International Journal of Systematic and Evolutionary Microbiology (2013), 63, 1930–1933
DOI 10.1099/ijs.0.046631-0
Phylogenetic placement of two previously
described intranuclear bacteria from the ciliate
Paramecium bursaria (Protozoa, Ciliophora):
‘Holospora acuminata’ and ‘Holospora curviuscula’
Maria S. Rautian and Natalia D. Wackerow-Kouzova
Correspondence
Natalia D. Wackerow-Kouzova
Department of Genetics and Selection, Faculty of Biology and Soil Science, Saint Petersburg State
University, 7-9 Universitetskaya nab., St. Petersburg 199034, Russia
[email protected]
‘Holospora acuminata’ infects micronuclei of Paramecium bursaria (Protozoa, Ciliophora),
whereas ‘Holospora curviuscula’ infects the macronucleus in other clones of the same host
species. Because these micro-organisms have not been cultivated, their description has been
based only on some morphological properties and host and nuclear specificities. One16S rRNA
gene sequence of ‘H. curviuscula’ is present in databases. The systematic position of the
representative strain of ‘H. curviuscula’, strain MC-3, was determined in this study. Moreover, for
the first time, two strains of ‘H. acuminata’, KBN10-1 and AC61-10, were investigated.
Phylogenetic analysis indicated that all three strains belonged to the genus Holospora, family
Holosporaceae, order Rickettsiales within the Alphaproteobacteria.
Holosporas, together with members of the genus Caedibacter, belong to a group of the so-called rickettsia-like
endosymbionts (RLE group). Members of the genus Holospora are obligate intranuclear bacteria and can only be
found in the nuclei of their host Paramecium cells. In
contrast with other prokaryotic symbionts of Paramecium,
holosporas possess complicated life cycles with infectious
and reproductive stages. After division of the host cell, only
infectious forms (IFs) are released into the medium and
may infect new host cells, while the reproductive forms
(RFs) remain in the host nucleus. Some RFs may then
develop into further IFs.
Currently, the genus Caedibacter includes both the cytoplasmic and nuclear symbionts of ciliates and amoebae.
Members of the genus Caedibacter are toxic to susceptible
strains of paramecia and thereby confer a killer trait to
their host. Within the RLE group, ‘Paraholospora nucleivisitans’, the recently described intracellular symbiont of
Paramecium sexaurelia, exhibits the highest similarity to
holosporas (Eschbach et al., 2009).
The genus Holospora currently comprises nine named bacterial
species which have been isolated from the nuclei [macronucleus (Ma) or micronucleus (Mi)] of different Paramecium
species: Holospora undulata, H. elegans, H. caryophila, H.
obtusa, ‘H. acuminata’, ‘H. recta’, ‘H. curviuscula’, ‘H. bacillata’
Abbreviations: IF, infectious form; Ma, macronucleus; Mi, micronucleus;
RF, reproductive form; RLE, rickettsia-like endosymbionts.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences of strains MC-3, AC61-10 and KBN10-1 are KC164378–
KC164380, respectively.
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and ‘H. curvata’. They are obligate symbionts that have not
yet been cultured. The phylogenetic positions of H. obtusa
(Amann et al., 1991) and H. undulata (Boscaro et al., 2012)
have been determined. Lang et al. (2005) also found that H.
obtusa is the closest bacterial relative of mitochondria
known to date.
The aim of present work was to study the phylogenetic
relationships of two holospora species, namely ‘H.
acuminata’ and ‘H. curviuscula’, Mi and Ma symbionts,
respectively, of Paramecium bursaria. Both species have
been phenotypically described previously (Ossipov et al.,
1980; Borchsenius et al., 1983). We used strains AC61-10
and MC-3 on which the first description of the two species
was made.
Clones of P. bursaria used in this study were identified by
morphological properties (Kudo, 1966). The presence of
bacterial symbionts was confirmed by direct microscopic
observations using differential interference contrast (microscope Polyvar, Reichert-Yung). We used three infected
clones of P. bursaria. Two of these, AC61-10 and MC-3, were
from the Culture Collection of Ciliates and their Symbionts
(CCCS) of the Department of Invertebrate Zoology, Saint
Petersburg State University, Russia. The third live specimen
was from a small pool near St Petersburg (59.9u N 29.3u E)
encompassing a clone of P. bursaria infected by strain
KBN10-1. Identification of the symbionts was conducted
according to Görtz & Schmidt (2005).
As these ciliates also feed on bacteria, bacterial rRNA can
be detected not only in the nuclei but also in the food
vacuoles of the host; thus, macronuclei need to be isolated
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Intranuclear symbionts of infusorians
to obtain the symbionts (Vakkerov-Kouzova & Rautian,
2011). In this study, we isolated the IFs from Ma and Mi in
a two-step Percoll (Sigma) gradient. Percoll densitygradient centrifugation is optimum for their purification
because Percoll has no osmotic effect and the purification
procedure does not involve the pelleting of the organisms
until the final stage (Tamura et al., 1982). Holosporainfected cells were treated by 0.01 % Nonidet 40 (Sigma),
then the homogenate was centrifuged at 200 g for 10 min.
The supernatant was mixed with 50 % Percoll and
centrifuged at 8000 g for 20 min. Two bands were formed
in the centrifuge tube. The upper band (near the top)
consisted of cell debris. The lower band (near the bottom)
consisted of bacteria. This pellet was resuspended in TE
(Tris/HCl 10 mM, EDTA 2 mM, pH 7.5) and was
centrifuged for 6 min at 5000 g after addition of 75 %
Percoll. Small bacteria, including the food bacteria and RFs
of Holospora were found at the top of tube, while IFs were
at the bottom. Purified IFs (107 to 10 8 c.f.u. ml21) were
treated with SDS (final concentration 1 %) and proteinase
K (20 mg ml21) overnight. DNA was extracted by the
conventional phenol/chloroform method (Grimont &
Grimont, 1995). PCR amplification targeting the 16S
rRNA gene using thermocycler Mastercycler (Eppendorf)
and purification of PCR products were carried out as
described previously (Vakkerov-Kouzova & Rautian, 2011).
However, we developed the additional primers for sequencing to yield an expected amplicons of approximately
1500 bp: RI1 59-TGACGGGCGGTGTGTACA-39 (Escherichia
coli positions 1398–1381) and RI2 59-ACTCCTACGGGAGGCAGC-39 (E. coli positions 329–346).
A BLAST analysis of the sequences obtained was run through
the NCBI website (http://www.ncbi.nlm.nih.gov/). Phylogenetic analysis was performed using the DNASTAR (Madison,
WI) and MEGA version 5.05 (Tamura et al., 2011) software
packages. Distances (distance options according to the
Kimura two-parameter model; Kimura, 1980) and clustering were based on the neighbour-joining (Saitou & Nei,
1987) and maximum-likelihood (Kishino & Hasegawa,
1989) methods. Bootstrap analysis (500 resamplings) was
used to evaluate the topology of the phylogenetic tree
(Felsenstein, 1985).
Results revealed the presence of Gram-negative non-motile
rods inhabiting nuclei of P. bursaria (Fig. 1). Bacteria were
present specifically in the Mi or Ma of Paramecium clones.
‘H. acuminata’ KBN10-1 and ‘H. acuminata’ AC61-10 were
present in Mi of P. bursaria. RFs were short fusiform rod;
IFs were 5.0–8.060.5–0.6 mm, straight rods, with both
ends tapered. ‘H. curviuscula’ MC-3 was present specifically
in the Ma of P. bursaria. RFs were short, spindle shaped,
0.7–0.862–3 mm; IFs were 0.4–0.564.0–10.0 mm, slightly
curved rods with tapered ends.
No toxic effects of Holospora-bearing paramecia on paramecia lacking the symbiont were observed. The killer-effect
on symbiont-free paramecia has been shown for members
of the closely related genus Caedibacter due to the presence
http://ijs.sgmjournals.org
Fig. 1. Light microscopy of symbionts in nuclei of host infusorians:
(a) ‘H. acuminata’ KBN-10 in Mi of P. bursaria; (b) ‘H. curviuscula’
MC-3 in Ma of P. bursaria. Ma, Macronucleus; Mi, micronucleus;
IFs, infectious forms. Bars, 30 mm.
of R bodies in a symbiont (Preer, 1977). R bodies are
associated with spherical phage-like structures or covalently closed circular DNA plasmids. In contrast, no
plasmids or phage genomes were found in Holospora (data
not shown).
In this study, the 16S rRNA gene sequences of the
representative strains ‘H. curviuscula’ MC-3 (1415 bp)
and ‘H. acuminata’ AC61-10 (1394 bp), and a new strain
‘H. acuminata’ KBN10-1 (1394 bp) were determined. A
BLAST analysis of the sequences revealed the degree of 16S
rRNA gene sequence similarity with members of recognized taxa. Results indicated that all of the samples
belonged to the family Holosporaceae, order Rickettsiales
in the class Alphaproteobacteria. The differences between
the rrs sequence of H. obtusa (X58198) and those of this
study were 4.00 % (56 bp) for ‘H acuminata’ AC61-10 and
3.26 % (46 bp) for ‘H. curviuscula’ MC-3. Both strains of
‘H. acuminata’ (AC61-10 and KBN10-1) had the same
sequence. Strains ‘H. curviuscula’ MC-3 and ‘H. curviuscula’ 02AZ16 (accession no. JF713683) differed from each
other only by 4 nt positions (0.28 %). Application of
16S rRNA gene molecular phylogeny attributed both ‘H.
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M. S. Rautian and N. D. Wackerow-Kouzova
87
100
83
100
100
Holospora obtusa 88Ti (JF713682)
Holospora obtusa 27aG3 (HE797905)
Holospora obtusa (X58198)
Holospora undulata StB (HE797906)
‘Holospora acuminata’ AC61-10 (KC164379)
‘Holospora curviuscula’ MC -3 (KC164378)
‘Holospora curviuscula’ 02AZ16 (JF713683)
‘Candidatus Gortzia infectiva’ TS-a (HE797909)
62
99
88
‘Paraholospora nucleivisitans’ (EU652696)
Caedibacter caryophilus C221T (X71837)
100
99
100
‘Candidatus Caedibacter acanthamoebae’ HN -3 (AF132138)
‘Candidatus Paracaedibacter acanthamoebae’ UWC9 (AF132137)
‘Candidatus Paracaedibacter symbiosus’ UWET39 (AF132139)
99
100
100
88
Anaplasma bovis SG176HL (EU181143)
Anaplasma phagocytophilum USG3 (AY055469)
Anaplasmataceae
Ehrlichia canis Nero (EU439944)
Ehrlichia chaffeensis Arkansas T (NR037059)
Orientia tsutsugamushi Karp T (NR025860)
Rickettsia bellii RML 369-C (U11014)
100
40
100
0.02
43
64
Rickettsia prowazekii Breinl T (NR044656)
Rickettsia akari MK T (NR029154)
Rickettsiaceae
Rickettsia canadensis 2678T (NR029155)
Rickettsia massiliae Mtu1T (NR025919)
Rickettsia rickettsii RT (NR028018)
Fig. 2. Maximum-likelihood phylogenetic tree based on the 16S rRNA gene sequences of representative strains ‘H. curviuscula’
MC-3 and ‘H. acuminata’ AC61-10, and members of related taxa within the family Holosporaceae. Sequences belonging to
members of the families Rickettsiaceae and Anaplasmataceae were used as the outgroup. Bar, 0.02 substitutions per nucleotide
position. New sequences are in bold. The neighbour-joining tree showed essentially the same topology (data not shown).
curviuscula’ and ‘H. acuminata’ to the genus Holospora
(Fig. 2). All three species H. obtusa, ‘H. curviuscula’ and ‘H.
acuminata’ formed compact group in an order Rickettsiales.
Inside this group, the greatest similarity was found between
‘H. acuminata’ and ‘H. curviuscula’. Holosporas, together
with ‘Paraholospora nucleivisitans’ and members of the
genus Caedibacter form the family Holosporaceae (Fig. 2).
Within the family, ‘Candidatus Gortzia infectiva’, the macronuclear symbiont of Paramecium jenningsi, exhibits the highest
similarity to members of the genus Holospora (Boscaro et al.,
2012). A better understanding of the Holospora evolution
will help in the future to elucidate the mechanisms of
transmission and virulence of the rickettsiae, as well as the
evolution of symbiotic relations and, in general, the
evolution of the eukaryotic cell.
ACKNOWLEDGEMENTS
This work was supported by the Russian Foundation for Basic
Research (10-04-01188a).
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