ERSITY IV  N S Assam University Journal of Science & Technology : ISSN 0975-2773 U I L M C

H A

S A Biological and Environmental Sciences

R S A Vol. 7 Number I 101-113, 2011

PCR Based Molecular Characterization of with Special Emphasis on Non-Heterocystous Filamentous Cyanobacteria Gunapati Oinam, O.N.Tiwari* and G.D. Sharma** Microbial Bioprospecting Laboratory Institute of Bioresources and Sustainable Development Takyelpat, Imphal-795001, Manipur, INDIA ** Department of Life science, Assam University, Silchar, Assam *Corresponding author email : [email protected]

Abstract Cyanobacteria have an ancient history dating almost 3.5 billion years and diversified extensively to become one of the most successful and ecologically significant organisms on earth, with respect to longevity of lineage and impact on earth’s early environment. Despite the availability of various monograph based on morphological and ecological variants, the identification and classification of cyanobacteria remain a difficult and confusing task leading to uncertain identifications. Therefore, molecular approaches based on PCR techniques and DNA fingerprinting have been adopted for taxonomical studies. Molecular markers such as RAPD, RFLP and AFLP are used for the PCR techniques. Keywords: Non-heterocystous, cyanobacteria, molecular characterization.

Introduction Cyanobacteria are an ancient group of prokaryotic aided by the presence of exopolysaccharides such microorganisms exhibiting the general as mucilage and / or a firm sheath. The presence characteristics of gram-negative . They or absence of a heterocyst is an important feature are unique among the prokaryotes in possessing separating genera. However, gas vacuoles, the capacity of oxygenic photosynthesis. structures that aid buoyancy, are found in species Cyanobacteria are a morphologically diverse group of many different genera. Rather surprisingly, the ranging from unicellular to colonial and starting point for the morphologically less complex filamentous forms. Taxonomically, cyanobacteria forms is much earlier, although there was of are grouped into unicellular forms that divide by course little understanding of their diversity at that binary or multiple fission and filamentous forms time. A number of “floras” summarizing the that are non heterocystous or differentiate known species of cyanobacteria in particular heterocysts in non branching or branching regions have been published during the 20th filaments. Unicells cyanobacteria may divide in century. Several of these provide a lot of one, two or three planes. Some unicells dividing in information about species occurring elsewhere in one plane show asymmetric division; in the world. A review by Castenholz (1992) and Chamaesiphon the smaller cells glide down to the sister volume (Bryant, 1994) to the present the base of the larger cell and repeats of this one make clear how important is an understanding process eventually give rise to a colony of many of cyanobacterial molecular biology not just as an cells. The resulting colonies can often be seen as aid to , but for interpreting ecological brownish spots on submerged rocks. Maintenance phenomena in general. Suboptimal light and of the colonial structure in many of these forms is nutrient conditions result in a number of responses

- 101 - PCR Based Molecular Characterization of...... that strongly influence the physiology of the cell. shallow marine habitats, calcareous sediments The responses can be striking or subtle and deposit continuously on the surface of the mat, subsequent changes take place rapidly or very and the cyanophytes move up through the slowly. Cyanobacteria tend to show resistance to sediments during the day (Gebelein, 1969). In multiple environmental stresses, and it is probable some cases the layers are diurnal, resulting from that the response pathways to the different stimuli phototrophic movement of the cyanophytes. overlap. The ability of some cyanobacteria to Stromatolitic formations became less and less withstand extremes of UV radiation and frequent and diverse after the Cambrian, and the desiccation is aided by their capacity for efficient most common explanation for this is that the DNA repair. It will be of interest to understand if, evolution in the Cambrian of microbe eating and how, such repair plays a role in other responses metazoans made it impossible for the large to environmental stimuli such as nutrient limitation cyanophytic mats to build up. and cyanophage infection. Ecology of cyanophytes Origin and early evolution of cyanobacteria The cyanophytes as a group adapted, in the The evolution of the cyanophytes: precambrian were found in a wide variety of habitats but today their distribution is more The evidence for the time and manner of origin restricted, in many habitats they must compete of cyanophytes is still meager, and most of it with eukaryotes. There is only one known type subject to varying interpretation. The cyanophytes of habitats on earth today where cyanophytes are arose between 3-4 billion years ago (Schopf, 1970) the exclusive O -evolving photosynthetic probably from a photosynthetic bacterium. After 2 organisms, and that is in thermal springs (Brock, the discovery of gliding photosynthetic bacterium 1970). However, even in the thermal springs Chloroflexis (Pierson and Castenholz, 1971) is a cyanophytes are not always successful. In acid reasonable candidate as a forerunner of the springs with pH less than 4.0, cyanophytes are cyanophytes. Cyanophytes were probably never found, irrespective of temperature. In responsible for a marked increase in the O 2 neutral and alkaline thermal springs, cyanophytes concentration of the atmosphere during the are the exclusive O -evolving photosynthetic Precambrian. Another reason why a cyanophytes 2 organisms at temperatures above 55-60°C, and is the best candidate for the first O -evolving 2 are far in the dominance at temperatures down photosynthetic organism is that at least some to about 40°C. The upper temperature limit for cyanophytes are able to grow an aerobically, a cyanophytes is about 72-73°C and at temperatures property not normally associated with eukaryotic above this only non photosynthetic bacteria are algae (Stewart and Pearson, 1970). If the bulk of present. In other habitats, even in those in which the atmospheric O is indeed of biogenic origin, 2 cyanophytes are widely held to be successful, they the organisms first involved in O formation would 2 are never exclusive and often not dominant. The of course have developed in an anaerobic fixation of nitrogen is found under aerobic environment and would have had to be able to conditions in heterocystous cyanophytes and under live an aerobically. anaerobic condition also in some non Stromatolites and the evolution of the heterocystous forms (Stewart and Lex, 1970). The cyanophytes ability of a variety of cyanophytes to fix nitrogen Stromatolites are rocks consisting of many layers, in natural aquatic systems is well established. The most commonly developing from the mats of presence in cyanophytes of phycobilins as cyanophytes. The rocks may be either siliceous accessory photosynthetic pigments may confer a or calcareous, although the later are most considerable ecological advantage to them under common. The layers probably build as a result of conditions of low light intensity. Perhaps one of sediment trapping and carbonate precipitation by the most important factors controlling cyanophytes unicellular and filamentous cyanophytes. In development may be the ability of this group to

- 102 - PCR Based Molecular Characterization of......

grow under conditions of low O2 concentration. It circumstances probably occurs within a thin sheath might be in such a niche that the first eukaryotic casing or film of mucus which adheres to the alga could have developed. In such an alga, with substrate and is shed and left behind as a ‘mucous’ its photosynthetic apparatus concentrated in a trail. When measured microscopically, the velocity chloroplast, the cytoplasm surrounding the of movement without reversals varies with species chloroplast could have provided an effective pH and environmental conditions. In the buffer which would have served to keep H+ from rates of well over 2µm sec-1 are the very acid sensitive chlorophyll molecule. The command and range up to 11µm sec-1 (Halfen and cyanophytes, with its photosynthetic apparatus in Castenholz, 1971). There is no consistent the cell periphery, may be less well adapted for correlation between trichome diameter of different keeping H+ from chlorophyll. species and the velocity of gliding. Other non photosynthetic, filamentous prokaryotes and Mechanism of movements in cyanobacteria myxobacteria glide at rates usually less than 1.0 Motility in cyanobacteria is gliding in contrast to µm sec-1. In contrast, flagellated rod and spirilla swimming is movement in contact with a solid or bacteria commonly attain speeds of 20-30 µm sec- semi solid substrate, but without a visible change 1 and vibrios apparently reach speeds as high as in the shape of the organism. A filament may have 50-200 µm sec-1. When measurement are made considerable passive flexibility, but there does not on a macro scale, such as distance covered on an seem to be any possibility of steering. The agar surface over a period of a few hours, less relatively slow progress of gliding is accompanied than maximum rates are usually recorded. This is in some species by a rotation of the trichome along presumably because of the more or less frequent its axis which is either right handed or left handed. stops and reversals of individual trichome together This means simply that any point on the surface with the devious path followed by some on the of the trichome will trace either a right handed or surface of the substrate. left handed helix, but whichever it is, it will be The mechanism of motility: One of the earliest species specific and non interchangeable. In liquid suggested mechanism for gliding motility (Doetsch microscope mounts with or without a cover slip, and Hageage, 1968) has been supported and free ends of trichome are seen to jerk back and modified by Halfen and Castenholz (1971) that forth, wave to and fro, or oscillate by tracing a the is that numerous microfibrils cone. It appears that all of these oscillations are strung uninterrupted in helices around the exterior passive responses to the rotation of the trichome portion of the trichome are the propulsive when any part of it is able to bear against a organelles which move the chain of cell against substrate and glide. Consequently, the whole an elastic sheath or other suitable substrate by a trichome rotates and, since they are seldom rapid succession of unidirectional waves with straight threads they will appear to be swinging submicroscopic amplitude. The origin of wave their free ends around. Rotation and accompanying propagation may shift from one pole to the other, oscillations are the usual features of gliding reversing the direction of wave movement and of trichome of most oscillatoriaceae, but not of most gliding. Unicellular species or those with trichome hormogonia and gliding trichome of heterocystous of more disjointed cell could have a similar system groups. but continuous for only a single cell length. Species Gliding behaviour: Gliding is movement in that do not rotate as they glide should have contact with a solid or semi solid surface without longitudinal microfibrils which are not helically flagella like propulsive organs. In most cases it is strung. a smooth, non jerking movement resembling Orientated movements: Phototaxis is only somewhat the progress of a snail. In many cases tactic response of cyanobacteria that has received the movement is continuous in one direction for much attention. Photo topotaxis is the orientation prolonged periods; in others frequent or regular and movement towards (+) or away from (-) the reversals occur. Gliding of trichome in almost all

- 103 - PCR Based Molecular Characterization of...... incident light. Photo phobotaxis is the reversal of after weakening the cell wall by penicillin direction of movement following a sudden change treatment. The intact vesicles were then purified from high to low light intensity (+) or from low to by a process of repeated, centrifugally high light (-) (Nultsch 1965). The general term accelerated flotation, followed by combination of phototaxis can mean any of these responses. It is membrane filtration, molecular sieving and liquid often difficult to distinguish topotactic and polymer partitioning (Buckland and Walsby, 1971). phobotactic reactions. Functions of gas vesicles: Of the three Gas vacuoles in cyanobacteria functions that gas vacuoles might fulfill that of storing gas is no longer tenable. The gas vesicle Fine structure of gas vesicles: The fine membranes are far too permeable to retain any structure of gas vacuoles has been investigated in particular gas. It has been pointed out that the a total of eleven species from the genera two other functions, providing light shielding and Microcystis, Oscillatoria, Trichodesmium, buoyancy do not depend on the presence of gas Anabaena, Aphanizomenon, Nostoc and in the vesicles, but rather on these structures being Gloeotrichia and in each case they have been kept free of liquid or solids. found to comprise gas vesicles of the same basis morphology originally described by Bowen and Light shielding: The peculiar optical quantities Jensen (1965). The vesicles have the form of of gas vacuoles have suggested (Waaland et al., hollow, cylindrical tubes with conical caps at each 1971) that they might provide light shielding for end. The tubes are possibly of uniform diameter, cells exposed to high light at water surfaces. Fuhs usually given as being about 70 nm the reported (1968) has pointed out that in light microscope extreme being 65 nm for Oscillatoria rubescens gas vacuoles are poor amplitude objects on and 75-85 nm for Trichodesmium erythraeum. account of their low absorptivity, but the large Various synonyms have been used to describe gas refractive index difference between their contents vesicles in cyanobacteria but it is suggested that and the surrounding cytoplasm makes them the original term should be retained. Gas cylinder excellent phase objects. The path length of light aptly described these structures in cyanobacteria passing through a gas vacuole may differ from but is inapplicable to homologous structure of that passing through the neighbouring cytoplasm different shape found in various bacteria (Walsby by more than ¼ wavelength and result in ‘false 1972). In many species the gas vacuole are reversal’ so that the vacuole looks brighter instead irregular in form and distributed throughout the of darker than the background. With high cell but in some algae they have a characteristic resolution optics, gas vacuoles appear almost shape and position. invisible under bright field illumination but with low numerical apertures they are characterized by Chemical composition of gas vesicles: diffraction fringes. Chemical analyses have now been made on gas vesicles isolated from cyanobacteria and the Light intensity in cyanobacteria: Within certain results have confirmed the idea, suggested by limits, an inverse correlation between light intensity electron microscopy, that the gas vesicles and photo pigment content is general among membrane is fundamentally different from typical photosynthetic organisms. As might be expected, unit membrane. Quantitative preparations of highly the ratio of phycocyanin to chlorophyll can vary purified intact vesicles have been obtained by widely in response to simultaneous change in light Walsby and Buckland (1969) from the filamentous intensity, temperature and carbon dioxide partial alga Anabaena flosaquae. To avoid collapsing pressure. In chromatically adapting strains, growth the gas vesicles, the cell were lysed by method in green light stimulates the synthesis of the red which did not involve exposing the preparations coloured phycoerythrin whereas when growth to pressure, the filamentous alga being disrupted occurs in red light it is the blue protein, by osmotic shrinkage in hypertonic sucrose phycocyanin, which is the dominant biliprotein. solution and the unicellular form by osmotic shock After a brief ‘red’ suspension immediately,

- 104 - PCR Based Molecular Characterization of...... phycocyanin alone is synthesized whereas after a size indicates a probable artificiality in the grouping brief ‘green’ irradiation, phycoerythrin and , a conclusion further supported by phycocyanin are both synthesizes. These effects the degree of disparity in some sequences of 800- are repeatedly reversible; the last 6 irradiation 900 continuous nucleotides of 16S rRNA from 11 determines the pattern of biliprotein synthesis in strains of 7 genera of this group (Giovannoni et the dark. al., 1988). The triviality of some generic distinctions used here should also be emphasized. Modern approaches and concepts on Often only one characteristic is used, a oscillatoriales characteristic that may be the result of a slight It includes all filamentous cyanobacteria that difference in genetic code. In the “Geitlerian” undergo binary fission in a single plane and that system, generic distinction in the oscillatoriales is produce “vegetative” cells only and heterocysts based primarily on the diversity of sheaths or their and akinetes do not occur. The terminal cell of absence. Although still used as a characteristic in some species may be distinctly shaped but is the present system, knowledge of physiology, apparently still capable of photosynthesis. biochemistry, and nucleotide base sequence Sometimes the terminal cell is tapered and with a homologies will eventually determine degree of cap or calyptra, but in a few forms the taper may relatedness. Members of oscillatoriales occur in include several sub terminal cells as well. The an enormous diversity of habitats: freshwater and terminal cell in some may never divide and marine, both as plankton, mats, and periphyton. trichomes may be flexible or semi rigid. In some Terrestrial crusts, mats, and turfs are also cases, the entire trichome is wound into a loose or common. The order oscillatoriales is characterized tight spiral; in others, only terminal portions of the into eight genera namely, Spirulina, Arthrospira, trichome may be openly spiraled. An apparent Oscillatoria, Lyngbya, Pseudanabaena, sheath may be present, but even species without Starria, Crinalium and Microcoleus. an easily visible sheath leave behind at least a very Spirulina thin, gossamer sheath when moving by gliding. When short fragments of a few cells separate from Filamentous organisms that divide exclusively by the remainder of the trichome near the free, open binary fission and in one plane but that grow in end of a sheath, these free trichomes may glide the form of a tight to nearly tight coiled right or out, eventually forming new sheaths. Although left handed helix. The cross walls are thin and trichomes without apparent sheaths also fragment, are invisible or nearly so with light microscopy. a separable, migrating, hormogonial phase is The trichome does not truly rotate but moves along difficult to distinguish, since all lengths of trichome the outer surface of the helix. Free ends not in are generally motile Movement forward or contact with substrate may oscillate. Variations backward may or may not be accompanied by a in the tightness of the trichome helix occur in both right or left handed rotation of the trichome. Spirulina and Arthrospira (Jeeji Bai and Fragmentation of trichomes occurs in some forms Seshadri, 1980; Hindak, 1985). The mole % G + where a cell loses much of its contents and dies. C of the DNA of the reference strain (PCC 6313) In some cases, there appears to be an orderly is 54, and the genome size is 1.53 x 109 daltons. sacrificial death of these cells (necridial cells) The members of this genus have a worldwide which determines the sites of trichome breakage distribution in freshwater, marine, and brackish (Ciferri, 1983). waters. The range in mole % G + C of the DNA is large Arthrospira (40-67) and the range of genome sizes is also great Filamentous organisms that divide exclusively by (2.14-5.19 x 109 daltons), but all sizes are generally binary fission and in one plane. The entire trichome less than those in the Nostocales or is arranged as an open helix in which transverse Stigonematales (Herdman et al., 1979b). The walls may be seen via light microscopy. Cells are range in DNA base composition and in genome

- 105 - PCR Based Molecular Characterization of...... generally shorter than broad to quadrate but are barrel shaped. The trichomes are usually straight occasionally elongate. Constrictions at cross walls and quite frequently short, consisting of only a may be present or absent. The mole % G + C of few to several cells. Single, detached cells are the DNA of the reference strain (PCC 7345) is frequent in most culture populations. Gliding 44.3. The life cycle of Arthrospira in laboratory motility occurs in trichomes and unicells, probably culture involves the breaking up of trichomes at without rotation. The mol% G + C of the DNA the sites of a necridium at intervals of every 4-5 ranges from about 42 to 47 (Guglielmi and Cohen- cells. Bazire, 1984) and the genome size is from 2.14 to 9 Oscillatoria 5.19 x 10 daltons. Microcoleus Filamentous organism that divide exclusively by binary fission and in one plane. The trichomes are These Oscillatorian type trichomes are straight to loosely sinuous near apices; flexible or characterized by the presence of a common, semirigid. Transverse septa are generally visible homogeneous sheath. It surrounds several parallel under light microscopy. Constrictions may or may trichomes that are often spirally and tightly not occur at cross walls, but the total indentation interwoven. never exceeds one eighth of the trichome Importance of cyanobacterial germplasm diameter. During fission the cytoplasmic membrane invaginates, with a thinner A germplasm is a collection of genetic resources peptidoglycan layer separating the new membranes for an organism. Genetic material, specially its of the daughter cells. Usually, sheaths are nearly specific molecular and chemical constitution that invisible, gossamer tubes that are shed as flattened comprises the physical basis of the inherited trials when the trichome moves on solid substrates. qualities of an organism and that is transmitted Occasionally, more visible sheath may build up on from one generation to another. There are several some trichomes, particularly during periods of reasons that could justify a collecting mission: the immobility in liquid culture (Chang, 1977). Several species is in danger of extinction or genetic species show “chromatic adaptation” (Tandeau erosion, users have expressed a need at the de Marsac, 1977). Some species, almost black in national or international level, and the diversity is colour, contain abundant C-PE and c-phycocyanin. missing from existing ex situ collections. The more diversity is conserved and made available Lyngbya for future use, the better the chances of meeting Filamentous organism that share the entire range tomorrow’s needs. of cellular types with Oscillatoria but which Conventional/ traditional methods for produce a distinct and persistent sheath. The systematics sheath may be thin but can be seen with phase contrast optics, particularly when it extends beyond Traditionally, the classification of cyanobacteria the terminal cell of the trichome. The trichome has been based on morphological characters such diameters range from 1ìm to about 80 ìm. The as trichome width, cell size, division planes, shape mol% G + C of the DNA is 43.4 and the genome and arrangement, pigmentation and the presence size is about 4.58 x 109 daltons. of characters such as gas vacuoles and a sheath (Baker, 1992; Komarek and Anagnostidis, 1989). Pseudanabaena The importance of structural and developmental Filamentous organism that divide exclusively by characters for classification of both field and binary fission and in one plane and that has cultured material makes it essential for conspicuous constrictions at the cross-walls; in researchers to isolate and purify the specimen of most strains, constriction cuts into about half or interest under optimal cultural conditions and more of the diameter of the trichome. Cells are provide it with a taxonomic identity. Many longer than broad to iso diametric and are often cyanobacteria are conspicuous in nature and

- 106 - PCR Based Molecular Characterization of...... collection of samples is therefore greatly modifications of previously published formulae and facilitated. some are derived from analysis of water in the native habitat and ecological considerations. Direct Observation Enrichment media This is most suitable for samples that contain good number of organisms. Rice field floodwater Most of the defined culture media are of known carrying suspended soil particles are filtered on chemical composition and are suitable for a membrane filters and the population is smeared particular community and can be used for on the slide and observed under microscope. For enriching additional algal groups by adding a variety proper enumeration of small cells of cyanobacteria, of components like lake or sea water, soil extracts microscopy as well as epifluorescence microscopy and other special substance. Such enrichment is also used to distinguish between cyanobacteria media stimulate diverse nutritional groups of and small sized bacteria. This method is based on species and have advantage of supporting the the chlorophyll-a fluorescence of algae that growth of large number of algal species. distinguishes them from bacteria. Combination of Plating techniques excitation and suppression filters may be used to differentiate between phycocyanin and In this method, selection of medium and its phycoerythrin (Hawes and Davey, 1989). Since concentration is very important. Care should be cultured samples give better performance, there given to pH and temperature of the culture is a need to improve it for field application. procedure because some cyanobacteria prefer to grow above 30°C. Best comparisons can be made Isolation and purification by counting the colony forming units (CFUs). Many cyanobacteria present in axenic culture can Purification be isolated by the liquid enrichment technique, which imposed a positives selection on those a. Repeated liquid subculture: This technique members of the population most of them to has been successfully used when a natural proliferate in the medium and under the culture collection is particularly rich in specific conditions (light, temperature) provided. cyanobacteria. Direct isolation b. Fragmentation: Homogenization of filaments with a glass homogenizer for 5-10 minutes The field samples are examined under the allows short filaments of 4-8 cells long to be microscope immediately on arrival in the laboratory obtained. Individual colonies can be obtained to evaluate the composition of cyanobacterial when suspension is streaked on agar plates species. Depending on the consistency of the crude containing suitable medium. material, the sample for examination is placed on c. Antibiotics: These can be used to kill certain the slide with the platinum loop or pasteur pipette. contaminants which cannot be removed by Once the sample has been thoroughly examined other means. and its composition recorded, aliquot are transferred directly with proper precautions, to d. Ultra violet radiation: This method has been solid media. After streaking the deposited crude widely used to obtain some strains including material on solid media, the plates are examined those frequently used culture of under binocular microscope in order to isolate cyanobacteria. cyanobacterial forms without inoculation. e. Higher temperature incubation: Thermophilic forms can be isolated by Culture media enrichment at about 40°C. For enumeration of algae in the soil, the following Maintenance procedure is adopted. There are many recipes for media for cultivation of cyanobacteria under All strains are maintained by sub culturing in liquid laboratory conditions. Most of them are the media and on agar slants. Some strains including - 107 - PCR Based Molecular Characterization of...... those frequently used in the inoculation of paddy Immobilization fields are maintained on suitable carriers. Strains Immobilization can be carried out either by physical properties may change during repetitive sub means such as adsorption or entrapment of the culturing after long term cultivation under cells in a gel or foam matrix, or by chemical laboratory conditions. Therefore, following methods such as covalent binding. The methods can be adopted for the conservation of advantages of immobilization are, stabilization of cyanobacteria. the catalytic activity resulting in increased product Frequency of transfer formation, ease of its separation from the medium and re-use of catalysts for extended periods of Cultures are transferred at different intervals time. depending on the maintenance conditions and species. Unicellular and filamentous non motile Biochemical and physiological may be transferred once every three to six months. characterization Flagellated species required more frequent Photosynthesis, N fixation, NH excretion, transfers. 2 3 pigment profile, carbohydrates, total soluble Preservation proteins and N-assimilatory enzymes like nitrogenase, nitrate reductase and glutamine The primary purpose of preserving cultures is to synthetase activity plays key role for maintain cyanobacterial population in a viable state characterization. for considerably longer period. During preservation, all the physiological processes of an organism are Measurement of pigments considerably slowed down, without affecting This is useful in quantifying surface growth viability. A preserved culture can be reactivated dominated by cyanobacteria (Davey, 1988; whenever desired by providing suitable growth Whitton and Roger, 1989) but less suited for sparse conditions. For preservation, most algae do well populations spread over the upper part of the soil at less temperature (15-20°C), with few column. The organisms can be frozen also to avoid exceptions where algae prefer higher degradation of pigments. The use of television temperatures for survival. image analysis with epifluorescence microscopy Lyophilization for cyanobacteria and other microorganisms has also been described (Wynn- Williams, 1990). Freeze drying or lyophilization is a technique where Satellite sensor images offer a completely drying is achieved by avoiding the liquid state, different approach to quantifying surface crusts. through sublimation. In this process, the ice crystals This method is similar to those used for studying forms in cells are directly converted into water semiarid vegetation index (Karnieli et al., 1996). vapours through evacuation at low temperature. Since the lyophilized culture are hygroscopic and Molecular approaches for classification show loss in viability when exposed to molecular Limitations of phenotypic characters have oxygen, they should be preserved in evacuated highlighted the requirement for more reliable sealed glass ampules such sample show very little methods and promoted molecular approaches in metabolic activity and remain viable for many cyanobacterial taxonomy, including DNA base years. composition (Kaneko et al., 2001), DNA Cryopreservation hybridizations (Kondo et al., 2000), gene sequencing (Nubel et al., 1997) and PCR Preservation of an organism at low temperature fingerprinting (Rasmussen and Svenning, 1998; in a deep freeze at -20°C to -80°C or in liquid Versalovic et al., 1991). Cyanobacterial specific nitrogen is good and effective methods. methods not requiring axenic cultures are of utmost importance since such cultures are difficult to obtain (Choi et al., 2008). PCR based techniques

- 108 - PCR Based Molecular Characterization of...... proved to be more reliable than the conventional (complementary to the template) are coupled to ones in various aspects of identification of the the primer on the 3' side (the polymerase adds cyanobacterial population and to uncover cryptic dNTP’s from 5' to 3', reading the template from variations of strains or closely related species. The 3' to 5' side, bases are added complementary to polymerase chain reaction (PCR) is a technique the template). widely used in molecular biology. It derives its name PCR amplification and sequencing of 16S from one of its key components, a DNA rRNA and rpoC1 genes polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, Since universal primers for direct sequencing of the DNA generated is used as a template for 16S rRNA genes are usually designed to be used replication. With PCR it is possible to amplify a with axenic cultures, and available primers for single or few copies of a piece of DNA across rpoC1 amplification and sequencing are highly several orders of magnitude, generating millions degenerate, specific primers were selected or or more copies of the DNA piece. PCR can be designed in order to obtain clean sequences for extensively modified to perform a wide array of both genes without the need for a cloning step. genetic manipulations. Developed in 1984 by Kary Restriction fragment length polymorphisms Mullis, PCR is now a common and often (RFLPs) indispensable technique used in medical and biological research labs for a variety of The restriction fragment length polymorphisms applications. (RFLPs) of particular PCR products can provide signature profiles specific to the genus, species, The purpose of PCR is to make a huge number of or even strain. Genetic characterization of copies of a gene. This is necessary to have enough cyanobacterial strains has been undertaken using starting template for sequencing. There are three RFLPs of the 16S rRNA gene (16S-ARDRA) major steps in a PCR, which are repeated for 30 (Lyra et al., 1997) and of the intergenic transcribed or 40 cycles. This is done on an automated cycler, spacer region (ITS-ARDRA) (West and Adams, which can heat and cool the tubes with the reaction 1997). Furthermore, amplification of the 16S–23S mixture in a very short time. During the rRNA ITS, which has been shown to be variable denaturation, the double strand melts open to single in length (Rocap et al., 2002; Iteman et al., 2002; stranded DNA, all enzymatic reactions stop. The Neilan, 2002, Laloui et al., 2002) and number in primers are jiggling around, caused by the cyanobacteria, can also be used as an identification brownian motion. Ionic bonds are constantly tool. Neilan et al., (1995) have also found formed and broken between the single stranded heterogeneity in the cluster containing mostly primer and the single stranded template. The more heterocystous planktonic strains of Anabaena and stable bonds last a little bit longer (primers that fit Aphanizomenon genera by using PCR/RFLP of exactly) and on that little piece of double stranded the phycocyanin locus with intergenic spacers. DNA (template and primer), the polymerase can RFLP has revealed the genetic proximity of the attach and starts copying the template. Once there Aphanizomenon with Anabaena and Nodularia are a few bases built in, the ionic bond is so strong with Nostoc. Also, it was found that the neurotoxic between the template and the primer, that it does starins of Anabaena were identical while the not break anymore. Elongation is the ideal working hepatotoxic strains formed a heterogenous group. temperature for the polymerase. The primers, where there are a few bases built in, already have Random amplified polymorphic DNA (RAPD) a stronger ionic attraction to the template than the Random amplified polymorphic DNA (RAPD) forces breaking these attractions. Primers that are markers are DNA fragments from PCR on positions with no exact match get loose again amplification of random segments of genomic (because of the higher temperature) and don’t give DNA with single primer of arbitrary nucleotide an extension of the fragment. The bases sequence. Unlike traditional PCR analysis, RAPD

- 109 - PCR Based Molecular Characterization of...... does not require any specific knowledge of the AFLP when compared to other marker DNA sequence of the target organism: the technologies including randomly amplified identical 10-mer primers will or will not amplify a polymorphic DNA (RAPD), restriction fragment segment of DNA, depending on positions that are length polymorphism (RFLP), and microsatellites. complementary to the primers’ sequence. AFLP not only has higher reproducibility, Furthermore, by RAPD, polymorphisms can be resolution, and sensitivity at the whole genome easily analyzed by small amounts of template level compared to other techniques, but it also has DNA. RAPD was used to generate unique and the capability to amplify between 50 and 100 identifying profiles for members of cyanobacterial fragments at one time. In addition, no prior genera Anabaena and Microcystis. This method sequence information is needed for amplification is based on the combination of the two 10- (Meudth and Clarke 2007). As a result, AFLP meroligonucleotides in a single PCR and provided has become extremely beneficial in the study of specific and repeatable DNA fingerprints which taxa including bacteria, fungi, and plants, where made it possible to discriminate among all toxigenic much is still unknown about the genomic makeup cyanobacteria studied, to the taxonomic ranks of of various organisms. AFLP analysis is based on genera, species and strains. Analysis of DNA selective amplification of DNA restriction typing results obtained by this method clearly fragments. It is technically similar to restriction discriminates between genera Anabaena and fragment length polymorphism analysis, except that Microcystis. Several amplified DNA fragments only a subset of the fragments are displayed and which were expected to be markers for a the number of fragments generated can be particular taxon with identical allozyme genotype controlled by primer extensions. The advantage were also observed on RAPD patterns. The good of AFLP over other techniques is that multiple accordance between the RAPD and allozyme bands are derived from all over the genome. This divergency, indicate the high reliability of RAPD prevents over interpretation or is interpretation due analysis for the easy and rapid discrimination of to point mutations or single locus recombination, cyanobacteria. which may affect other genotypic characteristics. The main disadvantage of AFLP markers is that Amplified fragment length polymorphism alleles are not easily recognized (Majer et al., (AFLP) 1998). The utility, repeatability and efficiency of AFLP-PCR is a highly sensitive method for the AFLP technique are leading to broader detecting polymorphisms in DNA. The procedure application of this technique in the analysis of is divided into three steps: i) Digestion of total cyanobacterial populations. cellular DNA with one or more restriction enzymes DNA fingerprinting based on STRR and and ligation of restriction half site specific adaptors LTRR sequences to all restriction fragments. ii) Selective amplification of some of these fragments with two Repetitive sequences constitute an important part PCR primers that have corresponding adaptor and of the prokaryotic genome (Van Belkum et al., restriction site specific sequences. iii) 1998). Despite their unknown function, and lack Electrophoretic separation of amplicons on a gel of knowledge about how they are maintained and matrix, followed by visualisation of the band dispersed, the presence, widespread distribution pattern. The AFLP technology has the capability and high conservation of these sequences make to detect various polymorphisms in different them methodologically important for DNA genomic regions simultaneously. It is also highly fingerprinting and allow their use as an alternative sensitive and reproducible. As a result, AFLP has for the identification of species or strains and in become widely used for the identification of diversity studies among related prokaryotes and genetic variation in strains or closely related for identification (fingerprinting) of microorganisms species of plants, fungi, animals, bacteria and in general. However it has been suggested that cyanobacteria. There are many advantages to they may regulate transcription termination or be

- 110 - PCR Based Molecular Characterization of...... the target of DNA binding proteins responsible As it can be difficult and time-consuming to for chromosomal maintenance in the cell. In the establish axenic cultures of cyanobacteria, the particular case of cyanobacteria, a family of developed PCR method with STRR or LTRR repetitive sequences, the short tandemly repeated primers provides a useful method for studying the repetitive sequences (STRRs), has been described diversity of cyanobacteria in the natural (Mazel et al., 1990). These heptanucleotide environment, whether free living or symbiotic. A sequences have been identified in several universal marker for DNA fingerprinting is the cyanobacterial genera and species, so far mostly oligonucleotide csM13. It has already been tested in heterocystous cyanobacteria (Zheng et al., in a small number of cyanobacteria (Valerio et 1999; Wilson et al., 2000; Teaumroong et al., 2002; al., 2005), and has a demonstrated ability even to Lyra et al., 2005) but also in some non discriminate strains of the same species. heterocystous ones. Furthermore, a 37 bp long Techniques based on the enterobacterial repetitive tandemly repeated repetitive sequence (LTRR) intergeneric consensus (ERIC) have also been has also been identified in some cyanobacterial used for identification and discrimination purposes species (Prasanna et al., 2006). Analysis of in some cyanobacteria (Lyra et al., 2001; Bruno STRRs and LTRRs has been described as a et al., 2006). However, the method based on powerful tool for taxonomic studies. Moreover, STRR and LTRR sequences is accurate in the specificity of these sequences has made STRRs distinguishing and classifying even closely related useful even for non axenic cyanobacterial cultures. strains of cyanobacteria.

References

Baker, P. (1992). Identification of common noxious Choi, G.G., Bae, M.S., Ahn, C.Y. and Oh, H.M. (2008). cyanobacteria Part II– Chroococales and Induction of axenic culture of Arthrospira (Spirulina) Oscillatoriales: urban water research association of platensis based on antibiotic sensitivity of Australia. Research Report no. 46. contaminating bacteria. Biotechnol Lett. 30: 87–92. Bowen, C.C. and Jensen, T.E. (1965). Blue green algae: Ciferri, O. (1983). Spirulina, the edible microorganism. fine structure of the gas vacuoles. Science 147: 1460- Microbiol Rev. 47: 551-578. 1462. Davey, M.C. (1988). Ecology of terrestrial algae of the Brock, T.D. (1970). High temperature systems. A Rev fell field ecosystems of signy island. South Orkney Ecol System. 1: 191-220. Islands. Br Antartica Surv Bull. pp. 69-74. Bruno, L., Billi, D., Albertano, P. and Urzi, C. (2006). Doetsch, R.N. and Hageage, G.J. (1968). Motility in Genetic characterization of epilithic cyanobacteria and prokaryotic organisms: problems, points of view, and their associated bacteria. Geomicrobiol J. 23: 293–299. perspectives. Biol Rev. 43: 317-362. Bryant, D.A. (1994). The Molecular Biology of Fuhs, G.W. (1968). Cytology of the blue green algae: cyanobacteria. Kluwer Academic Publishers, Light-microscopic aspects. In: Jackson D.E (Ed.) Algae, Dordrecht, The Netherlands. p-881. Man and the Environment. pp. 213-233. Buckland, B. and Walsby, A.E. (1971). A study of the Gebelein, C.D. (1969). Distribution, morphology, and strength and stability of gas vesicles isolated from a accretion rate of recent subtidal algal stromatolites, blue green alga. Arch Microbiol. 79: 327–337. Bermuda. J Sebium Petrol. 39: 49-69. Castenholz, R.W. (1992). Species usage, concept, and Giovannoni, S.J., Turner, S.G., Olsen, J., Barns, S., Lane, evolution in the cyanobacteria. J. Phycol. 28: 737-745. D.J. and Pace, N.R. (1988). Evolutionary relationships Chang, K.C. (1977). “Public Archaeology in China.” among cyanobacteria and green chloroplasts. J Paleo-Anthropology in the Peoples Republic of Bacteriol August . 170(8): 3584-3592. China. Edited by W.W. Howells & Patricia Jones Guglielmi, G. and Cohen-Bazire, G. (1984). Etude Tuchitari. CSCPRC Report 4 (National Academy of taxonomique d’un genre de cyanobactérie Sciences): pp 130–139.

- 111 - PCR Based Molecular Characterization of......

Oscillatoriacée: le genre Pseudanabaena Lauterborn. FLP of the internal transcribed spacer of the ribosomal I. Etude ultrastructurale. Protistologica. 20: 377•-391. operon. Microbiol. 148: 453–465. Halfen, L.N. and Castenholz, R.W. (1971). Gliding Lyra, C., Hantula, J., Vainio, E., Rapala, J., Rouhiainen, motility in the blue green alga, Oscillatoria princeps. L. and Sivonen, K. (1997). Characterization of J Phycol. 7: 133-145. cyanobacteria by SDS-PAGE of whole-cell proteins Hawes, I. and Davey, M.C. (1989). Use of florochrome and PCR/RFLP of the 16S rRNA gene. Arch Microbiol. auramine for determination of cell viability in 168: 176–184. filamentous and thalloid algae. Phycologia. 28: 518- Lyra, C., Suomalainen, S., Gugger, M., Vezie, C., 523. Sundman, P., Paulin, L. and Sivonen, K. (2001). Herdman, M., Janvier, M., Waterbury, J.B., Rippka, R. Molecular characterization of planktic cyanobacteria and Stanier, R.Y. (1979b). Deoxyribonucleic acid base of Anabaena, Aphanizomenon, Microcystis and composition of cyanobacteria. J. Gen Microbiol. 111 : Planktothrix genera. Int J Syst Evol Microbiol. 51: 63-•71. 513–526. Hindak, F. (1985). Morphology of trichomes in Lyra, C., Laamanen, M., Lehtima ki, J.M., Surakka, A. Spirulina fusiformis Voronichin from lake Bogaria, and Sivonen, K. (2005). Benthic cyanobacteria of the Kenya. Arch Hydrobiol, Suppl. 71, Algol Stud. 38/39, genus Nodularia are non toxic, without gas vacuoles, 201. able to glide and genetically more diverse than planktonic Nodularia. Int J Syst Evol Microbiol. 55: Iteman, I., Rippka, R., Tandeau de Marsac, N. and 555–568. Herdman, M. (2002). rDNA analyses of planktonic heterocystous cyanobacteria, including members of Majer, D., Lewis, B.G. and Mithen, R. (1998). the genera Anabaenopsis and Cyanospira. Microbiol. Genetic variation among field isolates of Pyrenopeziza 148: 481–496. brassicae. Plant Pathol. 47: 22-28. Jeeji Bai, N. and Seshadri, C.V. (1980). On coiling and Mazel, D., Houmard, J., Castets, A.M. and Tandeau de uncoiling of trichomes in the genus Spirulina. Arch Marsac, N. (1990). Highly repetitive DNA sequences Hydrobiol, Suppl. 60, Algol Stud. pp. 26-32. in cyanobacterial genomes. J Bacteriol. 172: 2755– 2761. Karnieli, A., Shachak, M., Tsoar, H., Zaady, E., Kaufman, Y., Denin, A. and Porter, W. (1996). The effect of Meudt, H.M. and Clarke, A.C. (March 2007). “Almost macrophytes on the spectral reflectance of vegetation forgotten or latest practice? AFLP applications, in semiarid regions. Remote Sensing of Environment. analyses and advances”. Trends Plant Sci. 12 (3): 57: 88-96. 106–17. Kaneko, T., Nakamura, Y., Wolk, C.P., Kuritz, T., Neilan, B.A., Jacobs, D. and Goodman, A.E. (1995). Sasamoto, Watanabe, A., Iriguchi, A., Ishikama, K., Genetic diversity and phylogeny of toxic cyanobacteria Kawashima, K. and other authors. (2001). Complete determined by DNA polymorphisms with the genomic sequence of the filamentous nitrogen fixing phycocyanin locus. Appl Enviro Microbiol. 61: 3875- cyanobacterium Anabaena sp. strain PCC 7120. DNA 3883. Res. 8: 205–213. Neilan, B.A. (2002). The molecular evolution and DNA Komarek, J. and Anagnostidis, K. (1989). Modern profiling of toxic cyanobacteria. Mol Biol. 4: 1–11. approach to the classification system of Cyanophytes. Nubel, U., Garcial-Pichel, F. and Muyzer, G. (1997). PCR 4. Nostocales. Algol Stud. 56:247–345. primers to amplify 16S rRNA genes from cyanobacteria. Kondo, R., Yoshida, T., Yusi, Y. and Hiroishi, S. (2000). Appl Environ Microbiol. 63: 3327–3332. DNA–DNA reassociation among a bloom-forming Nultsch, W. (1965). Light reaction systems in cyanobacterial genus, Microcystis. Int J Syst Evol cyanophyceae. Photochem Photobiol. 4: 613-619. Microbiol. 50:767–770. Pierson, B.K. and Castenholz, R.W. (1971). Laloui, W., Palinska, K.A., Rippka, R., Partensky, F., Bacteriochlorophylls in gliding, filamentous Tandeau de Marsac, N., Herdman, M. and Iteman, I. prokaryotes from hot springs. Nature New Biol. 233: (2002). Genotyping of axenic and non axenic isolates 25-27. of the genus Prochlorococcus and the OMF- Prasanna, R., Kumar, R., Sood, A., Prasanna, B.M. and ‘Synechococcus’ clade by size, sequence analysis or Singh, P.K. (2006). Morphological, physiochemical and

- 112 - PCR Based Molecular Characterization of...... molecular characterization of Anabaena strains. Van Belkum, A., Scherer, S., van Alphen, L. and Microbiol Res. 161: 187–202. Verbrugh, H. (1998). Short-sequence DNA repeats in Rasmussen, U. and Svenning, M.M. (1998). prokaryotic genomes. Microbiol Mol Biol Rev. 62: Fingerprinting of cyanobacteria based on PCR with 275–293. primers derived from short and long tandemly repeated Versalovic, J., Koeuth, T. and Lupski, J.R. (1991). repetitive sequences. Appl Environ Microbiol. 64: 265– Distribution of repetitive DNA sequences in eubacteria 272. and application to fingerprinting of bacterial genomes. Rocap, G., Distel, D.L., Waterbury, J.B. and Chisholm, Nucleic Acids Res. 19: 6823–6831. S.W. (2002). Resolution of Prochlorococcus and Waaland, J.R., Waaland, S.D. and Branton, D. (1971). Synechococcus ecotypes by using 16S–23S ribosomal Gas vacuoles. Light shielding in blue green algae. J DNA internal transcribed spacer sequences. Appl Cell Boil. 48:212-215. Environ Microbiol. 68: 1180–1191. Walsby, A.E. (1972). Structure and function of gas Rudi, K., Skulberg, O.M., Larsen, F. and Jakobsen, K.S. vacuoles. Bact Rev. 36: 1-32. (1997). Strain characterization and classification of Walsby, A.E. and Buckland, B. (1969). Isolation and oxyphotobacteria in clone cultures on the basis of 16S purification to intact gas vesicles from a blue green rRNA sequences from the variable regions V6, V7, and alga. Nature. 224: 716-717. V8. Appl Environ Microbiol. 63: 2593–2599. West, N.J. and Adams, D.G. (1997). Phenotypic and Schopf, J.W. (1970). Precambrian microorganisms and genotypic comparison of symbiotic and free living evolutionary events prior to the origin of vascular cyanobacteria from a single field site. Appl Environ plants. Biol Rev. 45: 319-352. Microbiol. 63: 4479–4484. Stewart, W.D.P. and Lex, M. (1970). Nitrogenase activity Whitton, B.A. and Roger, P.A. (1989). Use of blue green in the blue green alga Plectonema boryanum strain algae and Azolla in rice culture. In: Campbell R and 594. Arch Mikrobiol. 73: 250-260. Macdonald RM (eds) Microbial inoculation of crop Stewart, W.D.P. and Pearson, H.W. (1970). Effects of plants. IRS Press at Oxford. 25: 89-100. aerobic and anaerobic conditions on growth and Wilson, K.M., Schembri, M.A., Baker, P.D. and Saint, metabolism of blue green algae. Proc R Soc. 175: 293- C.P. (2000). Molecular characterization of the toxic 311. cyanobacterium Cylindrospermopsis raciborskii and Tandeau de Marsac, N. (1977). Occurrence and nature design of a species specific PCR. Appl Environ of chromatic adaptation in cyanobacteria. Journal of Microbiol. 66: 332–338. Bacteriology. 130: 82-91. Wynn-Williams, D.D. (1990): Microbial colonization Teaumroong, N., Innok, S., Chunleuchanon, S. and processes in Antarctic fell field soils - an experimental Boonkerd, N. (2002). Diversity of nitrogen fixing overview. In Proc. National Institute of Polar research cyanobacteria under various ecosystems of Thailand. symposium on Polar Biology. 3: 164-178. I. Morphology, physiology and genetic diversity. World Zheng, W.W., Nilsson, M., Bergman, B. and J Microbiol Biotechnol. 18: 673–682. Rasmussen, U. (1999). Genetic diversity and Valerio, E., Pereira, P., Saker, M.L., Franca, S. and classification of cyanobacteria in different Azolla Tenreiro, R. (2005). Molecular characterization of species by the use of PCR fingerprinting. Theor Appl Cylindrospermopsis raciborskii strains isolated from Genet. 99: 1187– 1193. Portuguese freshwaters. Harmful Algae. 4: 1044–1052

- 113 -