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Protistology 4 (4), 311-317 (2006/7) PPQtlStQlQ&y
Genomic dactyloscopy of Chlorella sp., symbionts of Paramecium bursaria
Irina N. Gaponova,1 Eugeny E. Andronov,2 Aleksandra V. Migunova,2 Konstantin P. Vorobyev,1 Elena P Chizhevskaja 2 and Konstantin V. Kvitko 2
1 Department of Genetics, All Russia Research Institute for Agricultural Microbiology, Pushkin-8, St.-Petersburg, Russia
2 Laboratory of Microbiology, Biological Research Institute of St. Petersburg State University, Stary Petehof St. Petersburg, Russia
Summary
The exon nucleotide sequences of the last part of the 18S rRNA gene in the northern and the southern ecotypes of zoochlorella strains (in the interval from 1315 to 1766 sites in accordance with sequence X56105 of Chlorella kessleri strain SAG-211-11g) were compared with the exon sequence of this gene in different Chlorella strains. These two ecotypes were shown to be the closest neighbours of Ch. vulgaris, Ch. soroki^aM, Ch. lobophora. All strains of northern and southern ecotypes were different from free-living Chlorella vulgaris in having introns in the first part of the 18S rRNA gene. The southern zoochlorellae had at least one more intron in middle part of the gene. We assume that in the course of evolution the ancestors of the northern zoochlorellae (Ch. vulgaris) got an intron (of the size about 330 nucleotides in the interval of 106-1315 b.p. of the 18S RNA gene), and southern zoochlorellae have got several introns, one similar to that of the northern ecotype in size and position and the others in the part of the gene, 1276-1766 (the size 647 nucleotides) and 495 b.p (according to the data of Hoshira et al., 2004, 2005) at the end of the gene.
Key words: Chlorella, PCR, 18S rRNA, phylogeny, introns, ecotypes of zoochlorella
Introduction
Unicellular green algae, zoochlorellae, are a part
of the symbiotic system "Paramecium bursaria -Chlorella sp. - PBCV virus (Chlorovirus, Phycodna-
viridae)". The cytoplasm of a Paramecium bursaria individual may contain several hundreds of zoochlorella cells. Each algal cell is enclosed in an individual perialgal vacuole, protecting it both from the ciliate's digestive enzymes and from contacts with the virus.
© 2007 by Russia, Protistology
According to their sensitivity to specific viruses, zoochlorella strains isolated from ciliates were divided into two types: NC64A and Pbi (Van Etten, 2003). We refer to them, respectively, as the "southern" and the "northern" ecotype (Kvitko et al., 1996, 2001, 2004; Migunova et al., 1996, 1999, 2000). It is not clear whether these two zoochlorella ecotypes belong to the same species or to different species. In cell morphology, the northern and southern Chlorella sp. are close to Chlorella vulgaris (Reisser et al., 1988), whereas in cell wall structure they are close to the Ch. vulgaris/Ch. sorokiniana group. Various strains of the same ecotype are identical in numerous physiological-biochemical parameters (Kessler et al., 1991) and other characteristics, such as sugars secretions, size of protein markers, 8 isoenzyme patterns (Linz et al., 1999) and surface antigens (Migunova et al., 1992), but strains of separate ecotypes are different. Genomic dactyloscopy by UPPCR-patterns (Migunova, 2002) allows one to divide zoochlorella strains into two different types (the northern and the southern ecotype) or, probably, even into two different species. The aim of our investigation was to reveal differences and similarities between zoochlorella ecotypes at the genomic level.
Material and Methods
Algal strains and culture conditions
We used virus-sensitive and virus-resistant Chlorella sp. strains isolated from P. bursaria as well as free-living Ch. vulgaris from the CALU collection of the Biological Research Institute (St. Petersburg State University) (Table 1). All strains were grown in standard BBL medium diluted to 1:4 (1.25 g of gelatin, 0.75 g of meat extract and 15 g of agar for 1 l of water), in tubes with agar slants at 15-28°C. Illumination was 2000-4000 lux.
For strain cultivation mineral media BBM was used: NaNO3 - 250 mg/ml, KH2PO4 - 175 mg/ml, K2HPO4 - 75 mg/ml, CaCl2H2O - 25 mg/ml, MgSO4
- 75 mg/ml, NaCl - 25 mg/ml, microelements - 1 ml); BBM + AP (10 % aminopeptid); BPS: BBM+peptone
- 1 g/l, sucrose - 5 g/l. For preparation of solid nutrient medium BPS, 15 g/l of agar was taken. Flasks with a volume of250-300 ml filled with 200 ml of liquid media BBM, enriched with AP (10 %), or Petri dishes with solid BPS nutrient medium were used for cultivation. They were kept at 15-28°C and illuminated by luminescent lamps (2000-4000 lux).
DNA MANIPULATION
DNA isolation from Chlorella strains was performed with "Nucleon PhytoPure" plant and fungal DNA extraction kits (Amersham) according to the producer's
instructions. 18S rRNA fragments were amplified using MyCycler (BioRad) thermocycler and DyNAzyme II DNA Polymerase (Finnzymes). The primers used are listed in Table 2. All PCRs were performed with the following temperature profile: an initial denaturation at 95°C for 3 min, 30 cycles of denaturation (30 s at 94°C), annealing (30 s at 55°C), extension (1 min at 72°C) and final extension at 72°C for 3 min. Amplified DNA was examined by electrophoresis in 1% agarose gel.
Aliquots of the DNA amplified were digested with HaeIII and MspI restriction endonucleases. The restriction fragments were separated in 4% agarose gels, stained with ethidium bromide and photographed with the Kodak EDAS290 system. 100 b.p. DNA ladder was used as a molecular weight marker. For sequencing, the fragments amplified were cloned in pTZ57R/T plasmid, "Ins T/A clone TM PCR Products Cloning Kit" (Fermentas) was used. The fragments cloned were sequenced with ABI-310 DNA sequencer according to the producer's instructions. RFLP tree was constructed with the help of MEGA version 3.0 (Saitou and Nei, 1987; Felsenstein, 1985).
Nucleotide sequences determination
The sequences obtained were submitted to the Gene Bank (accession numbers AY876290-AY876301). Intron locations were determined by aligning sequences obtained with the 18S rRNA gene of Chlorella kessleri strain SAG 211-11g (accession X56105) by using ClustalX program (Thompson et al., 1997). For 18S rRNA tree construction all introns were excised from sequences. The tree including some Chlorella representatives was constructed by simple estimation (number of differences) and the neighbor-joining method using MEGA version 3.0 (Saitou and Nei, 1987; Felsenstein, 1985). Bootstrapping with 1000 samplings was used to evaluate clusterization.
Results
According to the results of PCR amplification with 3F/4R and 6F/HR primers, the Chlorella strains investigated can be divided into three groups by the size of the fragments amplified (Fig.1 and 3). Comparison between the free-living Chlorella vulgaris strain, the northern ecotype and the southern ecotype has shown that the latter group had an insertion (more than 300 b.p.) between positions 106 and 1296 ns (Fig.1).
To study genetic relatedness of the strains, the restriction analysis of 18S rRNA gene fragments amplified with primers 3F/4R was performed. MspI and HaeIII restriction fragments turned out to be strictly specific for the three Chlorella groups studied. Moreover,
Table 1. Strain description.
Strain abbreviation Year of isolation, provenance, author and characteristics of the strain
Strains sensitive to NC64A type viruses (Van Etten, 2003), or southern ecotype (Kvitko et al., 1996, 2004)
NC64A 1963; the USA; M. Karakashjan and L. Muskatin. Received from J.L. Van-Etten. The type strain for the southern ecotype.
Ac-21SCp21 1988; subclone of str. NC64A (Migunove et al., 1002). Selected as resistant (able to growth on solid media with addition of streptomicin, canavanin, P-alanin).
211-6 1934; the USA; Loeffer strain. Received from Goetingen in 1990, SAG-211-6
N-1-A 1986, the USA, R. Meints (state Nebraska), received from J.L.Van-Etten
Strains sensitive to Pbi type viruses (Van Etten, 2003), or the northern ecotype (Kvitko et al., 1996, 2004)
OCH 1985; Karelia, Loukhsky area, Lake Cherlivoe; E. Kraeva
ОСН cr4; ОСН cr 6 1999; subclones of str. OCH (Canavanin-resistant mutants); M.J. Prokosheva and A.V. Migunova.
OS-1 and OS-6 1999; Karelia, Loukhsky area, island Sredny; M.J. Prokosheva and A.V. Migunova
241-80 1974; Germany, Goettingen, a pond in the Botanical garden; W. Koch
Pbi 1974, Germany, Goettingen, the same pond in the Botanical garden; W. Reisser. The type strain for the northern ecotype
Strains of free-living Chlorella vulgaris insensitive to viruses of both types
CALU-183 1892; M. Beijerinck. The type strain of Chlorella vulgaris. Received from Cambridge Collection in 1964. SAG211-11 b=UTEX259=CCAP211-11 b=CALU 183
CALU-157 1962. from loamy ground of r. Kuban’, Russia. Isolated and identified (as Chlorella vulgaris) by B.V. Gromov.
HaeIII enzyme allowed the determination of genotype differences in free-living Chlorella vulgaris strains and in the northern ecotype strains (Fig. 2).
However, the level of homology inside zoochlorella groups was very high. The difference in restriction patterns between the ecotypes might be determined by insertions of introns.
Zoochlorella strains of southern ecotype, in their turn, had an insertion (more than 500 b.p.) between positions 1315 till 1750 compared to the free-living Chlorella vulgaris and zoochlorella strains of north
ecotype (Fig. 3). The insertions detected are presumably introns, that can be found occasionally in 18S rRNA genes of different Chlorella species (Huss et al., 1999).
Presence or absence of introns in these two positions can be used to distinguish between free-living Chlorella vulgaris (no introns), zoochlorella of the northern ecotype (one intron) and zoochlorella of the southern ecotype (two introns). However, presence of introns in other positions in the end of the 18S rRNA gene of the strains in question cannot be ruled out. To do so, nucleotide sequences of the 18S rRNA gene of
Table 2. Primers used in the work.
Primer Nucleotide sequence Location*1 Reference
1F 5’-WACCT GGTT GAT CCT GCCAGT-3’ 1-21 Huss et al., 1999
3F 5’-AACTGCGAATGCCTCATTAAA-3’ 86-106 this work
6F 5’-ATGGCCGTTCTTAGTTGGTG-3’ 1276-1295 this work
2R 5’-GTAGGTGAACCTGCAGAAGGATC-3’ 1773-1795 this work
4R 5’-GGTTGCCTTGTCAGGTTGAT-3’ 1296-1315 this work
5R 5’-AAACGGCTACCACATCCAAG-3’ 399-418 this work
HR 5’-GGAGAAGTCGT AACAAG- 3 ’ 1750-1776 Huss et al., 1999
*’ Here and in the following text all positions are given in accordance with sequence of 18S rRNA strain SAG-211-11g (X56105).
Fig. 1. Amplificates of the first part of the 18S rRNA gene (from 106 till 1296 sites) of free-living Chlorella vulgaris (F), southern (S) and northern (N) zoochlorella strains. Only CALU-157 and CALU-183 do not have introns in this part of the gene. Markers - 6BstE-III, Ladder 100 b.p.
Chlorella strains belonging to separate species were aligned with the gene sequence of the Ch. kessleri strain SAG 211-11g lacking introns. Besides, the BLAST programme revealed, after intron sequences of the southern zoochlorellae (strains NC64A, Ac21ckp21 and N-1-A) were used as query, a zoochlorella strain So13-7k of the Japanese origin (Hoshina et al., 2GG4), whose 18S rRNA gene had 3 introns. Altogether, the search produced 4 types of introns, with different localization sites. Table 3 presents the list of symbiotic Chlorella strains containing introns that were investigated in the present work.
The above difference in the number of introns between zoochlorella strains allows us to make an
assumption about the origin of the northern and the southern ecotype at the level of the 18S rRNA gene. The 18S rRNA gene of zoochlorellae appears to have originated from the homologous intron-lacking gene of free-living Chlorella by means of consecutive inclusion first of one intron (northern zoochlorellae) and then of other introns (southern zoochlorellae) (Fig. 4).
According to T. Yamada (Yamada et al., 1994), intron introduction into the 18S rRNA gene of zoochlorellae can be caused, directly or indirectly, by transfer by the third party involved in symbiosis, the virus. BLAST search demonstrated the identity of the rRNA fragment sequenced (1142 b.p.) with 18S rRNA gene fragments ofsix Chlorella isolated from Paramecium bursaria in Japan (accessions AB162912-AB162917, Hoshina et al., 2GG4). This fact, in combination with the presence of an additional intron between positions 1G6 and 1296, gives us reasons to suppose that the strains of the southern ecotype studied in our work are close relatives of the Chlorella strains from Japan.
By the program BLAST, using as inquiry intron sequences of southern zoochlorellae, strains NC64A, Ac21SCp21 and N-1-A, we have found out that zoochlorella strains, So13-Zk (and some others) of Japanese origin (AB162 912-AB162917; Hoshina et al., 2GG4, 2GG5), containing three introns, are highly similar to 18S rRNA gene fragments of southern zoochlorellae, studied in this work.
On the basis of 18S rRNA gene sequences (the 3' exon part), a genetic tree was constructed
Fig. 2. Similarity tree of the southern and the northern zoochlorella ecotype and Chlorella vulgaris, inferred from restriction pattern of the first part of the 18S rRNA gene sequence (106-1296 ns, accession numbers AY876290-AY876301).
Fig. 3. Amplificates of the middle part of 18S RNA gene (from 1315 till 1766 sites). Only southern zoochlorella (group S) have the insertion. Markers - 6BstE III, Ladder 100 b.p.
by the neighbor-joining method (Fig. 5). The tree topology together with the bootstrap values shows that all the strains of the northern and the southern ecotype are closest to 3 species of Chlorella (sensu stricto): Ch. vulgaris, Ch. lobophora and Ch. sorokoniana. While the northern zoochlorella strains are closer to Ch. vulgaris and Ch. sorokiniana, the southern strains are closer to Ch. lobophora and form, together with the Japanese strain So13-7k, a separate cluster with a high bootstrap value. These findings suggest that the northern and the southern ecotypes may belong to two different species. We will be able to tell whether this is indeed so after complete rRNA sequencing.
Acknowledgments
The work was supported by the Russian Foundation for Basic Research (projects 02-04-48676, 05-04-49239 and 05-04-9274) and in part by the Program of Ministry of Science and Education (grant 2.2.3.1.4148), CRDF BRHE 4056 (for E.E. Andronov). The authors are grateful to Natalia V. Lentsman for editing the English version of the manuscript.
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51
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Address for correspondence: Konstantin V. Kvitko. Laboratory of Microbiology, Biological Research Institute of St. Petersburg State University, Oranienbaumskoye sh. 2, Stary Peterhof, 198004, St. Petersburg, Russia. Email: konstantin.kvitko@paloma.spbu.ru
Editorial responsibility: Alexander Yudin
