BioResource Now ! Vol.9 No.2 BioResource Now ! Issue Number 9 February2013

Masanobu Kawachi (National Institute for Environmental Studies） Reprinting and reduplication of any content of Introduction to this newsletter is prohibited. Resource Center Collection, Preservation, and Future Tasks of All the contents are protected by the Japanese No.43 copyright law and international regulations. Various Algal Resources P1 - 2 Database Resources Database of Escherichia coli Download the PDF version of this newsletter at of This Month http://www.shigen.nig.ac.jp/shigen/news/ “NBRP E.coli Strain” P2

Introduction to Resource Center〈NO. 43〉 Collection, Preservation, and Future Tasks of Masanobu Kawachi , Head Various Algal Resources Biodiversity Resource Conservation Section, Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies

Various Algae for algal cultures could not be controlled. The differences among algae appear as Algae are living organisms that are While being anxious about the restoration variations in their cytoarchitecture and characterized by “oxygenic .” of infrastructures, we must adopt urgent physiological, biochemical, and biological All living organisms that perform “oxygenic measures, e.g., moving stock cultures to characteristics. The diversity of algae, photosynthesis,” except terrestrial sunny places during daytime. The which extends over different classes of such as mosses, ferns, and seed plants, temperature in a liquid nitrogen refrigerator, , is the reason why algae are belong to the category of algae. Even in which frozen cultures were kept, could attractive as research resources. terrestrial plants that differentiated from be maintained within a safety zone even algae have evolved from algae belonging during a long-term power outage, and Resources for Elucidating the to the class Charophyceae (National frozen cultures were not affected by the Mechanism underlying Evolution BioResource Project [NBRP]-Algae earthquake. A lesson learnt from the Great and Formation of Chloroplasts conserves 81 and 256 strains). Hanshin-Awaji Earthquake is that frozen Algae do live in normal waterscape cultures have been mutually stored in Kobe environments, e.g., lakes, mashes, coasts, University and the NIES since 2008. This and oceans, as well as in extreme mutual storage system was a great relief to environments, e.g., hot springs, snow, ice, us. These 2 earthquake disasters taught us high-salinity waters, and arid zones. Many that risk diversification is important to algae are known to coexist with other prevent the loss of precious resources. living organisms such as corals and lichens. Various living organisms, Characteristics of Algae from the including prokaryotes, eukaryotes, Viewpoint of Phylogenetic Diversity unicellular organisms, and multicellular Recent molecular phylogenetic studies marine plants, that possess complicated revealed that algae are composed of systems and life histories, belong to the groups, which greatly differ from each class algae. The NBRP- Algae collects, other in their phylogenetic profiles. At conserves, and distributes various algae present, eukaryotes are composed of at Fig. 2: Cryptophytes (A, provided by Ms. Mayumi Sato), Chlorarachniophytes (B, NIES-624), Paulinella resources. least 9 groups (Fig. 1). chromatophora (C), (D, provided by Ms. Haruyo Yamaguchi), and (E, Structure of NBRP-Algae N211 N1035 N624 N674 NIES-483). The National Institute for Environmental N547 In algae, the presence of chloroplasts that Studies (NIES), a central resource center, Rhizaria N330 Chlorarachniophytes Alveolata are peculiar to each group has been collects, conserves, and distributes confirmed. Cryptophytes (NBRP-Algae Rhodophyta Chlorophyta Stramenopiles Dinoflagellate resources of micro algae, endangered Plants (narrow sense) Chromerida conserves 21 species and 46 strains) (Fig. algae, and protists that are phylogenet- ２A) greatly differ from Chlorarachniophytes ically close to algae. Kobe University, an (Terrestrial plants) Heterokontophyta Glaucophytes (NBRP-Algae conserves 5 species and 6 N345 allocation resource center, is in charge of Cryptophytes strains) (Fig. 2B) in their phylogenetic collecting, conserving, and distributing Haptophytes profiles. Remnants of eukaryotic nuclei Euglenoids macro algae. The University of Tsukuba, Amoebozoa (nucleomorphs) have been found in another allocation resource center, is newly chloroplasts of Cryptophytes and (Metazoa) (Fungi) in charge of collecting algae resources with Chlorarachniophytes. It is known that Opisthokonta Excavates N837 high utility values and preparing additional N1442 N286 chloroplasts in Cryptophytes and information such as classification Chlorarachniophytes are derived from information. Hokkaido University, a third Fig.1: Phyletic relationship among eukaryotes; those in Rhodophyta and Chlorophyta, modifications based on Baldauf (2003). Colored branches allocation resource center, is in charge of indicate algae and terrestrial plants. The numbers respectively. It is also known that in a backing up important strains. attached to the photographs are NIES strain numbers. nucleomorph in Cryptophytes, whose genome size is 0.55 Mb, approximately Many eukaryotes are microorganisms. The 600 genes code for proteins. The What We Learned from Disastrous presence of algae has been confirmed in all Earthquakes existence of nucleomorphs directly of these groups, except Amoebozoa and indicates the origin of chloroplasts and the The Great East Japan Earthquake that Opisthokonta. In this typical dendrogram, evolution process of organelles. occurred on March 11, 2011, greatly colored branches of algae are interspersed. affected algae conservation facilities of the The reason for this interspersion is The alga Paulinella chromatophora (NIES- NIES. Infrastructures such as power, water, considered to be that chloroplasts have 2635) (Fig. 2C) has a chloroplast (it is and gas supply systems could not be used been independently acquired in each group. called cyanelle for differentiation from other for a long time, and operations, including Algae that are able to perform photo- chloroplasts). Its origin differs from that of subcultures, were difficult to perform. synthesis greatly differ from each other in chloroplasts in other algae. It is interesting Lighting and temperature that are required their evolution and phylogenetic profiles. that the photosynthesis-related genes To the next page BioResource Now ! Vol.9 No.2

remained in the cyanelle, while they have Chlorella vulgaris (NIES-2170) (Fig. 3A) and rates are low. In the future, we will make already shifted to the nuclear genome in Euglena gracilis (NIES-48) (Fig. 3B) are every effort to examine conditions to other chloroplasts. Therefore, the cyanelle typical algae that have been used in various improve the survival rates of these strains is considered to represent a stage during research fields, including photosynthesis and to establish an inspection method, by which the chloroplast shifted to become an studies, physiological and biochemical which these strains can be stably organelle. studies, and research and development cryopreserved, even though their survival In various other algae and protists, inter- studies aiming at their practical use. Recently, rates are low. By exchanging information esting phenomena such as intracellular Euglena gracilis has been frequently used and technologies with researchers who symbiosis and kleptoplasty have been in research and development projects belong to various conservation facilities in observed. For example, Hatena arenicola aiming at its practical use for biofuels and the NBRP, we attempt to develop a new (Fig. 2D), a protist, seems to be a foods. Dunaliella tertiolecta (NIES-2258) cryopreservation technology. alga possessing green chloroplasts. (Fig. 3C) is a species of Chlorophyta and However, this protist “incorporates” a free- has adapted to high-salinity environments. Future Task of NBRP-Algae, No. 2 living prasinophyte alga into its cell, By maintaining a high-salinity environment, Enhancement of Model Organisms changes the size and shape of the alga, mass cultivation of this species can be and uses the alga as if it was a chloroplast. achieved in open ponds, similar to pure In algae, many interesting phenomena This prasinophyte alga was found to be a cultures. Although the proliferation potency have been observed, e.g., mineralization※1, species that belongs to the is not very high, Botryococcus braunii sex pheromone production, toxin produc- Nephroselmis (NBRP-Algae conserves 5 (NIES-836) (Fig. 3E) that accumulates oil tion, and various cell movements. Model species and 15 strains) (Fig. 2E). How can (which is similar to fuel oil) in its algal organisms used to investigate these Hatena arenicola change and control biomass and Haematococcus lacustris biological phenomena and mechanisms Nephroselmis in its cell? Has a part of the (NIES-144) (Fig. 3F) that produces must be further enhanced. The NBRP-Algae genome of Nephroselmis already been astaxanthin (which is a substance useful for has also been developing its own model shifted to that of Hatena arenicola? Hatena human health) have often been used in the organism using cyanobacteria, which live arenicola is an interesting object to field of practical research in recent years. in many different environments and elucidate the initial process of chloroplast possess various interesting light-related formation. physiological functions, e.g., photosynthesis, Future Task of NBRP-Algae, No. 1 circadian rhythm, and . Using 95 Some algae and achromatic protists that Serious Dilemma Due to Diversity are phylogenetically close to algae acquire species and 706 strains of cyanobacteria chloroplasts again after having secondarily Although the diversity of algae is an conserved by the NBRP, we have been lost them. Thus, algae are materials advantage of algae as resources, it selecting strains useful for molecular- suitable to study the evolution and formation sometimes becomes the cause of worry biological research by investigating their process of an organelle, a chloroplast. when conserving resources. In the NIES, of genetic modification abilities. the 2,339 available strains, 786 strains Resources for Study and Practical have been cryopreserved and 1,553 strains Use of Photosynthesis have been sub-cultured (approximately 2/3 Among algal resources, there are species of the available strains). For these sub- that have excellent proliferation potency; cultured strains, 76 types of media and 7 they proliferate very densely and are easy types of containers are used and various to handle in experiments. temperatures and light conditions are Fig. 4: Cyanobacteria, into which a plasmid containing applied. The subculture interval varies the green fluorescent protein (GFP) gene has been according to the strain, and weekly growth introduced using conjugation with Escherichia coli: (A) an optical microscope image, (B) a chlorophyll fluorescence inspections and regular sterility tests are image, and (C) a GFP image. The cell without indispensable. We have been sub-culturing chlorophyll fluorescence in the center is Escherichia coli. various strains using the knowledge that These images were provided by Mr. Yohei Shimura. has been gained by our experiences over a ※1 Mineralization : Extracellular structures composed long time. However, some strains die out of minerals have been found in algae, e.g., calcium almost every year. carbonate in coccolithophorids (Fig. 5A, NIES-2696), siliceous substances in diatoms (Fig. 5B, NIES-2363), Some strains, which have been sub- and iron oxide + cultured, cannot be cryopreserved using manganese oxide in euglenoids (Fig. 5C, Fig. 3: Chlorella vulgaris (A), Euglena gracilis (B), current technologies and some can be obtained from a A B C Dunaliella tertiolecta (C), a farm of Dunaliella in Israel cryopreserved, even though their survival (D), Botryococcus braunii (E), and Haematococcus natural pond). Fig. 5: Mineralization lacustris (F)

Database of This Month Contact Address Resources Database of Escherichia coli “NBRP E.coli Strain” Genetic Resource Center, National Institute of Genetics 1111 Yata, Mishima-shi, Shizuoka 411-8540, Japan DB name：NBRP E.coli Strain Tel.: 055-981-6885 (Yamazaki) URL: http://www.shigen.nig.ac.jp/ecoli/strain/ : Language: Japanese, English E-mail [email protected] Original contents: Mutants, clones Exhaustive mutants (gene-disrupted strains, large Editor's Note deletion mutants, transposon insertion mutants) This month, Mr. Kawachi, who has been the representative of Phages, cloning vectors the organization of algae resources since the third stage of the Features: Various genome-wide mutants are being prepared NBRP, kindly wrote about the characteristics and and can be ordered through the Internet. The entire attractiveness of algae as living organisms. The diversity is process from the application procedure to the distribution said to be an advantage of algae as resources. However, I can be tracked through the Internet. understand that to deal with various conservation conditions is Because of the cooperation with Profiling of E. coli a difficult task for the organization that aims to conserve algal Chromosome (PEC), a genome database, related strains resources. A gallery of beautiful algal images can be accessed can be accessed from physical maps. at www.shigen.nig.ac.jp/algae/ (Y. Y.) . Number of strains: 22,725 The description of each collection is substantial. Number of genes: 6,926 DB construction group: NBRP E. coli Strain, NBRP Information BioResource Information Number of papers: 276 Management organization: Genetic Resource Center, NIG （As of February 2013） Year of DB publication: 1999 Year of last DB update: 2013 (NBRP) www.nbrp.jp/ Comment from a practicing developer: Fourteen years have passed since the first publication, and this (SHIGEN) www.shigen.nig.ac.jp/ database is in a stable state now. I, as a developer of this database, am pleased to know that the number of (WGR) www.shigen.nig.ac.jp/wgr/ users has been increasing as the number of types of resources has been steadily increasing. We have also (JGR) www.shigen.nig.ac.jp/wgr/jgr/jgrUrlList.jsp been developing the Profiling of E. coli Chromosome (PEC), the development of which was initiated earlier than that of NBRP E. coli Strain. Therefore, by functionally connecting these 2 databases, users can make the most use of their contents. This resource database seems to be operated by a single server. However, in order to allow users to follow the entire process from the application procedure to the distribution via the Internet, multiple servers are connected BioResource Now ! with each other in this database. We receive some positive feedback from our users through the many inquiries Issue Number 9 February 2013 sent to us via the Contact us link. We will continuously improve this database, so please feel free to contact us. "translated by ASL translatiaon service and proofread by Sharoh Yip"