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Protistology 6 (1), 39—44 (2009)
Protistology
An atypical Chlorella symbiont from Paramecium bursaria
Konstantin Vorobyev1, Evgeny Andronov2, Maria Rautian1, Inna Skoblo1, Alexandra Migunova1 and Konstantin Kvitko1
1 Biological Research Institute ofSt.Petersburg State University, St.Petersburg, Stary Peterhof, Russia,
2 Laboratory ofGenetics and Selection of Microorganisms, All-Russian Research Institute for Agriculture Microbiology, St.Petersburg, Pushkin, Russia
Summary
A useful approach to rapid identification of Chlorella strains in a single cell of Paramecium bursaria was developed. A “nested” PCR-based method with primers flanking intron insertion sites in the 18S rRNA gene of Chlorella ensured fast screening of large P. bursaria collections for the presence of the two basic types of Chlorella endosymbionts without isolating them in pure culture. The method applied to several Paramecium isolates showed that the P. bursaria clone T-24-5 from Tajikistan presumably bears an atypical Chlorella symbiont possessing an atypical intron in the 18S rRNA gene. This sequence was cloned and sequenced. BLAST analyses of the intron sequence cloned showed the presence of136-bp stretch highly similar to the Chlorella virus intron fragment in major capsid protein Vp54 gene. The similarity value indicated a common origin of the sequences and suggested that the horizontal transfer of this DNA fragment had happened between the chlorella and the virus attacking it.
Key words: Paramecium bursaria, Chlorella, PBCV, 18S rRNA gene, group I intron Abbreviations: PBCV — Paramecium bursaria Chlorella virus
Introduction
Unicellular green algae, also called zoochlo-rellae, are part of the triple symbiotic system “Paramecium bursaria — Chlorella sp. — PBCV virus (Chlorovirus, Phycodnaviridae)” (Migunova et al., 1999). One cell of P. bursaria contains several
hundred green algae in the cytoplasm, as forage or endosymbionts. 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. Apart from virus protection, P. bursaria supplies its endosymbionts with some vitamins and nitrogen-containing organic substances. The
© 2009 by Russia, Protistology
Table 1. Paramecium bursaria clones used in this work.
Clone Origin Collection
G12 Russia, St. Petersburg St. Petersburg University, K.P. Vorobyev
E1 Russia, St. Petersburg St. Petersburg University, K.P. Vorobyev
PA-2-2 Russia, Altay Biological Research Institute of St. Petersburg University, M.S. Rautian
26-1 Russia, Novgorod Biological Research Institute of St. Petersburg University, M.S. Rautian
T-24-5 Tajikistan, Dushanbe Biological Research Institute of St. Petersburg University, M.S. Rautian
Bal Ukraine, Balaklawa St Petersburg University, Migunova
Cs-2 Japan Biological Research Institute of St. Petersburg University, M.S. Rautian
Ard-5 USA Biological Research Institute of St. Petersburg University, M.S. Rautian
Ard-16 USA Biological Research Institute of St. Petersburg University, M.S. Rautian
algae, in turn, produce for their host photosynthetic products — oxygen and sugars (maltose and glucose), so the symbiosis is practically obligate. Zoochlorella strains isolated from ciliates were divided into two types: “Southern” and “Northern” according to prefered temperature conditions and specific viruses hosted by them. Viruses of “Southern” type infect only “Southern” type of symbiotic Chlorella, whereas “Northern” type of symbiotic Chlorella is resistant to these viruses, and vice versa (Linz et al., 1999; Migunova et al., 1999, 2000; Van Etten, 2003, Kvitko et al., 2001, 2004).
All symbiotic Chlorella types (northern and southern ecotypes) and intron-bearing free-living forms are highly diverse regarding the number of introns and their location in the 18S rRNA gene (Hoshina et al., 2004, 2005; Gaponova et al., 2006; Vorobyev et al., 2006, 2007) (Fig. 1). Thus, introns in the 18S rRNA gene of Chlorella are good candidates for a taxonomic marker (in the “presence-absence” style); they are also a promising area for evolutionary investigations.
The total DNA from a Paramecium culture was already used for symbiotic Chlorella detection by Tanaka with colleagues (2002), but to simplify identification of Chlorella species in P. bursaria we have developed a “nested” PCR-based technique allowing identification of Chlorella ecotypes in a single cell of this ciliate. Such a method could be very useful for screening large collections of Paramecium (and other protists) for Chlorella symbionts, since it does not require the time-consuming isolation of zoochlorella in pure culture. Initially, we intended to report only the details of the method developed. However, when the method was applied to a Paramecium collection an unusual algae was detected. The 18S rRNA gene ofthis putative Chlorella symbi-
ont had an atypical intron containing 136-bp stretch highly similar to Chlorella virus intron fragment in major capsid protein Vp54 gene. The last finding turned out to be the main object of the article.
Material and Methods
The ciliates (Table 1) were cultivated at room temperature on lettuce medium inoculated with Enterobacter aerogenes at 15-22° and illuminated by luminescent lamps (2000-4000 lux) (Sonneborn 1970). Pure Chlorella DNA representing the southern (Chlorella sp. strain NC64A (USA, Nebraska University)) and the northern (Chlorella sp. strain SAG 241-80 (Germany, SAG collection)) ecotype were used as control in each experiment.
Primer design. As the quantity of DNA extracted from a single cell of Paramecium is very low, the “nested” PCR was used for amplification of the target sequences. Three primer pairs have been designed (Tab. 2, Fig. 1). Primers 3F/4R were used in the first round of the nested PCR. Primers SEV_EX_F/SEV_EX_R and YUG_EX_F/ YUG_EX_R flanking introns in the “Northern” and the “Southern” Chlorella ecotypes were used in the second round of the nested PCR, allowing ecotypes identification depending on fragment sizes (longer fragments with intron and shorter intronless ones). According to the results of the “in-silica” test, all the primers designed should fit perfectly for amplification of the target sequence in almost all known Chlorella — both free-living ones and Paramecium inhabitants.
DNA manipulation. From one to five cells of P. bursaria containing symbiotic Chlorella were taken with the pipet tip from the liquid culture medium,
18S rRNA gene
758 - 1081
327 648 496
1170 - 1496 1933 - 2580 2751 - 3246
Fig. 1. Localization and size (bp) of introns in two basic types of symbiotic Chlorella. N is the northern ecotype symbiotic chlorella with a single intron, and S is the southern ecotype of chlorella with three introns. Arrows show 18S rDNA primers used for detection of Chlorella spp. Intron insertion sites are given according to C. kessleri strain SAG-211-11g (X56105).
washed thoroughly in sterile water and placed in a PCR-tube. Thirty ^l of milliQ water and glass beads (d=0,35 mm, 1/3 oftotal milliQ water volume) were added. The mix was homogenized (two times for 60 seconds) in FastPrep 24 system (MP Biomedicals) with maximal intensity. The suspension was incubated at 95oC for 5 min, shortly vortexed and centrifuged at 16000g for 2 min. Seven microliters of the supernatant was used as a DNA-matrix in the 50 ^l PCR-mix with 3F / 4R primers pair with standard thermal cycle (3 min at 95oC, then 30 cycles — 30 s at 94 oC, 30 s at 56 oC and 1 min at 72 oC and additional extension for 5 min at 72 oC). All 18S rRNA fragments were amplified in MyCycler (BioRad) thermocycler with Taq polymerase and buffer supplied (Helicon, Moscow, Russia). One ^l of the PCR product resulting from the first round was used as the DNA matrix in the second PCR round with two primer pairs flanking “northern” and “southern” intron insertion sites. This second step was carried out under the same conditions. Pure Chlorella DNA representing southern and northern ecotypes was used as control in each experiment.
For sequencing, the fragment ofinterest (T-24-5 strain) was excised from gel, purified and cloned into pTZ57R/T plasmid (Ins T/A clone TM PCR Products Cloning Kit, Fermentas). The cloned fragment (three different clones) was sequenced with M13 standard primers by using Beckman Coulter CEQ 8000 sequenator, according to the manufacturer instruction. The sequence obtained was submitted to GenBank (EU281549).
Sequence analyses. To determine intron size and its location, the sequence was aligned with intronless 18S rRNA gene of Chlorella kessleri strain SAG-211-11g (X56105). BLAST analyses were used for nucleotide similarity search (Altschul et al., 1990).
Results
The method developed works well with a single P. bursariacell (data not shown), but for the better reproducibility in experiments we used up to five cells. The method in this modification was applied to seven different clones of P. bursaria ofvarious origins
Table 2. Primers used in this work.
Primer Sequence Reference
3F 4R SEV_EX_F SEV_EX_R YUG_EX_F YUG_EX_R 5’-AACTGCGAATGGCTCATTAAA 5’-ATCAACCTGACAAGGCAACC 5’-ATTCCAGCTCCAATAGCGT 5’-CCCGACYGTCCCTWTTAA 5’-GAAAGAYGAACYDCTGCGAAAG 5’-ACAAGGCAACCCACCAAC Kvitko et al., 2006 Kvitko et al., 2006 This work This work This work This work
Fig. 2. 18S rRNA gene fragments corresponding to symbiotic and free-living Chlorella amplified in the second round of PCR from different Paramecium clones. 1-7 lanes — clones of P. bursaria, 241-80 and NC64A — controls, M — marker Ladder 100 bp.
(Table 1). As a result of “nested” PCR, one or two amplified fragments were detected (Fig. 2).
Fig. 2 represents the results of screening of seven P. bursaria clones for different ecotypes of symbiotic Chlorella. Ciliate clones are clearly distinguished by the products specific for the “Northern” (lanes # 3, 4, 6 — primers SEV_EX_F/R) or “Southern” (lanes # 2, 5, 7 — primers YUG_EX_F/R) ecotype of symbiotic Chlorella. The sizes of these products correspond to the sizes of intron-bearing 18S rRNA gene ofthe “Northern” and the “Southern” symbiotic Chlorella ecotypes (600 and 650 bp correspondingly). In several cases (lanes # 1, 3, 4) there were additional shorter bands specific for intronless fragments, presumably originating from nonsymbiotic Chlorella species inhabiting Paramecium cells.
In T-24-5 P. bursaria clone we detected a “strange” fragment of 18S rRNA gene with an unusual molecular size (~ 850 bp) amplified with SEV_EX_F / SEV_EX_R pair primers (Fig. 2, line 1). It is not the only “strangeness” of this fragment. This P. bursaria clone was isolated in Tajikistan, where only the “Southern” Chlorella ecotype has been found so far. Nevertheless, the PCR product detected presumably contains an intron with a location characteristic of the “Northern” ecotype. For a more precise analysis, this DNA fragment was purified, cloned and sequenced. Its alignment with the intronless 18S rRNA gene of Chlorella kessleri strain SAG-211-11g (X56105) demonstrated intron presence in the fragment analyzed.
The location ofthe intron detected did not differ from that in the “Northern” type, but the T-24-5 intron size was significantly larger (554 bp) and its nucleotide sequence had nothing in common with the intron of the “Northern” type. Finally, the 16 bp direct repeat flanking the intron was identified. It is of special interest that the repeat identified is an imperfect one (single nucleotide deletion/insertion). It means that the exact intron boundaries could be established only after sequencing the corresponding cDNA. In summary, our results presumably indicated detection of a new type of symbiotic Chlorella that differs from the “Southern” and the “Northern” ecotype. BLAST search (Altschul et al., 1990) showed that the closest identified relatives of the exon part of the cloned sequence are the well-known symbiotic Chlorella sp. NC64A (DQ057341) and several free-living Chlorella species. The intron sequence did not show any significant similarity to the known sequences when standard BLAST search parameters were applied (except three distantly similar fungal ribosomal sequences with the similarity region from 33 to 113 bp). The most interesting was the result of BLAST analysis of the intron sequence against the virus database with “soft” parametres (“somewhat similar sequences” search). In this analysis we detected the presence of a 136-bp region highly similar (expectation 10-16, identities 108/140 (77%)) to the fragment ofgroup I self-splicing intron of major capsid protein Vp54 gene of Chlorella virus strain CVT2 (AB006978) (Fig 3). It is especially
Fig. 3. Similarity region of intron sequence with Chlorella virus major protein Vp54 gene.
interesting that the virus heading the BLAST “hit-list” is the component of triple symbiotic system P. bursaria — Chlorella — PBCV virus. All the other virus candidates had rather/significantly lower similarities (expectations more than 10-3).
Further analysis of the virus similarity region revealed wide distribution of somewhat similar stretches in different introns in 18S rRNA genes ofdiffer-ent algae (Lobochlamys segnis AJ410456; Coelastrum pseudomicroporum AF388381; Chlamydomonada sp. AY220563; Pediastrum boryanum AY536060, etc.).
Discussion
An efficient approach has been developed for rapid identification of Chlorella spp. in a single P. bursaria cell. This “nested” PCR-based method with primers flanking intron insertion sites in the 18S rRNA gene of Chlorella sp. ensures an extremely fast screening of large P. bursaria collections for the presence of Chlorella endosymbionts belonging to different ecotypes without isolation of algae in pure culture. The presence of additional bands suggests that Chlorella population in a particular ciliate cell is genetically heterogeneous (because of presence of nonsymbiotic Chlorella) and confirms results of Hoshina with colleagues (2008). For more precise analysis these fragments should be cloned and sequenced, but it was not the aim of the present work.
When the method was used for screening of P. bursaria collection, a putative symbiotic Chlorella sp. inhabiting T-24-5 P. bursaria clone was identi-
fied. It is possible that we detected a new, so far unknown type of symbiotic chlorella. An interesting feature ofthis symbiont is the presence of18S rRNA gene intron with 136 bp region highly similar to the Chlorella virus intron fragment. This virus is exactly the one that attacks symbiotic Chlorella.
Virus intron-like sequences were found in rRNA genes of various organisms including green algae, red algae, yeasts, fungi and protozoa, suggesting horizontal gene transfer between these distantly related organisms (Lambowitz et al., 1993; Yamada et al., 1994; Nishida et al., 1998). This work presents the first detection of such homologous region in symbiotic Chlorella. The sequence similarity and expectation value indicate a common origin of the sequences and suggests that the horizontal transfer of this DNA fragment had happened between the chlorella and the virus attacking it.
At the moment the work on isolation of this zoochlorella strain (from T-24-5 P. bursaria clone) in pure culture is underway. Further investigation could prove or disprove the symbiotic state of the Chlorella detected, determine the exact boundaries of the exon sequence of 18S rRNA gene (after sequencing the corresponding cDNA) and answer some other important questions.
Acknowledgements
The work was supported by the Russian Foundation for Basic Research (projects nos. 05-04-9274, 07-04-01755 and 08-04-01430). We thank I.Y. Khudyakov for fruitful scientific discussion and assistance in the manuscript preparation.
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Address for correspondence: Konstantin P. Vorobyev. St. Petersburg State University, Department of Microbiology, 16-th line, 33, St. Petersburg, Russia, e-mail: [email protected]
