Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

17

Original

Analysis of an intron intervening the SSU rDNA of
Chlorella sp. T-24-5, a photobiont of Paramecium
bursaria
Ryo HOSHINA
Department of Biomedical Science, College of Life Sciences, Ritsumeikan University,
Kusatsu, Shiga 525-8577, JapanSUMMARY
The intronic sequence intervening the small subunit ribosomal DNA (SSU rDNA) of Chlorella sp.
T-24-5, an atypical photobiont of Paramecium bursaria was examined. The position of the insertion was
found to be 10 nucleotides upstream from that of a major P. bursaria photobiont, Micractinium reisseri(S651), and was found to be a novel insertion site (S641; the numbering reflects the homologous positionin the rRNA gene of Escherichia coli: S = SSU rRNA). A secondary structure diagram showed that the
intron is classified as a group I intron (subgroup IC), characterized by an extended P5 helix. Phylogenetic
analyses could not reveal its evolutionary relationships with other introns, but were suggestive of amonophyletic relationship with introns of some trebouxiophytes. These introns all share the insertion
position S641, and their sequences are extremely conserved and are likely to have spread recently. The
intron of Chlorella sp. T-24-5 had twelve-nucleotide sequence repeats lying at the head of the intron andafter the insertion, which may play a role in intron invasion.
Key words: Group I intron, S641, Photobiont

INTRODUCTION
The green ciliate Paramecium bursaria(Ehrenberg) Focker is one of the best-studied pro-

Tel: +81-77-566-1111/Fax: +81-77-561-5203G
E-mail: wwhoseena@hotmail.com
Present address: Department of Environmental Science and Engineering, Graduate School of Scienceand Engineering, Yamaguchi University, Yamaguchi 753-8512, Japan
Received: 1 February 2012; Accepted: 19 May
2012.

tists because of its observable endosymbiosis. The
symbiotic relationship can be restarted—algaeremoved P. bursaria can absorb and fix algae as
new photobionts (Kodama and Fujishima, 2009
and references therein). Despite the ability of P.
bursaria to re-establish symbiosis, the diversity of
its photobionts is limited. Nearly 50 strains of photobionts have been genetically identified; however,most of them belong to either Chlorella variabilis
Shihira et Krauss or Micractinium reisseri Hoshina, Iwataki et Imamura (Chlorellaceae, Trebouxiophyceae) (Linz et al., 1999; Kvitko et al.,18

Novel intron of a Paramecium symbiotic alga

2001; Hoshina et al., 2004, 2005, 2010; Gaponova
et al., 2007; Summerer et al., 2008; Hoshina andImamura, 2008a; Vorobyev et al., 2009; Pröscholdet al., 2011).
To determine which photobiont P. bursaria
possesses, algal DNA amplifications directly fromP. bursaria extracts have been introduced(Hoshina and Imamura, 2009a; Vorobyev et al.,2009). These methods are based on unique intron
insertions at different sites in the small subunit
ribosomal DNA (SSU rDNA) of these photobionts; polymorphisms in the lengths of PCR products containing (or not containing) these introns
can be a useful tool for symbiont identification(intron insertion sites, see Hoshina et al., 2010).
Recently, Vorobyev et al. (2009) reported an
atypical photobiont SSU rDNA sequence from P.
bursaria collected from Tajikistan. This sequence(Chlorella sp. T-24-5, GenBank accession number
EU281549) included an intron at nearly the same
position as in M. reisseri (described as “Northern”
ecotype), but the length and sequence were different (Vorobyev et al., 2009). They stated that the
intron sequence did not show any significant similarity to other sequences using a BLAST search fornucleotide, whereas it was highly similar to thegroup I intron intervening the major capsid protein
Vp54 gene (AB006978) of C. variabilis virus(CvV) under conditions of “somewhat similar sequences” in the viral nucleotide database.
Group I introns are a distinct RNA group thatfunction as enzymes, splicing themselves out of
precursor RNA transcripts and ligating exons. Another distinctive characteristic of group I introns istheir mobility. Phylogenetic analyses have indicated that introns at homologous gene sites are related(position family), even among distantly related
host organisms. This phenomenon is linked to intron spreading mechanisms (i.e. homing or reverse
splicing). When an intron at a gene locus infects adifferent organism, the new intron will be inserted
into the same locus where it was originally located(for the general characteristics of group I introns,see Cech, 2002; Haugen et al., 2005; Nielsen andJohansen, 2009 and the references therein). GroupI introns are classified into subgroups IA throughIE based on their structural diversity and phylogeny, and nuclear encoding introns belong to either
subgroup IC or IE (Cannone et al., 2002).
The photobionts of P. bursaria, C. variabilis
has four subgroup IC and four IE introns and M.
reisseri has two IE introns in their nuclear rDNA.
These introns are interesting for two reasons. First,
some viruses possess group I introns, of which the
only eukaryotic virus is CvV (Zhou et al., 2008).
This virus infects only C. variabilis, which is aphotobiont of P. bursaria. Since their introns areclassified as IC type (like the introns intervening
eukaryotic nuclear rDNA), some researchers havesuggested that viruses play a role in intron transfer(Yamada et al., 1994; Bhattacharya et al., 1996;
Nishida et al., 1998; Friedl et al., 2000). Second,
subgroup IE introns commonly inserted into C.
variabilis and M. reisseri are monophyletic andindependent from other IE intron lineages. However, the insertion positions are straggled in theirrDNA, and this cannot be explained by existing
intron transmission mechanisms. In other terms,
unknown intron transmissions could have occurredin P. bursaria (Hoshina and Imamura, 2009b).
This study examined the sequence, structure, andphylogenetic relationship of the Chlorella sp. T-24
-5 intron.
MATERIALS AND METHODS
Structure prediction
A secondary structure diagram of the Chlorella sp. T-24-5 intron (EU281549) was preparedaccording to previously reported models (e.g.
Lehnert et al., 1996; Haugen et al., 2002). The
structure of the central catalytic intron core (e.g.
from P3 to P7) was solved previously; the remaining helices, predicted using the Mfold (Mathews etal., 1999; Zuker, 2003) web server (http://mfold.rna.albany.edu/?q=mfold), were subsequent-Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

19

Fig. 1. Flanking sequences and secondary structure prediction of the
Chlorella sp. T-24-5 SSU rDNA
group I intron. A. Differences in intron annotations between Vorobyev etal. (1999) and this study. The replicated sequence —TACTTTGAGTAA—is in bold. P1 and P10 are pairingexon segments constructing helices P1
and P10 in the secondary structure.
The arrowhead indicates the S651
insertion position. B. Structure diagram of the intron. Capital lettersrepresent the sequence of the intron;
lower case letters indicate flanking
exon sequences. Conserved sequence
elements P, Q, R and S are shaded.
Arrows point to the 5’ and 3’ splicesites.Gly appended to the core.
Phylogenetic analyses
Intron sequences were aligned through juxtaposition, taking into account the secondary structures. Although some unalignable regions remained between ingroup and outgroup (fungal
L2449 introns) taxa, this study emphasized thealignment accuracy between ingroup taxa. A totalof 154 aligned positions (138 parsimonyinformative) were selected for IC intron analysis.
These selected positions were limited conservedbase paring elements P3 to P7 and the comparatively conserved base paring elements (determinedin this study) of P.2.1, P5a, P5b, P8, P9, and P9.1.
The sequence alignments are available from theauthor upon request.
Two phylogenetic trees were constructedusing the p-distance neighbor-joining (NJ) method
in MEGA 5 (Tamura et al. 2011) and the maximum likelihood (ML) method in PAUP 4.0b10(Sinauer Associates, MA). Based on the Akaike’s
Information Criterion, the best-fit evolutionarymodel for ML analysis was determined via Modeltest 3.7 (Posada and Crandall, 1998), which selected the TrN + I + G evolutionary model with thefollowing parameters: substitution-rate matrix of
AC = 1, AG = 2.5291, AT = 1, CG = 1, CT =4.0889, and GT = 1; proportion of sites assumed to
be invariable = 0.0522; rates for variable sites assumed to follow a gamma distribution with shapeparameter = 0.7832; and number of rate categories= 4. With these settings, a heuristic search was
performed using the neighbor-joining tree as thestarting tree and a nearest-neighbor interchange
swapping algorithm. Bootstrap probabilities were20

Novel intron of a Paramecium symbiotic alga
Fig. 2. Comparisons of Chlorella
sp. T-24-5 sequence with C. variabilis and M. reisseri under the
intron annotation presented here.
The nucleotide number corresponds to the sequence coordinateof Chlorella sp. T-24-5 (EU281549). Nucleotides between
201 and 710 are omitted. Intron
sequence of M. reisseri is excluded. Differences from the uppermost variant are shown by nucleotide, identical nucleotides by adot, and a dash donates a missing
nucleotides. The arrows indicate
start and end points of the intron(SSU rDNA: <1..172,726..>857,
Intron: 173..725).

computed for 1000 (NJ) and 100 (ML) replicateswith these settings.
RESULTS AND DISCUSSION
A large insertion in a nuclear rRNA gene is
generally categorized as a group I intron. The intron sequence annotated by Vorobyev et al. (2009)deviates from the fundamental rules for group I
introns; that is, the last exon nucleotide (insertion
point) is T (U) and the last intron nucleotide is G
(Burke et al., 1987; Lambowitz and Belfort, 1993)(Fig. 1A). Upon closer inspection of the insertion,
the same twelve-nucleotide sequences, —
TACTTTGAGTAA—, was found flanking theinsertion. This sequence repeat did not follow thecorrect intron annotation by Vorobyev et al.
(2009). An annotation pattern was highlighted
compared to some Chlorella rDNA sequences. It is
possible that the intron started with —
TACTTTGAGTAA— and ended before the second —TACTTTGAGTAA— (intron sequencelength of 553 nucleotides) since this annotation
follows the fundamental rules for group I introns,
as discussed above (Fig. 1A). Under this annotation, there was no gap when compared to otherchlorellacean exon sequences (Fig. 2). The exon

sequence (having removed the first four nucleotides [nucleotide numbers 1–4] and the last nucleotide [857]), which is 299 nucleotides in length,
differed from some Chlorella (including C. variabilis) and Micractinium species by one transition,and differed from M. reisseri by one transition and
one transversion.
Figure 1B shows the secondary structure
diagram of the intron. It was constructed by tracing
the well-known core region (as a ribozyme) andadding physically folded peripheral loops. The
structure contained base paring helices P1–P10.
The extended P5 helix is typical for IC introns.
The P1 helix was constructed with a 5’ flanking
exon sequence to compensate for the internal guidesequence (IGS). Similarly, the P10 helix was constructed with a 3’ flanking exon sequence. These
helices play an important role in excising the intron from rRNA (Cech, 1990; Suh et al., 1999).
These results demonstrated that the above annotation is more reasonable. Therefore, the presumableinsertion position is ten nucleotides upstream fromthat of M. reisseri (S651) (Fig. 1A). This insertion
position has never been reported as an intron insertion site, and this study tentatively proposes the
position to be S641, compared to Escherichia colirRNA (the numbering reflects their homologous

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

21
Fig. 3. Annotations for introns
similar to the Chlorella sp. T24-5 S641 intron. A. Annotation results considering thefundamental rules for group I
introns (see text) and secondary structures. Sequence repeats lying at the head of theintron and after the insertion
are in bold. B. Structure predictions for the introns; P1 andP10 pairing segments are only
shown. Capital letters represent
the sequence of the intron;
lower case letters indicate
flanking exon sequences. Arrows point to the 5’ and 3’splice sites.G

positions in the E. coli rRNA gene; S = SSUrRNA). The intron has characteristics of an extended, degenerative P9.1 helix and a long P9.2b
(P9.2b is the original name in this study). The area
similar to the viral intron (Vorobyev et al., 2009)
corresponds to helices P4 and P5, although it does
not have a structurally distinctive feature.
Surprisingly, a BLAST search identified
trebouxiophytes with similar introns, although
Vorobyev et al. (2009) did not identify any significantly similar introns. These sequences have been
published recently (Luo et al., 2010; Pažoutová etal., 2010; Krienitz and Bock, 2011). This study
examined these sequences; the annotation results
are shown in Figs. 3A and B. The introns wereinserted in the same position (S641), and the repeat
sequences were commonly found at the intron
heads and 3’ flanking exon sequences, although
the lengths of the repeat sequences varied.
The phylogenetic relationships of IC introns
were determined via NJ and ML analyses. Whenmultiple parameters are expected to estimate highly associated variances for short alignment data
(i.e. short and highly divergent sequences like thisintron alignment), the parameter-rich ML analyses
may give incorrect topologies (Nei et al., 1998).
The “simple” models such as the single parameter

NJ method are potentially useful for these cases(Bruno and Halpern, 1999; Piontkivska, 2004).
Most of previous works used NJ method to construct the phylogenetic tree of introns(Bhattacharya et al., 2005; Haugen et al., 2004b;
Nikoh and Fukatsu, 2001). By contrast, some literatures indicated the superiority of ML method withappropriate parameters (Holder and Lewis, 2003;
Som, 2009). The present study, therefore, shows
both simple NJ and parameter-rich ML trees (Figs.
4A and B). The trees were rooted with fungal
L2449 introns that have been indicated the introns
with properties intermediate between IC and IE(Hoshina and Imamura, 2009b). Topologies of NJ
and ML trees were somewhat different from eachother. In NJ, IC introns were diverged into S516
introns of pelagophytes and rhodophytes and theothers (Clade A). ML tree indicated fugal andgreen algal S1506 introns diverged one by one,and the others (Clade B) involved the S516 introns. These S1506 introns were scattered in NJ
tree. Bootstrap supports for the branches in CladeA (NJ) or Clade B (ML) were basically low. CladeC was indicated by both analyses, but bootstrap
values of ML was below 50%.
Subgroup IC intron analyses have been advanced with focus on fungal rDNA introns. In fun-

22

Novel intron of a Paramecium symbiotic alga

Fig. 4. GPhylogenetic trees of selected IC introns. The numerals at each node are bootstrap probabilities of NJ/ME(above the node) and ML/MP (below the node) analyses; only values with >50% support are shown. Three clades
are named A, B and C (see text). The letters in brackets indicate taxonomic affiliations: Vc, Chlorophyceae; Vt,
Trebouxiophyceae; Vu, Ulvophyceae; Vz, Zygnemophyceae; A, ascomycetes; C, ciliates; P, pelagophytes; R,rhodophytes; S, plasmodial slime molds. Inserted positions are given on the right side of thick vertical bars or after
brackets. A. P-distance neighbor-joining (NJ) tree. B. Maximum likelihood (ML) tree using the TrN + I + G evolutionary model.G

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

23

B

Fig. 4—continued.

gal intron phylogeny, introns are separated into
intron group that share an insertion site (position
family), although relationships of those groups areunclear (e.g. Bhattacharya et al. 2005). There may
be only one phylogenetic study covering compre-

hensive green algal IC introns, where definedgroups were hardly indicated (Hoshina andImamura, 2008b). The present study also could not
resolve this problem. Perhaps, irregular evolutionary events have occurred among the IC introns

24Novel intron of a Paramecium symbiotic algasuch as transpositional intron copy within a genome seen in subgroup IE introns (Hoshina andImamura, 2009b). Indeed, some mildly related
introns of single species (C. variabilis and Dictyochloris pulchra Deason and Herndon) can be seenin Clade C.
Although phylogenetic relationships of theintrons in Clade A (Fig. 4A) or Clade B (Fig. 4B)
are unclear, the monophyly of the introns sharing
S641 insertion site are supported with higher ormoderate bootstrap values. Whereas, S641 introns
have a tenuous connection with viral introns, although the alignment data set used in the phylogenetic analyses includes regions P4, P5, and 5’ side
sequence of P5a and P5b. Also, S641 introns andthe introns of C. variabilis were not phylogenetically related.
With the genome analyses by Blanc et al.
(2010), Chlorella variabilis (46.2 Mb) became thefirst genome sequenced species in the Trebouxiophyceae. This study revealed surprisingobservations with respect to genes involved in cellwall metabolism. The Chlorella cell wall contains
chitin and chitosan. These genes were derived
from CvV and are not related to those of higher
plants (Blanc et al., 2010). Thus, it is not surprising if the viral intron was transferred into Chlorella genome.
The P4 and P5a, b and c helices of the Chlorella sp. T-24-5 intron are similar to those of CvV.
Approximately 70% of the sequences are identicalto their P5a, b and c helices (comparatively variable region). However, the monophyletic relationship between them could not be determined fromthis study (Figs. 4A and B). To determine whether
CvV truly mediates intron transfer requires further
investigation.
This study identified more reasonable insertion position (novel insertion site S641) of Chlorella sp. T-24-5 intron (Fig. 1A), and the intron
constructed a monophyletic clade with those same
insertion positions (Figs. 3A, 3B, 4A and 4B).
Such a close relationship between introns inserted

at the same site is common (known as a position
family), and is the result of intron spreading mechanisms. Introns may spread via homing or reverse
splicing. In homing, the intron encodes a homing
endonuclease (HE) gene at the terminus of a peripheral helix. HE recognises specific sequences ofdouble-stranded DNA, 14–40 bp in length, andthen cleaves the target site and inserts the intron.
Reverse splicing is the inverse process of intron
splicing at the RNA level. This pathway also requires between four and six specific nucleotides
upstream of the insertion site, which the intron
recognizes as the optimal site for self-insertion.
Therefore, group I intron movement typically occurs at homologous sites due to these sequence
recognition mechanisms. Consequently, the number of insertion sites is fairly limited (it is exceptional that the insertion sites in green algal IC introns are diverse), and introns at homologous sites
are often more closely related than introns at different sites (for a review, see Haugen et al., 2005).
Namely, the S641 position family can be explainedby either homing or reverse splicing. HE genes
were not found in these S641 introns. The introns
that include the HE gene are a tiny minority, whichwere once fixed in the population, the HE genes nolonger have active functions and were ultimately
lost (Goddard and Burt, 1999; Haugen et al.,2004a). Instead, long sequence insertions (>50
nucleotides) at peripheral helices, as seen in P9.1of the Chlorella sp. T-24-5 intron, can be regarded
as HE gene remnants (Haugen et al., 2004a).
Meanwhile, the origin of the S641 introns
remains an important question. Members of theS641 position family exhibit extremely strong sequence conservation. In the core regions of P, Q, R
and S, and pairing region P3, the S641 introns had
67 invariants and two 75% conserved positions outof 69 juxtaposed positions (data are not shown).
This indicates that these introns have undergone a
recent, rapid spread (see Wikmark et al., 2007).
The first invasion to position S641 may relate to
the sequence repeat, as shown in Figs. 1A and B.

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

Specific twelve-nucleotide-long sequences emerge
stochastically once per 16 Mb (412). This is unlikely to be coincidental, and may play an as yet unidentified role in intron invasion. This study could
not determine whether P. bursaria underwent intron spread or invasion to S641.
ACKNOWLEDGEMENTS
This study was supported by an Environmental Research Grant (103180) from The Sumitomo
Foundation.
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