Algae (Order Chroococcales) R

BACTEROLOGICAL REVIEWS, June 1971, p. 171-205 Vol. 35, No. 2 Copyright © 1971 American Society for Microbiology Printed in U.S.A. Purification and Properties of Unicellular Blue-Green Algae (Order Chroococcales) R. Y. STANIER, R. KUNISAWA, M. MANDEL, AND G. COHEN-BAZIRE Department of Bacteriology and Immunology, University of California, Berkeley, California 94720, and The University ofTexas, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 INTRODUCTION ............................................................ 171 MATERIALS AND METHODS ............................................... 173 Sources of Strains Examined .................................................. 173 Media and Conditions of Cultivation ........................................... 173 Enrichment, Isolation, and Purification of Unicellular Blue-Green Algae 176 Measurement of Absorption Spectra ............................................ 176 Determination of Fatty-Acid Composition ....................................... 176 Extraction of DNA .......................................................... 176 Determination of Mean DNA Base Composition ................................. 177 ASSIGNMENT OF STRAINS TO TYPOLOGICAL GROUPS 177 DNA BASE COMPOSITION ................................................. 181 PHENOTYPIC PROPERTIES ................................................ 184 Motility................................................................... 184 Growth in Darkness and Utilization of Organic Compounds ........................ 184 Nitrogen Fixation ............................................................ 191 Maximal Temperature for Growth ............................................. 191 Sensitivity to Light ......................................................... 192 Penicillin Sensitivity .......................................................... 192 Cellular Absorption Spectra ................................................... 192 Cellular Fatty-Acid Composition ....... ....................................... 193 PROBLEM OF GENERIC NOMENCLATURE IN THE ORDER CHROOCOC- CALES .................................................................. 194 POSSIBLE CORRESPONDENCES BETWEEN TYPOLOGICAL GROUPS AND TRADITIONAL FORM GENERA IN CHROOCOCCACEAE 195 MERISMOPEDIA: A GROWTH FORM OF APHANOCAPSA? ................ 197 GENERA CHLOROGLOEA AND CHLOROGLOEOPSIS ...................... 199 SPECIATION IN CHROOCOCCACEAE ....................................... 199 Subdivisions in Typological Group I (Synechococcus) ......... .................... 199 Subdivisions in Typological Group II (Aphanocapsa, Gloeocapsa, Microcystis) ... .... 201 CONCLUDING COMMENTS 202 LITERATURE CITED ........................................................ 203 To the memory of E. G. Pringsheim for many decades (16, 58, 69, 74) and was solved (1881-1970). only when the structure of the cell in these two groups could be studied at the level of resolution INTRODUCTION afforded by the electron microscope. Electron Blue-green algae occupy an anomalous position microscopic investigations, together with analyti- in the biological world. They are treated by cal data on cell wall composition and ribosomal botanists as a division (or class) of algae because structure, have now revealed the common de- they are photoautotrophs that use water as an nominators, which are fundamental: bacteria and electron donor and contain the two photopig- blue-green algae are the only organisms with cells ments (chlorophyll a and (-carotene) that are of the procaryotic type (6, 10, 21, 47, 70). the chemical hallmarks of plant photosynthesis. The designation blue-green algae is therefore However, it has long been recognized that in misleading, although this common name is now cellular and organismal respects they resemble the so firmly established that its use can probably bacteria. This was first noted by Cohn (14), who never be eradicated. These organisms are not subsequently proposed (15) to place the two with groups in a common division, the Schizophytae, algae; their taxonomic association eucaryotic even though their shared properties then appeared algal groups is an anachronism, formally equiva- to be negative ones. The problem of defining in lent to classifying the bacteria as a constituent positive terms the properties that are shared by group of the fungi or the protozoa. In view of bacteria and blue-green algae remained intractable their cellular structure, blue-green algae can now 171 172 STANIER ET AL. BACTERIOL. REV. be recognized as a major group of bacteria, filamentous forms, since most of the unicellular distinguished from other photosynthetic bacteria members of the group are immotile and, therefore, by the nature of their pigment system and by will form small, compact colonies on solid media. their performance of aerobic photosynthesis. The most successful pioneer in the purification of Their properties are still very poorly known. blue-green algae, M. B. Allen (1), isolated one Much of the available information about them unicellular strain, the organism known as Ana- lies in the domain of natural history, not biology. cystis nidtilans, by standard plating methods. Her The structural diversity of blue-green algae is strain remained for many years the sole repre- considerable, and for this reason algologists have sentative of the Chroococcales available in pure been able to develop elaborate taxonomic treat- culture, and for this reason it has been widely used ments of the group based solely on structural in physiological and biochemical investigations properties, determined through microscopic (see 33). A few additional unicellular strains have examination of field collections (19, 28). Most been purified from marine sources by Van Baalen algal taxonomists recognize several different (73), and Castenholz (11) has purified several orders. The unicellular blue-green algae are thermophilic unicellular strains from hot springs. assigned to a single order, Chroococcales, con- Our work on this problem began about 8 years taining two families, Chroococcaceae and Ento- ago when M. M. Allen (2) succeeded in purifying physalidaceae (28). Members of the Chroococ- by plating methods some unicellular strains that caceae are coccoid or rod-shaped organisms which had been received as unialgal cultures from other multiply by binary fission, and they may form laboratories. This convinced us that purification loose colonies in which the constituent cells are is not difficult, provided that suitable material is held together by a common slime layer or by available in the form of unialgal cultures or crude sheaths. In structural terms, they appear to be cultures that contain a preponderance of uni- counterparts of unicellular true bacteria. Mem- cellular blue-green algae. However, these or- bers of the Entophysalidaceae are coccoid organisms typically occur in nature as minor com- ganisms, distinguished primarily by their colonial ponents of mixed algal populations; since their growth habit. They grow as dense, parenchyma- nutritional requirements do not differ signifi- tous masses of cells, typically found on the sur- cantly from those of many other algae, they face of moist rocks. cannot be specifically enriched from such mixed In the study of any microbial group, the transi- populations by nutritional selection. Natural tion from natural history to biology is largely populations that consist largely of unicellular dependent on the solution of a technical problem: blue-green algae may arise sporadically as water the isolation and maintenance of its members in blooms in lakes or ponds (28) but are of rela- pure culture. The present paucity of information tively constant occurrence only in hot springs, about blue-green algae reflects the difficulty that where they are composed of a few physiologically has been encountered in the purification of these highly specialized thermophilic forms (11). microorganisms. The total number of strains Allen and Stanier (5) discovered that many purified so far is small, and the pure strains now mesophilic blue-green algae, both filamentous available are far from representative of the evi- and unicellular, can be enriched successfully dent biological diversity of the group. Never- from mixed algal populations by temperature theless, the nutrient requirements of these or- selection, since their temperature maxima are ganisms are simple, and it is easy to obtain growth significantly higher than those of nearly all on solid media. The primary obstacle to obtaining eucaryotic algae from the same environments. pure cultures seems to lie in the synthesis by many When samples of soil or fresh water are placed blue-green algae of a copious extracellular slime in a suitable mineral medium and incubated in the layer, which harbors bacteria. light at 35 C, the population that develops con- Some filamentous blue-green algae have been sists almost exclusively of blue-green algae. This purified by taking advantage of their vigorous enrichment technique is particularly valuable for gliding movement and inducing phototactic the isolation of unicellular blue-green algae be- migration of trichomes or hormogonia over (or cause, if present in the inoculum, they eventually through) agar plates (41). This does not always far outnumber filamentous members of the group eliminate bacteria, however, and purification of as the enrichment culture develops. Experience some filamentous strains has been achieved only with its use for several years has shown that one by treatment of trichomes

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