon, were, investigated and related to selected climatic and hydrographic factors. Distribution was strongly influenced by seasonal patterns of rainfall resulting in the introduction of a large volume of fresh water into the estuary during fall and winter. Plankton assemblages in spring, summer and fall had fewer diatom species and exhibited a more rapid rate of change in species composition than in winter. Winter assemblages were further characterized by many pennate diatoms, apparently dislodged from the benthos during periods of high freshwater discharge and silt loads. A statistical measure of community difference indicated an increase in taxonomic homogeneity among assemblages throughout the estuary with the onset of the rainy season in late fall and a gradual transition to a more heterogeneous system again during late spring. Canonical correlation ordered 20 prominent diatom taxa along the salinity gradient and identified possible relationships among certain taxa and selected environmental variables, namely visible light energy and temperature. Redundancy in the species data given the environmental data was only 40%, emphasizing the difficulty in demonstrating a quantitive relationship between plankton dynamics in the field and concurrent measurements of chemical and physical variables.     

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.     

滤膜法测定水中大肠菌群简便方法的研究

本研究方法为ＩＳＯ９３０８－１规定的滤膜法测定水中大肠菌群的方法,该方法操作简便只需分离和证实两步实验即可得出结果;分离培养基上的阴阳性菌落颜色分明,易于分辩;所用培养基不含致癌物质。本研究方法的准确度高,回收率的均值为９４７％,批内变异系数小于１０％,检出限为５００ｍｌ水样可检出１个ＣＦＵ,检出率是传统方法的３１倍。本研究还探讨了分离培养基的ｐＨ值对菌落颜色的影响。本研究内容经查新在国内尚无报道。     

IDENTIFICATION OF THE CYTOKININ ISOPENTENYLADENINE IN A STRAIN OF ARTHRONEMA AFRICANUM (CYANOBACTERIA)

Isopentenyladenine was isolated from a strain of the cyanobacterium Arthronema africanum (Schwabe et Simons) Komárek et Lukavský using a combination of biological and chemophysical techniques, including the soybean callus bioassay, cation exchange resin chromatography, paper chromatography, HPLC, and GC-MS. Positive identification of the major biologically active compound was achieved.     

GEL PURIFICATION OF RED ALGAL GENOMIC DNA: AN INEXPENSIVE AND RAPID METHOD FOR THE ISOLATION OF POLYMERASE CHAIN REACTION-FRIENDLY DNA1

A simplified approach for the extraction of DNA from red algae in presented. Procedures are simple and fast, requiring a minimum of reagents and apparatus. The method involves an initial lysis step followed by an optional phenol/chloroform extraction. The final gel-purification step removes polymerase chain reaction-inhibiting polysaccharides from the DNA preparation. DNA is extracted as easily from dried algae as it is from snap-frozen, fresh material, thus greatly facilitating the collection and transport of algal samples.