FEMS Microbiology Letters 122 (1994) 109-114 Federation of European Microbiological Societies Published by Elsevier

109

FEMSLE 06159

Characterization of two new thermoacidophilic microalgae: Genome organization and comparison with Galdieria sulphuraria

David Moreira, Ana-Isabel L6pez-Archilla, Ricardo Amils * and Irma Marln

Centro de Biolog[a Molecular, Universidad Autdnoma de Madrid, Cantoblanco, 28049 Madrid, Spain

(Received 8 June 1994; revision received 12 July 1994; accepted 13 July 1994)

Abstract: Thermoacidophilic algae (Cyanidiaceae) constitute a taxonomic group with interesting phylogenetic and ecological implications. In this report, we have classified three thermoacidophilic microalgal isolates from Rio Tinto (Spain) using a combination of classical analysis of phenotypic features and the characterization of their electrophoretically determined karyotypes by means of pulsed-field gel electrophoresis. Using this technique, we have been able to demonstrate that thermoacidophilic algae genomes have the smallest genomes of all photosynthetic eukaryotes studied so far. In addition, we show that two of these Rio Tinto isolates may constitute new species within the genus Galdieria.

Key words: Galdieria sulphuraria; Thermoacidophilic algae; Acid environment; Pulsed-field gel electrophoresis; Electrophoretic karyotype

Introduction

Photosynthetic primary production in thermal acidic ecosystems is carried out by microalgae of the family Cyanidiaceae. Surprisingly, these eu- karyotic algae are the only photosynthetic organ- isms living in these extreme habitats, although prokaryotic microorganisms had traditionally been considered more adaptable to this kind of envi- ronment. The existence of some thermophilic cyanobacteria that can grow at temperatures above 45°C and even as high as 74°C [1], is well

* Corresponding author. Tel: 34 1 397 8077; Fax: 34 1 397 8344; E-mail: DMOREIRA@MVAX.CBM.UAM.ES.

known. This is the highest temperature at which photosynthesis has been reported to occur, but none of these bacteria tolerate acid pHs.

Sulfurous mining regions are suitable habitats for acidophilic microorganisms because sulfidic ores favour the establishment of low pHs, and these environments frequently exhibit high tem- peratures. Southwestern Spain is an important mining area due to the abundance of ferrous sulfide (pyrite) and other sulfur compounds. The Tinto river flows across this pyritic formation and exhibits, as characteristic features, a constant acidic pH and an extremely high content of heavy metals. Acidophilic algal populations are fre- quently found in this river and the neighbouring acid soils, several of them being thermophiles.

SSDI 0378-1097(94)00311-4

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The existence of thermoacidophilic algae was reported long ago, and the first isolate was classi- fied as Cyanidium caldarium [2]. Initially, the algal communities living in thermal acidic envi- ronments were considered to be formed only by members of this species [3]. However, later on, two other genera of the Cyanidiaceae were de- scribed [4,5], namely Cyanidioschyzon merolae and Galdieria sulphuraria, which thrive as mixed pop- ulations with C. caldarium. These three algae coexist in acidothermal habitats showing a world- wide biogeographic distribution [3,6].

Traditionally, numerical taxonomy has been based on the study of several phenotypic charac- ters. In our case, we have supplemented the study of the phenotypic characters of three thermoaci- dophilic microalgae isolates with their karyotyp- ing analysis using pulsed-field gel electrophoresis (PFGE). This technique, which allows resolution of DNA macromolecules, has made it possible to analyze complete microbial genomes. Elec- trophoretic karyotyping has proved to be of value in determining taxonomic relationships for sev- eral groups of eukaryotic microorganisms, such as tripanosomatids [7], different yeast taxa [8-10], fungi [11] and algae [12].

In this report, we describe the isolation and characterization of new thermoacidophilic algae, and their genomic analysis by means of PFGE, in order to demonstrate their relationship to the genus GaMieria.

Materials and Methods

Algal strains and media Galdieria sulphuraria strain 19.71 was obtained

from the G6ttingen University Algal Collection. The other algal strains were isolated from the mining region of Rio Tinto (Spain) as described below. All strains were cultured in modified As- cione's medium [13] supplemented with 0.5% dextrose and 0.05% malt extract, adjusted to pH 3 with H 2 S O 4.

Isolation of algae Algae samples were collected from the sedi-

ments and water of the Tinto river and from the

soils of different locations in the mining region. These samples were inoculated in liquid Ascione's medium and incubated for five weeks at 50 ° C with aeration, in order to enrich the cultures in thermoacidophiles. Aliquots of these cultures were spread on solid Ascione's medium and incu- bated at the described conditions until algal colonies were observed. Several colonies were respread on solid medium and individual colonies were inoculated in liquid medium to obtain pure cultures. Absence of bacteria and other contami- nants was verified by direct microscopical obser- vation.

Water chemical analysis Metal ionic contents of water (Fe 2+ and Fe3+;

Zn2+; Cu 2+) were determined by atomic absorp- tion spectrophotometry. SO4 z- concentration was valued from the turbidity data measured by means of a spectrophotometer.

Pigment extraction Algae cultures were centrifuged at 10000 X g

for 10 min and the harvested cells were stirred in acetone-methanol (7 : 2, v : v) at 4°C for 2 h. After removal of cell debris by centrifugation, ab- sorbance spectra were determined using a spec- trophotometer.

Preparation of intact chromosomal DNA Ceils were grown until late logarithmic phase

(5 X 10 7 cells ml - I ) in liquid medium and har- vested by centrifugation at 10 000 × g for 10 min, resuspended in PETT IV (10 mM Tris-HC1, 1 M NaC1) to wash the cells, and pelleted again under the same conditions. The cell pellet was resus- pended in protoplast forming medium [12] sup- plemented with 2% macerase (Sigma), 2% cellu- lase (Sigma) and 2% hemicellulase (Sigma) (w/v) and used to make plugs following the procedure described by Higashiyama et al. [12].

Pulsed-fieM gel electrophoresis Orthogonal-field alternation gel electrophore-

sis (OFAGE) and contour-clamped homogeneous electric field electrophoresis (CHEF) were per- formed using a Pharmacia LKB Pulsaphor sys- tem. Different size resolution windows were

achieved by using pulse times between 5 and 70 s. Typical running conditions were 330 V and 36 h for O F A G E gels, and 170 V and 36 h for C H E F gels. Sizes of algal chromosomes were deter- mined by comparison with the linear standards lambda D N A concatemers (Pharmacia) and Sac- charomyces cereuisiae YN295 chromosomes.

Results

Study site and algal habitats In this study, three algae strains were isolated

from the Rio Tinto mining region. Two strains, RT-M6 and RT-M9, were isolated from liquid samples collected from water drainages of leach- ing piles formed by complex polymetalic sulfides. Physicochemical characteristics of these samples are given in Table 1. The third strain, RT-I .1, was isolated from an acid soil area (pH 3) near the origin of the river. This alga forms a 2 -3 mm thick layer under the surface of amorphous gyp- sum crystals, and is found associated to several

A B

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bacteria and other acidophilic algae (Chloro- phyceae and Chrysophiceae) forming complex cryptoendoli thic communit ies in which this species is the most abundant one. The existence of such acidophilic cryptoendolithic communities has not yet been reported.

Morphological, structural and physiological charac- terization of Rio Tinto isolates

All three Rio Tinto isolates, as well as G. sulphuraria 17.91, share the following characteris- tics: (1) spherical shape with average sizes of 5.8 /zm for G. sulphuraria 17.91, 6 .0 /zm for RT-M6, and 6 . 2 / , m for RT-I .1 and RT-M9; (2) presence of several vacuoles and chloroplasts; (3) photosyntethic pigments: chlorophyll a and phycocyanin; (4) number of autospores: more than 4, usually between 8 and 16; and (5) facultative growth: photoautotrophic on min- eral medium and heterotrophic on medium sup- plemented with carbohydrates.

1 2 3 4 1 2 3 4 5

g

48.5J

Fig. 1. Separation of chromosomal DNAs of Galdieria species by OFAGE using 330 V for 36 h at different pulse times: I0 s (A); 50 s (B) and 70 s (C). Lanes: 1, lambda concaterners; 2, RT-M6; 3, RT-I.1; 4, G. sulphuraria 19.71; 5, S. cerevisiae. White arrowheads

indicate the position of RT-M6 and RT-I.1 larger chromosomes. Sizes are indicated in kilobase pairs.

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Table 1

Physicochemical characteristics of the sampling sites where RT-M6 and RT-I.1 were isolated

Isolate Site Water characteristics

[Fel g 1- ~ [Cu] g 1- l [Zn] g 1-1 [SO 4] g 1- l Conducti- Redox pH vity. mS potential.

mV

RT-M6 Water 2.14 0.2 0.24 12.75 11.89 419 2.5 drainage

RT-M9 Water 5.0 0.7 0.82 12.9 14.67 413 1.6 drainage

Comparative analysis of electrophoretic karyotypes. PFGE was carried out at different pulse times

in order to allow the resolution of the complete set of chromosomes that compose the genomes of RT-M6, RT-M9 and RT-I.1 isolates and G. sul- phuraria 17.91 collection strain (Fig. 1).

The total number of chromosomes was esti- mated from the number of bands, taking into account their relative intensities. We have esti- mated the chromosomal numbers as indicated in Table 2.

Genome sizes were calculated by summing the sizes of all the chromosomal bands found in each strain. G. sulphuraria 17.91 and RT-M6 show similar genome sizes while strains RT-M9 and RT-I.1 possess larger genomes (Table 2).

With the exception of a few chromosomes in certain strains, most of the chromosomes of these algae have sizes ranging from 100 to 420 kb. However, strains RT-M6, RT-M9 and RT-I.1 possess additional large chromosomes (of 514 and 930 kb). Strain RT-M6 also has another large chromosome of 538 kb. None of these three large chromosomes can be seen in G. sulphuraria.

Table 2

Genome characteristics of RT-M6, RT-M9, RT-I.1 and G. sulphuraria

Organism Chromosome Genome number size (mb)

G. sulphuraria 40 9.8 RT-M6 47 10.6 RT-I.1 57 14.2 RT-M9 57 14.2

Discussion

Morphological, structural and physiological features of the isolates RT-M6, RT-M9 and RT- 1.1 confirm their relationship to the genus GaMieria. In this sense, the analysis of their metabolic behaviour is very important: these al- gae can grow heterotrophically with a carbohy- drate supplement as well as autotrophically in a mineral medium exposed to light. These charac- teristics are exclusive to the genus Galdieria and differentiate it from the other genera of ther- moacidophilic algae, Cyanidioschyzon and Cyani- dium, which are obliged autotrophs [14].

Analysis of these phenotypic characteristics has shown that the three isolates and G. sulphuraria are undistinguishable at this level. Genomic anal- ysis is an efficient tool to differentiate microor- ganisms of similar phenotypic traits. For this pur- pose, it has been used successfully in the taxo- nomic description of several lower eukaryotic genera. For this reason, we have applied it to the study of the Rio Tinto isolates. We have deter- mined, by means of PFGE, some genomic charac- teristics of these algae: chromosome number, range of chromosome sizes and genome size. This karyotypic analysis corroborates the ascription of Rio Tinto isolates to the genus Galdieria, since their genomic characteristics are very similar to those of the collection strain, G. sulphuraria 19.71. A comparative analysis shows differences that allow us to affirm that they are distinct species, except in the case of RT-M9 and RT-I.1, which share identical electrophoretic karyotypes (data

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not shown). Thus, three Galdieria species can be distinguished: G. sulphuraria, RT-M6 and RT-I.1.

Electrophoretic karyotypes of the three species show the existence of chromosome length poly- morphism within the genus Galdieria: most Galdieria chromosomes have sizes in the range from 100 to 420 kb, while RT-M6 and RT-I .1 also have some larger chromosomes (Fig. 1). This resemblance of the electrophoretic karyotypes of RT-M6 and RT-I .1 suggests that both isolates are more related to each other than to G. sulphu- raria. Nevertheless, genome sizes are not in agreement with this hypothesis since G. sulphu- raria (9.8 mb) and RT-M6 (10.6 mb) have similar genome sizes, while RT-I .1 shows a larger genome of 14.2 mb.

On the other hand, genome sizes of the Galdieria species are one of the most interesting findings of this work. To our knowledge, G. sul- phuraria 17.91 has the smallest eukaryotic genome so far characterized. Other studied microalgae possess genome sizes approximately four times larger, e.g. Chlorella ellipsoidea C87 and C. vul- garis C169 show genomes in the range of 38-39 mb [12]. The genomes of Galdieria species are more similar in size to those of several cyanobac- teria, for instance Anabaena sp. strain PCC 7120, which has a circular chromosome of 6.37 mb [15]. Among the eukaryotes whose genome sizes have been determined, only yeasts have comparable values (11-14 mb). However, in spite of this re- semblance in genome size, the genome organiza- tion of Galdieria species is more complex than yeasts. The relatively small genomes of G. sulphu- raria 17.91, RT-M6 and RT-I .1 are organized into an unexpected high number of chromosomes (from 40 to 57), while yeasts show smaller chro- mosome numbers ranging from 3 in Schizosacch- haromyces pombe to i6 in Saccharomyces cere- visiae. These small chromosome numbers are fre- quent in other lower eukaryotes, such as in fila- mentous fungi, the basidiomycete mushroom genus Pleurotus [11], and in Chlorella. Thus, the genome organization of the genus Galdieria seems to be unusual among the lower eukaryotes at the level of genome organization because it combines a small genome with a high number of chromo- somes. Analysis of other thermoacidophilic algae

should clarify whether this kind of genome orga- nization is common among these algae or is ex- clusive to the genus Galdieria.

We can show that the photosynthetic popula- tion in extreme thermal acidic environments is more diverse than has been described before. We have isolated two new species of the genus Galdieria from the mining region of Rio Tinto and characterized them at the phenotypic and genomic levels, demonstrat ing that this is not a monotypic genus, but it consists of at least three different species (G. sulphuraria, and the new isolates "Galdieria RT-M6" and "Galdieria RT- 1.1"). Galdieria species can not only be found in a variety of already described thermal and acidic habitats, including mining regions such as Rio Tinto, but can also form complex acidophilic cryptoendolithic communities with other prokary- otic and eukaryotic species.

Acknowledgements

We thank Dr. P. L6pez-Garcia for critical reading of a first version of the manuscript. This work was supported by a grant of the CICYT (BIO91-0223-C03-01) and an EEC grant (BIO2- CT93-0274 D G 12 SSMA). We wish to thank Foundation Ram6n Areces for its support. D.M. and A.I.L. are fellows of the Ministerio de Edu- caci6n y Ciencia.

References

1 Brock, T.D. (1967) Life at high temperatures. Science 158, 1012-1019.

2 Allen, M.B. (1959) Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch. Mikrobiol. 32, 270-277.

3 Brock, T.D. (1978) Thermophilic microorganisms and life at high temperatures. In: The Genus Cyanidium. Chapter 9, pp. 255-302. Springer-Verlag. New York, Heidelberg, Berlin.

4 Seckbach, J., Fredrick, J.F. and Garbary, D.J. (1983) Auto- or exogenous origin of transitional algae: an appraisal. In: Endocytobiology II (Schenk, H.E.A. and Schwemmler, W., Eds.) pp. 247-262. Gruyter and Co., Berlin.

5 Merola, A., Castaldo, R., De Luca, P., Gambardella, R., Musacchio, A. and Taddei, R. (1981) Revision of Cyanid-

114

ium caldarium. Three species of acidophilic algae. G. Bot. ltal. 115, 189-195.

6 Seckbach, J. (1987) Evolution of eukaryotic cells via bridge algae: the Cyanidia connection. In: Endocytobiology III (Lee, J. and Fredrick, J.F., Eds.), pp. 424-437. Ann. NY Acad. Sci. 503.

7 Giannini, S., Schittini, M., Keithly, J.S., Warburton, P.W., Cantor, C.R. and Van Der Ploeg, L.H.T. (1986) Karyotype analysis of Leishmania species and its use in classification and clinical diagnosis. Science 232, 762-765.

8 Coetzee, D.J., Kock, J.L.F. and Pretorius, G.H.J. (1987) The value of orthogonal-field-alternation gel electrophore- sis as a rapid identification process for some species repre- senting the genus Saccharomyces. J. Microbiol. Methods 7, 219-225.

9 Viljoen, B.C., Kock, J.L.F., Miller, M. and Coetzee, D.J. (1988) The value of orthogonal-field-alternation gel elec- trophoresis and other criteria in the delimitation of anamorphic-teleomorphic relations. System. Appl. Micro- biol. 11, 305-311.

10 Viljoen, B.C., Kock, J.L.F. and Coetzee, D.J. (1988) Or-

thogonal-field-alternation gel electrophoresis banding pat- terns of some asporogenous yeasts and their respective ascosporogenous states. System. Appl. Microbiol. 10, 228- 230.

11 Sagawa, I. and Nagata, Y. (1992) Analysis of chromosomal DNA of mushrooms in genus Pleurotus by pulsed-field gel electrophoresis. J. Gen. Appl. Microbiol. 38, 47-52.

12 Higashiyama, T. and Yamada, T. (1991) Electrophoretic karyotyping and chromosomal mapping of Chlorella. Nu- cleic Acids Res. 19, 6191-6195.

13 Ascione, R., Southwick, W and Fresco, J.R. (1966) Labo- ratory culturing of a thermophilic alga at high tempera- ture. Science 153, 752-755.

14 Seckbach, J. (1991) Systematic problems with Cyanidium caldarium and Galdieria sulphuraria and their implications for molecular biology studies. J. Phycol. 27, 794-796.

15 Bancroft, 1., Wolk, C.P. and Oren, E.V. (1989) Physical and genetic maps of the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain 7120. J. Bacteriol. 171, 5940-5948.