Patterns of growth in coccoid, aggregate forming cyanobacteria

Ann. Bot. Fennici 35: 219–227 ISSN 0003-3847Helsinki 20 November 1998 © Finnish Zoological and Botanical Publishing Board 1998

Patterns of growth in coccoid, aggregate formingcyanobacteria

Katarzyna A. Palinska & Wolfgang E. Krumbein

Palinska, K. A. & Krumbein, W. E., Carl von Ossietzky University, ICBM, Geomicro-biology, P.O. Box 2503, D-26111 Oldenburg, Germany

Received 4 February 1998, accepted 18 August 1998

Several Merismopedia-like forms were observed and collected from the microbial matsat Mellum and Norderney Islands. The formation of giant cells and giant cell aggre-gates in otherwise smaller Merismopedia cultures was often observed in all cultures. Inthe course of this study it was possible to show, that accelerated and delayed divisionoccurs in Chroococcales not unlike the multiple fission pattern in Pleurocapsales. Onthe basis of the existence of the terms baeocyte and nanocyte for cell size decrease inone clone and our observation of delayed division in Merismopedia isolates we suggestthe following terms: (1) baeocyte for rapid, multiple fission resulting in very smallindividual cells (motile and non-motile), (2) nanocyte for accelerated division resultingin considerably smaller cell sizes, and (3) megacyte for considerably enlarged cell sizesupon delayed division. Nanocyte formation and megacyte formation when occurringunder stable environmental conditions may actually have been and still be misinter-preted as separate species in field samples and herbarium materials.

Key words: baeocyte, cyanobacteria, Merismopedia, nanocyte, taxonomy

classified in 3 orders: Chroococcales, Chamaesi-phonales and Pleurocapsales. Komarek and Anag-nostidis (1986) classified all coccoid “cyano-phytes” in a simple order Chroococcales with 7definable families (Microcystaceae, Chroococca-ceae, Entophysalidaceae, Chamaesiphonaceae,Dermocarpellaceae, Xenococcaceae and Hydro-coccaceae). Their arguments to do so were basedon the type of division, which is basically the samein all coccoid cyanobacteria. The bacteriologists— Rippka et al. (1979) and Bergey’s Manual(Castenholz 1989a, 1989b, Waterbury 1989,Waterbury & Rippka 1989, ) — divided coccoidor non-filamentous cyanobacteria into two orders:

INTRODUCTION

The cyanobacteria constitute one of the largestsub-groups of Gram-negative prokaryotes. As aresult of their traditional assignment to the algae,the classification of these organisms was devel-oped by phycologists, working under the rules ofthe botanical code. This consideration is supportedby their functional position in nature since cyano-bacteria as phototrophic organisms are importantprimary producers in almost all biotopes on theEarth.

According to Geitler (1932), considering theirtypes of reproduction, non-filamentous species are

220 Palinska & Krumbein • ANN. BOT. FENNICI 35 (1998)

Chroococcales (reproduction by binary fission inone, two or three planes or by budding), and Pleuro-capsales (multiple fission or combination of mul-tiple fission and binary fission).

The differences between “botanists” (Geitler,Komarek and Anagnostidis) and “bacteriologists”(Rippka, Castenholz, Waterbury) are that the for-mer used the keys and descriptions of cyanobac-teria in the field and herbaria, and the latter ac-cept only pure cultures. According to Bergey’sManual and the traditional botanical literature, thepresence or absence of cell aggregates and theircharacteristics, the number and regularity of theplanes of division, as well as statistically stabi-lized cell size clusters are the major characteris-tics used to determine genera and groups of coc-coid cyanobacteria. The presence of extracellularsheath layers in several coccoid taxa (Gloeobacter,Gloeothece and Gloeocapsa) in culture has provento be a stable feature as well. However, Synecho-cystis, Merismopedia, Microcystis and Eucapsis(in botanical sense) occur in cell aggregates innature but rarely in culture. Additionally, in prac-tice, it is often difficult to determine the numberof successive planes of division, and the presenceor absence of a glycocalyx.

Thus it seemed worthwhile to study the mor-phological, physiological, biochemical and mo-lecular aspects of coccoid cyanobacteria withoutbaeocytes (lacking rapid multiple fission), in thefield and laboratory. For this particular group ofcyanobacteria much less is known in terms of be-haviour in culture, and their taxonomic positionis still very confused.

MATERIAL AND METHODS

Origin, growth and culture conditions of thestrains

Field sampling was carried out in June 1991 on the Islandof Mellum; and from 1991 to 1995, each year in October,on the Norderney Island. Both sites are located in the Ger-man Bight of the North Sea.

For isolation, a small sample of a microbial mat mate-rial was used as an inoculum on a liquid medium. Enrich-ment was done in 100 ml Erlenmeyer flasks containing 50 mlof medium MN (Waterbury & Stanier 1978) of North Seasalinity, and 50 µg ml–1 cycloheximide (Serva Heidelberg).The flasks were placed in an illuminated incubator at verylow shaking speed (Gallenkamp, UK), at 800 lux and 20°C.

Coccoid cyanobacteria from the genera Synechocystis, Syne-chococcus, Merismopedia and Eucapsis appeared homo-geneously suspended in the medium, while the filamentouscyanobacteria showed, in general, heavy wall growth.

A second isolation method was the direct single cell orcell aggregate transfer into the liquid medium with the helpof inverted microscope (Zeiss Axiovert 10, Germany) equippedwith a micromanipulator (Zeiss, Germany). The CooperDish technique (Waterbury & Stanier 1978) gave very goodresults as well. In this case, small aggregates were placedon agar plates and observed microscopically until the nexttransfer was advisable.

The isolates were grown in MN, half-concentrated MN(0.5 × MN), double-concentrated MN (2 × MN) liquid me-dia prepared according to Waterbury and Stanier (modi-fied) (1978), and BG 11 according to Rippka et al. (1979).Cultures were kept at room temperature in Erlenmeyer flaskswithout further aeration and were illuminated with naturaldaylight or with Osram tungsten light tubes of 200 lux. Allisolates showed very good growth on a solid substrate suchas ∅0.5 mm glass beads (B. Braun Melsungen AG, Ger-many), sea sand (Merck, Germany), and glass fibber filters(Whatman 934-AH). Sterile glass beads or sea sand wereused as an adhesion substrate for the isolated strains andwere simply spread on agar plates. Glass fibber filters wereprepared by packing five ∅90 mm filters into the bottom ofa glass Petri dish (100 mm × 20 mm), covering the plate,and then sterilizing the plate by heating at 260°C for 30 min.After cooling, the filters were saturated with 20 ml of ster-ile liquid medium.

Microscopical investigations

Colony, aggregate, and cell morphology were examinedusing a Zeiss photomicroscope III. Pictures were taken onKodak EPY64 tungsten films. Cell and aggregate dimen-sions were measured with an ocular and an objective mi-crometer.

For scanning electron microscopy, the cell suspensionsor sediment grains with the attached cyanobacteria werespread onto poly-l-lysine coated glass slides (Tsutsui et al.1976) while small mat pieces were wrapped in thin blot-ting-paper (lens paper, neoLab Heidelberg), fixed in 4%glutaraldehyde (Fluka Chemie, Switzerland) in 0.1M so-dium phosphate buffer (pH 7.2), and washed two times inthis buffer. They were then dehydrated in a series of etha-nol-water solutions starting with 10% of ethanol, then pro-ceeding through seven steps to pure ethanol, with each steplasting 30 minutes. The dehydration series ended with twowashes in pure ethanol. Afterwards, the samples were criti-cal-point dried in an Balzers Union (CPD 010) apparatus,before gold sputtering (Balzers Union, SCD 030). The ob-jects were examined with a Zeiss DSM 940 or a Hitachi S-450 scanning electron microscope operated at 10 or 20 kVand with working distances of 7 to 9 mm. Pictures weretaken on Agfapan APX25 or Ilford FP4 films.

ANN. BOT. FENNICI 35 (1998) • Patterns of growth in coccoid cyanobacteria 221

RESULTS

Several Merismopedia-like forms were observedin the field in the Mellum microbial mats. Severalof these forms have been isolated in uni-cyanobac-terial culture (Stal & Krumbein 1985, Krumbein1987). One of the original isolates of 1984 is stillmaintained in Oldenburg under the strain assignmentOl 86. On the basis of morphological features, itwas attributed to the species Synechocystis sp.(Geitler name: Merismopedia punctata). To studythe phenomenon of the considerable differencesbetween the phenotype in the laboratory culture,and the phenotype (or ecophene) in its proper eco-logical niche, more intense, new observations andisolations were carried out in 1991.

Two types of Merismopedia-like forms wereisolated in these isolation attempts (Mellum 1991).One larger type (OL 202) with cell sizes between4.0–6.7 µm, and a smaller one (OL 201) with cellsizes varying between 1.9 and 3.8 µm. After sev-eral transfers in medium BG 11, strain Ol 201 lostcompletely the potential for aggregate formation,while the original cell cluster transferred continu-ously in medium MN maintained this capacity.The strain assignment Ol 201a was given to theisolate that now had to be regarded as Synecho-cystis sp. (no aggregate formation).

Strain OL 201, according to the classical as-signments of Geitler (1932), could be attributedto the species Merismopedia punctata. Strain OL

202 exhibits features known for M. elegans (sensuGeitler), and was grown on the BG 11 mediumwith sediment extract from the Wadden Sea (30 mlin 1 litre). The ability to grow in aggregates didnot change much under these laboratory condi-tions.

Seven Merismopedia-like cyanobacteria werecollected and isolated from the microbial mats atNorderney Island. After several subculturingsteps, five strains that are still kept as uni-cyano-bacterial cultures in our laboratory have remained.These strains could represented M. glauca, M. punc-tata and M. elegans sensu Geitler (1932).

The formation of giant cells (Figs. 1 and 2),and giant cell aggregates (Figs. 3 and 4) was fre-quently observed in all cultures isolated from Mel-lum (1984 and 1991), and from Norderney (1991–1995), especially in the first 8 transfers upon iso-lation. These abnormal cell sizes (2–3 times big-ger than common cells) are most probably resultsof irregular (possibly delayed) division in one ortwo planes. The occurrence of giant cells and cellaggregates in otherwise smaller Merismopediacultures further hint to the possibility that new,different conditions in the culture influence themorphology of the isolates. This, together withfield observations and molecular analyses ulti-mately points to the necessity of combining fieldobservation, culture observation, and moleculartechniques for identification of growth character-istics of individual cyanobacteria.

Fig. 1. Irregular divisionpattern with formation ofmegacyte, frequently ob-served in culture. [Bar = 25µm].


DISCUSSION

The composition and development of microbialmats in the intertidal sediments of the WaddenSea in the southern North Sea have already beenstudied for several years by members of the Geo-microbiology Division of ICBM at the Univer-sity of Oldenburg (Stal 1985, Stal & Krumbein1985, Gerdes et al. 1987, Krumbein et al. 1994,Palinska et al. 1996).

In the early stages of the microbial mat devel-opment, coccoid cyanobacteria such as Gloeocap-sa sp., Synechocystis sp., Synechococcus sp. andMerismopedia sp. were frequently observed. Theirecological position and importance in the mat aswell as their generic assignment is not yet under-stood. In order to get new isolates, we decided touse the classical sites, namely the microbial matsdescribed by Oerstedt (1841) for the Baltic, andby Schulz (1936) for the North Sea.

The genera Eucapsis, Merismopedia, Synecho-cystis and Synechococcus are usually expected tocomprise coccoid cyanobacteria reproducing bybinary fission (or regular division in contrast toe.g., multiple fission in the Pleurocapsaleans).They have been subdivided on the basis of thecell size, the division pattern and aggregate for-mation. In the classical nomenclature all the fourgenera belong to the order Chroococcales. Geitler

(1932) classified them into four different generawithin one family and order, while Komarek andAnagnostidis (1986) put them together in one fam-ily Microcystaceae instead of Chroococcaceaemaintaining, however, the Geitler’s order name.They included into this order all coccoid cyano-bacteria and introduced new families or familydivisions by separating those coccoid cyanobac-teria that have division planes perpendicular toeach other from those with irregular divisionplanes, a separation not applied so strictly by theGeitlerian system.

The approach of Komarek and Anagnostidis(1986) to the taxonomy of this complex group isa significant departure from the Geitlerian sys-tem. The most important difference between thetwo approaches is the departure from classifyingorganisms into different systematic groups onlyon the basis of their ability of forming or main-taining cell aggregates. The presence of cell ag-gregates seems to be an unreliable taxonomic char-acteristic. They, however, stressed the importanceof other features, namely a type of reproduction(binary or multiple fission, budding) or the orien-tation of the division planes (perpendicular or ir-regular). All these features, however, seem to bevariable when cultures of the group are studied.Holtkamp (1985) for example showed that the di-vision planes of Aphanothece halophytica are dis-

Fig. 2. SEM-photomicro-graph of a subculture ofMerismopedia-like isolate,exhibiting delayed divisionwith megacyte production.


torted considerably, and even to rich filamentousmorphologies under positive or negative osmoticstress. Golubic et al. (1996) showed highly vari-able cell forms and sizes in field material and cul-tures of Solentia sanguinea. Komarek and Lund(1990) and Palinska et al. (1996) found that theSpirulina/Arthrospira complex may also showconsiderable morphological variations through en-vironmental stress and other cell wall and divi-sion related factors (e.g. pore size and thicknessof cell walls in the punctured portions of the cellwall). The slime production in a culture is alsoaffected by the growth phase of the cyanobacteria,by the conditions under which they were grown,and by the degree of purity of the culture (Becker1992).

In addition, to our knowledge, practically noextended physiological work has been done andreported on isolates going back to Merismopediaor Eucapsis field “types” although in several cya-nobacteria collections benthic and planktonicforms are maintained.

The few data collected so far on the divisionpattern, slime formation, cell sizes, and aggregatebuild-up of this complex group of coccoid cyano-bacteria dividing in one, two or three regular orirregular planes do not allow far-ranging conclu-sions. Physiological and cultural experiments onisolates of the group seem to lack completely(Geitler 1932, Niell & Anadon 1978, Rippka etal. 1979, Stal & Krumbein 1985).

It has been shown in the cultural experimentswith three different isolates that irregular colonypatterns may emerge from regular ones within oneclone. Further, it has been shown that an origi-nally aggregate forming clone can transform irre-versibly into a culture that forms irregular aggre-gates or single cell homogeneously growing formswhich according to classical taxonomy should benamed Synechocystis sp. or Synechococcus sp.

Cell division in coccoid cyanobacteria exhib-its (generally speaking) “normal” binary cell di-vision or production of so called “spores” or“cytes”. “Endospores” or “nanocytes” (“nanno-cystes” in Bourrelly 1970) arise by rapid succes-sive division of a single cell into several smalldaughter cells. Waterbury and Stanier (1978) andothers use the term “baeocytes” instead of “endo-spores”, because the latter term already has a dif-ferent sense in bacteriology. Chadefaud (see Bou-relly 1970) proposed the term “coccospore” in-stead of “endospore”. Fritsch (1945) called daugh-ter cells which grow to the original cell size andare liberated singly “planococci”. Bourelly (1970)defines the “planococci” (“planocoques”) as mo-tile “coccospores”. Kaas (1983) proposed the term“monospore” for similar reproductive cells, whennon-motile, analogous to such in the Rhodophy-ceae.

The differences in cell diameters which havealready been frequently observed before (Palinska& Krumbein 1994) are not necessarily good taxo-

Fig. 3. An aggregate oforiginally 4 cells (right cor-ner). One of the cells divid-ed properly into 4 new cellsgiving rise to an aggregateof 16 cells; the upper rightpackage of 8 showing thefourth division while threecells of the original pack-age of four remained un-divided but formed giantcells. [Bar = 25 µm]


nomic characters. The formation of giant cells andgiant cell aggregates as an irregular division pat-tern was frequently observed especially in the firsteight transfers upon isolation. The formation ofgiant cells or abnormal, small cells or cell aggre-gates is certainly affected by an irregular divisionpattern caused by a delayed division or faster, ac-celerated division.

In the course of these studies, it was possibleto show that accelerated and delayed divisions oc-cur in this Chroococcales cyanobacteria group asopposed to the multiple fission pattern in Pleuro-capsalean cyanobacteria. Because of the great dif-ferences and two different terms used by Water-bury and Stanier (1978, baeocyte), and Komarekand Anagnostidis (1986, nanocyte), a new classi-fication of cells reflecting not only their sizes, butalso their division pattern is proposed. It is sug-gested to use nanocyte for cases of accelerateddivision and a new term megacyte for cases ofdelayed division in cell size differentiation of Me-rismopedia punctata, whereas baeocyte should berestricted to more or less rapid, multiple fissionfollowed by a release of motile or non-motile “pro-pagules” from the glycocalyx of the precursor cell,in the group of Pleurocapsales. This in turn, fur-ther differentiates the principles and developmentlines of the coccoid group of Merismopedia-likestrains from the Pleurocapsalean group, in whichusually only the single cell unit is motile.

The giant cells — in contrast to dwarf cells

called nanocytes by Komarek and Anagnostidis(1986) — are, thus, called megacytes while theterm baeocyte introduced by Waterbury and Sta-nier (1978) points rather to developmental cyclesin the reproduction. The occurrence of nanocytesand megacytes may be a consequence of adapta-tion to different field conditions and seasonal orannual limitations. Microbial mats of the “Farb-streifen-Sandwatt” type, which the Mellum andNorderney mats are, are a complex, dynamic sys-tem, and that is, why one will always witness theestablishment of several ecotypes or ecophenesof the same species type in terms of molecularand physiological characteristics.

The division between the two major groups ofcoccoid cyanobacteria, however, can still be main-tained. Further, since the time of Cohn (1853) andHansgirg from Prague (1892) it has still been dis-cussed if “swarmed” (“Schwärmerzellen”) cells(e.g., baeocytes sensu Waterbury) are to be foundin Section I (Rippka et al. 1979), especially in the“Synechocystis-Merismopedia” complex. Finally,it depends on the mode and time of separation ofa single nanocyte or megacyte whether the proc-ess can be called budding as well. Thereby, a junc-tion with Chamaesiphon, where usually only theterminal cells divide could be made.

In short, there is a remarkable difference be-tween megacyte and nanocyte as compared to anendospore or baeocyte formation and a normalbinary fission. We agree with Komarek and Anag-

Fig. 4. Pattern of delayeddivision in the aggregateforming Merismopediapunctata. Giant cell aggre-gates are clearly visible.[Bar = 25 µm].


nostidis (1986) who classified coccoid cyanobac-teria in a single, big order Chroococcales. We pro-pose, however, to consider ephemeral motility ofdaughter cells derived from a rapid multiple fis-sion as a fundamental taxonomic character, where-as motility of grown-up mature vegetative cells,cell aggregates and trichomes seems to be wide-spread and complex in character.

Finally the data presented point to the possi-bility that, in terms of the embracing taxonomy,cyanobacterial genera and species could be keptmuch more flexible in the strain histories. Themolecular taxonomy, thus, does not not make theidentification easier or replacie the older systems.It is adding a new tool and dimension to the ques-tions of taxonomy and taxonomy related physi-ological and ecological characteristics of the liv-ing world.

All the three genera Synechocystis, Merismo-pedia and Eucapsis are well distinguishable in na-ture: Synechocystis lives as solitary cells, Meris-mopedia forms rectangular flat, and Eucapsis cu-bic colonies. All the three genera are clearly dif-ferent in the reproduction type as well. Transi-tions were not found in natural populations, andthis is the reason why all these three genera arestill classified separately by classical taxonomists,although the situation in culture is entirely differ-ent. The slime layers very often disappear and theresulting strains grow as solitary cells. It happensoften in Merismopedia strains but also in Eucapsis,Microcystis and various Aphanocapsa isolates.Thus, if one obtains a strain of the “Synechocys-tis”-type where the cells grow solitary, it is verydifficult, if not impossible, to identify the genericclassification without the knowledge of the origi-nal material from the field i.e. the “strain history”.The Synechocystis and Eucapsis strains (popula-tions) unlike Synechocystis/Aphanocapsa/Meris-mopedia -strains, can be easily distinguished bythe study of division types in special chambers oron agar plates (Kovacik 1983). Maybe, the closegenotypic relatedness (or identity?) of these threegenera will be proved with the help of 16S rDNAanalysis.

The intrageneric taxonomy of Merismopediais still very foggy. According to traditional ap-proaches (e.g., Geitler 1932), the various speciesdiffer practically only in cell dimensions. Mod-ern studies on Merismopedia sp. are lacking com-

pletely. As it was shown in Results, the cell sizevariability is evidently wider than it is known inthe literature. Populations with more or less sta-ble cell sizes, but also other ones that by changesin cell diameter cover the range of several spe-cies, exist. From all these results another ques-tion arises: how to classify types, constantly dif-ferent in their various environments, but beingidentical according to 16S rDNA sequences (Pa-linska et al. 1996)? They evidently exist in na-ture, in different biotopes. In their environmentthey are morphologically well distinguishable, andplay a different role in the biotope.

The stability and existence of various differ-ent phenotypes in one uniform biotope (whichreally exists) must be explained at first. The mo-lecular methods are certainly to be used parallelor as supplement to the phenotypic characterisa-tion but never to replace it. Otherwise, the moreprecise and perhaps more reliable molecular datawould produce confusing results concerning theoccurrence of cyanobacterial genera and speciesin nature. This holds true not only for the pheno-menological aspects (relatively stable and partiallylargely different morphotypes in the field) but alsofor the processes these taxa regulate in a naturalenvironment (e.g., capability of different types ofphysiology, motility, fertility and genetic ex-change).

The molecular taxonomy thus, in the sense andstage we have it now, is unfortunately not makingthe identification easier or replacing the older sys-tems. It is adding a new tool and dimension to thequestions of taxonomy, and taxonomy relatedphysiological and ecological characteristics of theliving world.

The concept of species (and/or genera, strains,phenotypes etc.) in cyanobacteria, especially incoccoid ones, must be changed in the future, butthere are yet not enough data for this evaluation,and the modern molecular tools are not yet suffi-ciently developed. In some cases, we simply needto acknowledge the limits to our actual taxonomicpower, as methods and approaches in any scien-tific inquiry are not perfect.

It is very artificial to divide the cyanobacterialrealm into morphological, ecological, bacterio-logical, molecular or other genera or even spe-cies. Exactly this, however, is presently and un-fortunately, a fact. The current cyanobacterial tax-


onomy must be changed, and it is obvious thatonly the combined, polyphasic molecular — cy-tomorphological — ecological approach can beapplied.

CONCLUSION

Several Merismopedia-like forms were observedin the field in the microbial mats of the East FresianIslands. The formation of giant cells and giant cellaggregates was frequently observed in all isolatedcultures. The formation of giant cells or abnor-mal, small cells or cell aggregates is affected byan irregular division pattern, as a result of a de-layed or accelerated division. One should consideras well the effectiveness of cyanophages as a pos-sible factor causing changes in the division pat-tern. Unfortunately, there are still few studies onviral infections done in natural populations of cya-nobacteria.

In the course of these studies, it was possibleto show that accelerated and delayed divisions oc-cur in this cyanobacteria group as opposed to themultiple fission pattern in Pleurocapsalean cyano-bacteria. Because of the great differences, and twodifferent terms used by Waterbury and Stanier(1978; baeocyte) and Komarek and Anagnostidis(1986; nanocyte), a new classification of reflect-ing not only cells sizes, but also their division pat-terns is proposed. It is suggested to use nanocytefor cases of accelerated division, and a new termmegacyte for cases of delayed division in cell sizedifferentiation of Merismopedia punctata, where-as baeocyte should be restricted to more or lessrapid, multiple fission followed by release of mo-tile or non-motile “propagules” from the glycoca-lyx of the precursor cell, in the group of Pleurocap-sales.

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Patterns of growth in coccoid, aggregate forming cyanobacteria

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