Microbiology (2004), 150, 1245–1250
DOI 10.1099/mic.0.26923-0
Mitosomes of Entamoeba histolytica are abundant
mitochondrion-related remnant organelles that lack
a detectable organellar genome
Gloria León-Avila3 and Jorge Tovar
School of Biological Sciences, Royal Holloway, University of London, Egham TW20 0EX, UK
Correspondence
Jorge Tovar
[email protected]
Received 19 November 2003
Revised 20 January 2004
Accepted 20 January 2004
The existence of mitochondrion-related relict organelles (mitosomes) in the amitochondrial
human pathogen Entamoeba histolytica and the detection of extranuclear DNA-containing
cytoplasmic structures (EhKOs) has led to the suggestion that a remnant genome from the
original mitochondrial endosymbiont might have been retained in this organism. This study reports
on the mutually exclusive distribution of Cpn60 and extranuclear DNA in E. histolytica and on the
distribution of Cpn60-containing mitosomes in this parasite. In situ nick-translation coupled to
immunofluorescence microscopy failed to detect the presence of DNA in mitosomes, either in fixed
parasite trophozoites or in partially purified organellar fractions. These results indicate that a
remnant organellar genome has not been retained in E. histolytica mitosomes and demonstrate
unequivocally that EhKOs and mitosomes are distinct and unrelated cellular structures.
INTRODUCTION
Over the past few years mitochondrial remnant organelles
(mitosomes) have been identified in a number of amitochondrial parasitic protozoa (e.g. Giardia intestinalis,
Entamoeba histolytica, Trachipleistophora hominis and
Cryptosporidium parvum) (Mai et al., 1999; Tovar et al.,
1999, 2003; Williams et al., 2002; Riordan et al., 2003),
disproving the Archezoa hypothesis, which postulated that
amitochondrial eukaryotic organisms were the direct
descendants of the nucleated cell that hosted the original
mitochondrial endosymbiont (Cavalier-Smith, 1983, 1998).
It is now apparent that eukaryotic microbes that lack
typical mitochondria are not primitively amitochondrial
but are highly derived descendants of mitochondrioncontaining ancestors whose capacity for aerobic respiration
has been gradually lost during the course of evolution.
Because of their recent discovery, little is known about the
physiological functions of mitosomes. Some, such as those
of Entamoeba and Cryptosporidium, have been shown to
contain chaperonin Cpn60, a protein known to participate
in the refolding of imported proteins (Sigler et al., 1998;
Mai et al., 1999; Tovar et al., 1999; Riordan et al., 2003);
mitosomes of Trachipleistophora contain a mitochondrialtype Hsp70 (mtHsp70), a molecular motor that helps
internalize proteins into the organelle (Matouschek et al.,
2000; Williams et al., 2002). Other proteins have also been
3Present address: Departmento de Genética y Biologı́a Molecular,
Centro de Investigación y Estudios Avanzados I.P.N., San Pedro
Zacatenco, 07360, Mexico.
Abbreviation: EhKO, Entamoeba histolytica kinetoplast-like organelle.
0002-6923 G 2004 SGM
suggested as putative mitosomal components in various
amitochondrial lineages but their cellular localization has
not been demonstrated experimentally (Clark & Roger,
1995; Bakatselou et al., 2000, 2003; Katinka et al., 2001;
Morrison et al., 2001; Zhu & Keithly, 2002; Arisue et al.,
2002). Perhaps the most significant finding in relation to
the biology of mitosomes is the direct demonstration that
Giardia mitosomes function in the biosynthesis of molecular iron–sulphur (Fe–S) clusters and in their subsequent
incorporation into functional Fe–S proteins (Tovar et al.,
2003). Genes encoding several proteins involved in Fe–S
cluster metabolism have also been identified in the genomes
of several other amitochondrial organisms, including
Encephalitozoon cuniculi, Entamoeba histolytica, C. parvum
and Trichomonas vaginalis (Katinka et al., 2001; Tachezy
et al., 2001; LaGier et al., 2003; Bankier et al., 2003; http://
www.sanger.ac.uk/Projects/E_histolytica/ and http://www.
tigr.org/tdb/e2k1/eha1), leading to the suggestion that the
requirement for Fe–S proteins probably represents the
selective pressure driving the retention of the original
mitochondrial endosymbiont in all eukaryotic lineages
(Tovar et al., 2003; van der Giezen & Tovar, 2004; Embley
et al., 2003b).
The presence of a remnant organellar genome with prokaryotic phylogenetic affinity is perhaps the strongest
evidence for the bacterial ancestry of mitochondria. But
whether E. histolytica mitosomes have retained an ancestral
organellar genome has been a matter of debate and
controversy. Mai et al. (1999) and Tovar et al. (1999)
found no evidence for the presence of DNA in Cpn60containing mitosomes (syn. cryptons) using Hoechst dye
and propidium iodide staining, respectively. Further studies
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:18:41
Printed in Great Britain
1245
G. León-Avila and J. Tovar
by Ghosh et al. (2000), however, suggested the existence in
cryptons of a large organellar genome estimated at 2?2 % of
the nuclear genome (about 880 kb in size). This finding
fuelled speculation that mitosomes/cryptons might be the
same organelle as the DNA-containing cytoplasmic structures
described by Orozco and collaborators in E. histolytica –
termed kinetoplast-like organelles (EhKOs) (Orozco et al.,
1997). Despite mounting evidence for the autogenous
nature of EhKOs (i.e. originating from an internal cellular
compartment as opposed to having an external endosymbiotic origin) (Orozco et al., 1997; Rodrı́guez et al., 1998;
Luna-Arias et al., 1999; Solis et al., 2002; Marchat et al., 2003;
Mendoza et al., 2003), the question of whether or not the
DNA-containing EhKOs could be the same as the Cpn60containing mitosomes has remained unresolved.
Here, we have used laser scanning confocal microscopy and
a specific antibody against E. histolytica Cpn60 to show that
mitosomes are abundant organelles present in all parasite
trophozoites and that their distribution is different from
that of the DNA-containing EhKOs, which are only present
in a subset of the parasite population. We also use one of the
most powerful molecular techniques for the detection of
DNA – in situ nick-translation coupled to immunofluorescence microscopy – to demonstrate that the mitosomes of
E. histolytica lack a detectable organellar genome.
METHODS
Parasite culture. E. histolytica trophozoites were cultured axeni-
cally in YI-S medium with 15 % adult bovine serum as described by
Clark & Diamond (2002).
DNA detection. E. histolytica trophozoites were detached from culture tubes by incubation on ice and collected by centrifugation at
275 g for 10 min. Cells were washed in PBS, fixed in 4 % paraformaldehyde in PBS for 30 min at 37 uC and finally washed and resuspended in 1 ml PBS. Fixed cells were attached to polylysine-coated
glass slides, washed twice in PBS and permeabilized with 0?2 %
Triton X-100 at room temperature for 5 min. Following permeabilization, slides were rinsed twice in PBS and subjected to in situ nicktranslation using biotinylated dATP and a Nick-Translation System
(Invitrogen) as described by the manufacturer. Reactions were incubated for 4 h at room temperature and washed four times, blocked
with 1 % BSA in PBS and incubated for 1 h at 37 uC with streptavidin–
FITC conjugate diluted 1 : 200 (v/v) in blocking buffer.
Mitochondria selective stain. A 48 h monolayer of mouse
muscle cells C2C12 (ATCC, Manassas, VA, USA), growing in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10 % FCS, was treated with 0?25 % trypsin in TDE buffer (136 mM
NaCl, 50 mM KCl, 50 mM EDTA, 25 mM Tris pH 7?5) for 2 min,
detached by resuspension, centrifuged at 500 g for 10 min and
resuspended in 1?2 ml fresh DMEM complete medium. Cells
(8?06104) were applied on each glass slide and attached to the surface for 3 h at 37 uC in a 5 % CO2 incubator. Following removal of
medium and addition of 200 ml fresh medium containing 125 nM
MitoTraker Red (Molecular Probes), slides were incubated at 37 uC
for 30 min, washed twice in PBS and fixed in 4 % paraformaldehyde
for 30 min at 37 uC. Cells were permeabilized with 0?2 % Triton
X-100 at room temperature for 5 min and subjected to in situ nicktranslation as described above.
1246
Immunodetection of Cpn60. E. histolytica slides were blocked
with 1 % BSA in PBS (blocking buffer) for 30 min and incubated
with specific rabbit anti-Cpn60 antibodies overnight at 4 uC; samples
were washed repeatedly in PBS and further incubated with a goat antirabbit TRITC conjugate (Pierce) diluted 1 : 300 in blocking buffer.
Purification of microsomal fraction. Cell fractionation was done
according to Tovar et al. (1999), with some modification. The cells
were lysed by freeze–thawing and the microsomal fraction was
obtained by centrifugation at 100 000 g for 30 min at 4 uC. Aliquots
were fixed in 4 % paraformaldehyde in PBS, attached to glass slides
and permeabilized by treatment with acetone for 5 min at room
temperature. After repeated rinsing in PBS, fractions were subjected
to in situ nick-translation and anti-Cpn60 treatment as described
above.
Laser scanning confocal microscopy. After treatments, all slides
were washed five times in PBS and mounted with 15 ml VectaShield
mounting medium (Vector). Slides were observed under a scanning
confocal microscope (Radiance 2100; Bio-Rad) fitted with Argon
and HeNe laser beams. Images were collected using the BioRad LASERSHARP 2000 software and processed with POWERPOINT
version 7.0.
RESULTS
Laser scanning confocal immunomicroscopy of fixed
E. histolytica trophozoites treated with antibodies raised
against recombinant Cpn60 from the parasite (recEhCpn60)
show the compartmentalized distribution of the protein in
discrete foci distributed throughout the cytoplasm (Fig. 1).
Confocal optical slices of 500 nm thickness revealed that
Cpn60-containing mitosomes are smaller and more abundant than previously estimated by low-resolution fluorescence microscopy (Tovar et al., 1999). The number of
mitosomes in the trophozoites shown in Fig. 1(a) was 256
as estimated by counting labelled structures in stacks of
confocal optical slices of 500 nm thickness. Most organelles
were only observed in single optical slices, but some labelled
structures could be seen over two and occasionally three
confocal slices. Maximum projection images showed
considerable overlap of label due to the random distribution
of organelles throughout the cell cytoplasm. All trophozoites
in the parasite populations analysed displayed Cpn60containing mitosomes.
The putative presence of a remnant organellar genome in E.
histolytica mitosomes was investigated using one of the most
powerful techniques currently available for the detection of
DNA, in situ nick-translation coupled to immunofluorescence microscopy (Clemens & Johnson, 2000). Nuclear
DNA was readily detected in all trophozoites analysed,
whereas extranuclear DNA was only detected in a minority
percentage of the cell population (Fig. 2). Immunodetection
of Cpn60 in these cells confirmed the presence of multiple
mitosomes in each cell of the parasite population but no
co-localization of Cpn60 and DNA label was detected.
Computer-assisted magnification of mitosomes and of
DNA-containing structures in merged images further
demonstrated that the cellular distribution of Cpn60 and
of extranuclear DNA in E. histolytica is mutually exclusive
(Fig. 2b). To test the limits of detection of DNA by in situ
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:18:41
Microbiology 150
E. histolytica mitosomes lack a detectable genome
Fig. 1. Distribution of E. histolytica Cpn60 viewed by confocal immunofluorescence microscopy. Optical slice images of fixed
parasite trophozoites treated with anti-Cpn60 antibodies were collected and processed as indicated in Methods.
Fluorescence images of slices 2, 6 and 11 (0?5 mm thickness) from a single cell are depicted; the maximum projection images
of this cell (a) and that from a different field with four trophozoites (b) are shown alongside. The corresponding differential
interference contrast (DIC) transmission images are presented under each panel. Maximum projection panels were
superimposed on their respective DIC transmission images.
nick-translation we used mammalian muscle cells as a
control. The 16?3 kb mitochondrial genome of mouse
cells (Bayona-Bafaluy et al., 2003) was readily detected
by confocal microscopy (Fig. 2c), demonstrating that an
organellar genome as small as 16?3 kb in size can be readily
identified using this technique.
To further investigate the presence of extranuclear DNA in
E. histolytica, we prepared a mixed-membrane subcellular
fraction enriched in biological membranes and membranebounded organelles. Immunofluorescence microscopy of
the mixed-membrane fraction, incubated with anti-Cpn60
antibodies and subjected to de novo DNA biosynthesis by
in situ nick-translation, revealed the presence of DNAassociated and Cpn60-containing subcellular structures/
membranes (Fig. 3). However, no co-localization of Cpn60
and DNA labelling was detected, confirming that the cellular
distribution of Cpn60 and DNA is mutually exclusive in this
parasite.
DISCUSSION
The Archezoa hypothesis, now abandoned by its original
proposer (Cavalier-Smith, 2002), crumbled under the weight
of evidence for the presence of relict mitochondrial organelles
in several organisms representative of the major amitochondrial eukaryotic lineages (Archeoamiba, Metamonada,
Parabasalia and Microsporidia), rejecting the idea that those
organisms diverged from the main eukaryotic trunk prior to
the endosymbiotic acquisition of the mitochondrion.
http://mic.sgmjournals.org
This study provides evidence that E. histolytica mitosomes
are abundant mitochondrion-related organelles that lack a
detectable organellar genome. High-resolution confocal
microscopy revealed the presence of dozens of Cpn60containing mitosomes in every single parasite trophozoite.
The fact that most organelles were only seen in single optical
slices limits their size to a maximum of 500 nm (the
thickness of the optical slice). Larger labelled structures
spanning two or occasionally three optical slices observed in
some cells indicate the presence of either closely associated
mitosomes not resolved by the methodology employed or of
individual structures of 1?5 mm maximum diameter. These
are likely to represent the structures previously observed by
low-resolution fluorescence microscopy (Tovar et al., 1999).
The mutually exclusive distribution of Cpn60 and DNA
in confocal optical slices of parasite trophozoites demonstrates unequivocally that the Cpn60-containing mitosomes
and the DNA-containing EhKOs are distinct intracellular
entities of different provenance. De novo DNA biosynthesis
and Cpn60 labelling of partially purified membranebounded organelles confirmed the mutually exclusive
nature of Cpn60-containing and DNA-associated cell
structures. Detection of biotinylated DNA in the mixedmembrane fraction indicates the presence of cellular
structures that contain, or are associated with, DNA; these
structures can be taken as a positive control for the detection – given its presence – of a putative organellar genome
in Cpn60-containing mitosomes. The lack of a detectable
mitosomal genome is consistent with the absence of a
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:18:41
1247
G. León-Avila and J. Tovar
Fig. 2. Detection of DNA in E. histolytica and in control mammalian cells by in situ nick-translation. Fixed E. histolytica
trophozoites treated with anti-Cpn60 antibodies and fixed muscle cells pre-treated with MitoTracker Red were subjected to
DNA synthesis by in situ nick-translation using biotinylated dATP; de novo synthesized DNA was detected using streptavidin–
FITC and confocal microscopy. Panels depicting individual and merged confocal images are presented. (a) Single parasite
trophozoite representative of cells where no extranuclear DNA is observed (>65 % of the cell population). (b) Single
trophozoite representative of cells where extranuclear DNA is detected (<35 % of the cell population). Insets on the merged
image show computer-assisted magnification of indicated organelles highlighting the lack of co-localization between DNA and
Cpn60. (c) Control mouse muscle cells showing mitochondrial and nuclear genomes.
detectable organellar genome in the mitochondrion-related
hydrogenosomes of Trichomonas (Dyall & Johnson, 2000;
Clemens & Johnson, 2000; Embley et al., 2003a). Although
our failure to detect DNA in mitosomes does not formally
exclude the theoretical existence of a very small mitosomal
genome, it is clear that such remnant genome would have to
be significantly smaller than 16 kb to escape detection using
this methodology, as the 16?3 kb mouse mitochondrial
genome was readily detected in control experiments
(Clemens & Johnson, 2000; Bayona-Bafaluy et al., 2003).
In this respect, the reported existence of a large 880 kb
organellar genome in the Hsp60-containing crypton (syn.
mitosome) is puzzling (Ghosh et al., 2000).
1248
Our data suggest that the genome of the original mitochondrial endosymbiont in E. histolytica mitosomes has
been lost – like many other mitochondrial components –
through reductive evolution. Most genes present in the
genome of the original mitochondrial endosymbiont have
been transferred to the cell nucleus. Biosynthesis of nuclearencoded organellar proteins is thus carried out in the
cytoplasm, with subsequent targeting of these proteins into
their respective organelles through highly sophisticated
protein import mechanisms (Haucke & Schatz, 1997; Kunau
et al., 2001). It has recently been hypothesized that the
reason why all known mitochondria (and chloroplasts) have
retained a highly reduced but functional genome might be
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:18:41
Microbiology 150
E. histolytica mitosomes lack a detectable genome
Fig. 3. Immunodetection of DNA and
Cpn60 in a mixed-membrane fraction of E.
histolytica enriched in membrane-bounded
organelles. Aliquots of a mixed-membrane
parasite fraction obtained from cell-free
extracts fractionated by differential centrifugation were fixed and incubated with specific anti-Cpn60 antibodies. Slides were then
subjected to in situ nick-translation and
confocal immunofluorescence microscopy as
described in the legend to Fig. 2. No colocalization of DNA and Cpn60 label is
observed at low (a) or high (b) computerassisted magnification of different optical
fields.
that – as most of the remaining genes encode the structural
proteins responsible for the maintenance of the redox
balance in the cell – the expression of redox proteins needs
to be tightly regulated on site to avoid the potential toxic
effects of their electron transfer activities during ATP
biosynthesis (Allen & Raven, 1996; Race et al., 1999; Allen,
2003). It is proposed that such tight regulation requires the
co-location of genes and gene products for efficient
operation (Allen, 2003). In this respect, the fact that ATP
biosynthesis is no longer compartmentalized in E. histolytica
but occurs via substrate level phosphorylation in the cell
cytosol is highly relevant (Müller, 2003). The selective
pressure for the retention of a mitosomal genome would
have eased as the organelle was freed from the constraints of
electron transfer activities, a relaxation that may have led to
the complete loss of the mitosomal genome.
demonstration that EhKOs emanate from the cell nucleus
confirm the autogenous nuclear origin of EhKOs and
invalidate the suggestion that these cellular structures might
be self-replicating organelles of endosymbiotic origin. The
apparent biogenesis of minute vesicles from the cell nucleus
in the diplomonad G. intestinalis (Benchimol, 2002) – akin
to the process observed in E. histolytica (Solis et al., 2002) –
betrays the existence of an enigmatic biological phenomenon which may be unique to these parasites and which
deserves further investigation. Given the different origins,
cellular distribution and molecular composition of mitosomes and EhKOs, we conclude that mitosomes and EhKOs
of E. histolytica are distinct and unrelated subcellular
structures.
All known endosymbiosis-derived organelles, i.e. mitochondria, chloroplasts, mitosomes and hydrogenosomes, are
self-replicating entities that segregate faithfully into each
daughter cell following asexual cell division. In contrast, the
proportion of the parasite population harbouring EhKOs
appears to be cell-cycle-dependent, with as few as 10 and
20 % of the population displaying these structures at G2 and
S phases, respectively (Rodrı́guez et al., 1998). In experiments with unsynchronized parasite trophozoites the
percentage of the cell population harbouring extranuclear
DNA does not exceed 50 % (Orozco et al., 1997; Rodrı́guez
et al., 1998; Luna-Arias et al., 1999), a distribution not
consistent with that of an essential cellular organelle carrying out vital cellular functions. EhKOs have been shown to
emanate from the cell nucleus, to be cell-cycle-regulated and
to contain eukaryotic-type rRNA, a pyruvate : ferredoxin
oxidoreductase-like protein and a number of eukaryotictype transcriptional regulators, including a TATA boxbinding protein, an enhancer-binding protein and a
tumour-suppressor protein (Orozco et al., 1997; Rodrı́guez
et al., 1998; Luna-Arias et al., 1999; Solis et al., 2002;
Marchat et al., 2003; Mendoza et al., 2003). The presence
of eukaryotic-type nuclear components and the recent
This work was supported by a grant (111/C13820) from the BBSRC
to J. T.
http://mic.sgmjournals.org
ACKNOWLEDGEMENTS
REFERENCES
Allen, J. F. (2003). The function of genomes in bioenergetic
organelles. Philos Trans R Soc Lond B Biol Sci 358, 19–37.
Allen, J. F. & Raven, J. A. (1996). Free-radical-induced mutation vs
redox regulation: costs and benefits of genes in organelles. J Mol Evol
42, 482–492.
Arisue, N., Sánchez, L. B., Weiss, L. M., Müller, M. & Hashimoto, T.
(2002). Mitochondrial-type hsp70 genes of the amitochondriate
protists Giardia intestinalis, Entamoeba histolytica and two microsporidians. Parasitol Int 51, 9–16.
Bakatselou, C., Kidgell, C. & Clark, C. G. (2000). A mitochondrial-
type hsp70 gene of Entamoeba histolytica. Mol Biochem Parasitol 110,
177–182.
Bakatselou, C., Beste, D., Kadri, A. O., Somanath, S. & Clark, C. G.
(2003). Analysis of genes of mitochondrial origin in the genus
Entamoeba. J Eukaryot Microbiol 50, 210–214.
Bankier, A. T., Spriggs, H. F., Fartmann, B., Konfortov, B. A.,
Madera, M., Vogel, C., Teichmann, S. A., Ivens, A. & Dear, P. H.
(2003). Integrated mapping, chromosomal sequencing and sequence
analysis of Cryptosporidium parvum. Genome Res 13, 1787–1799.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:18:41
1249
G. León-Avila and J. Tovar
Bayona-Bafaluy, M. P., Acin-Perez, R., Mullikin, J. C. & 7 other
authors (2003). Revisiting the mouse mitochondrial DNA sequence.
CCAAT/enhancer-binding proteins are regulated through the cell
cycle. Exp Parasitol 103, 82–87.
Nucleic Acids Res 31, 5349–5355.
Matouschek, A., Pfanner, N. & Voos, W. (2000). Protein unfold-
Benchimol, M. (2002). A new set of vesicles in Giardia lamblia. Exp
ing by mitochondria. The Hsp70 import motor. EMBO Rep 1,
404–410.
Parasitol 102, 30–37.
Cavalier-Smith, T. (1983). A 6-kingdom classification and a unified
phylogeny. In Endocytobiology, vol. II, pp. 1027–1034. Berlin & New
York: Walter de Gruyter & Co.
Cavalier-Smith, T. (1998). A revised six-kingdom system of life. Biol
Rev Camb Philos Soc 73, 203–266.
Mendoza, L., Orozco, E., Rodriguez, M. A., Garcia-Rivera, G.,
Sanchez, T., Garcia, E. & Gariglio, P. (2003). Ehp53, an Entamoeba
histolytica protein, ancestor of the mammalian tumour suppressor
p53. Microbiology 149, 885–893.
Cavalier-Smith, T. (2002). The phagotrophic origin of eukaryotes
Morrison, H. G., Roger, A. J., Nystul, T. G., Gillin, F. D. & Sogin, M. L.
(2001). Giardia lamblia expresses a proteobacterial-like DnaK
and phylogenetic classification of Protozoa. Int J Syst Evol Microbiol
52, 297–354.
Müller, M. (2003). Energy metabolism. Part I: anaerobic protozoa. In
Clark, C. G. & Diamond, L. S. (2002). Methods for cultivation of
luminal parasitic protists of clinical importance. Clin Microbiol Rev
15, 329–341.
Clark, C. G. & Roger, A. J. (1995). Direct evidence for secondary loss
of mitochondria in Entamoeba histolytica. Proc Natl Acad Sci U S A
92, 6518–6521.
Clemens, D. L. & Johnson, P. J. (2000). Failure to detect DNA in
homolog. Mol Biol Evol 18, 530–541.
Molecular Medical Parasitology, pp. 125–139. Edited by J. Marr, T.
Nilsen & R. Komuniecki. London: Academic Press.
Orozco, E., Gharaibeh, R., Riverón, A. M., Delgadillo, D. M.,
Mercado, M., Sánchez, T., Gómez-Conde, E., Vargas, M. A. &
López-Revilla, R. (1997). A novel cytoplasmic structure containing
DNA networks in Entamoeba histolytica trophozoites. Mol Gen Genet
254, 250–257.
hydrogenosomes of Trichomonas vaginalis by nick translation and
immunomicroscopy. Mol Biochem Parasitol 106, 307–313.
Race, H. L., Herrmann, R. G. & Martin, W. (1999). Why have
Dyall, S. D. & Johnson, P. J. (2000). Origins of hydrogenosomes
Riordan, C. E., Ault, J. G., Langreth, S. G. & Keithly, J. S. (2003).
and mitochondria: evolution and organelle biogenesis. Curr Opin
Microbiol 3, 404–411.
Cryptosporidium parvum Cpn60 targets a relict organelle. Curr Genet
44, 138–147.
Embley, T. M., van der Giezen, M., Horner, D. S., Dyal, P. L. &
Foster, P. (2003a). Mitochondria and hydrogenosomes are two
Rodrı́guez, M. A., Garcı́a-Pérez, R. M., Mendoza, L., Sánchez, T.,
Guillén, N. & Orozco, E. (1998). The pyruvate : ferredoxin oxido-
forms of the same fundamental organelle. Philos Trans R Soc Lond B
Biol Sci 358, 191–204.
reductase enzyme is located in the plasma membrane and in a
cytoplasmic structure in Entamoeba. Microb Pathog 25, 1–10.
Embley, T. M., van der Giezen, M., Horner, D. S., Dyal, P. L., Bell, S.
& Foster, P. G. (2003b). Hydrogenosomes, mitochondria and early
Sigler, P. B., Xu, Z., Rye, H. S., Burston, S. G., Fenton, W. A. &
Horwich, A. L. (1998). Structure and function in GroEL-mediated
organelles retained genomes? Trends Genet 15, 364–370.
eukaryotic evolution. IUBMB Life 55, 387–395.
protein folding. Annu Rev Biochem 67, 581–608.
Ghosh, S., Field, J., Rogers, R., Hickman, M. & Samuelson, J.
(2000). The Entamoeba histolytica mitochondrion-derived organelle
Solis, F., Orozco, E., Córdova, L., Rivera, B., Luna-Arias, J. P.,
Gómez-Conde, E. & Rodrı́guez, M. A. (2002). Entamoeba histolytica:
(crypton) contains double-stranded DNA and appears to be bound
by a double membrane. Infect Immun 68, 4319–4322.
DNA carrier vesicles in nuclei and kinetoplast-like organelles
(EhkOs). Mol Genet Genomics 267, 622–628.
Haucke, V. & Schatz, G. (1997). Import of proteins into
Tachezy, J., Sánchez, L. B. & Müller, M. (2001). Mitochondrial type
mitochondria and chloroplasts. Trends Cell Biol 7, 103–106.
iron-sulfur cluster assembly in the amitochondriate eukaryotes
Trichomonas vaginalis and Giardia intestinalis, as indicated by the
phylogeny of IscS. Mol Biol Evol 18, 1919–1928.
Katinka, M. D., Duprat, S., Cornillot, E. & 14 other authors (2001).
Genome sequence and gene compaction of the eukaryote parasite
Encephalitozoon cuniculi. Nature 414, 450–453.
Kunau, W. H., Agne, B. & Girzalsky, W. (2001). The diversity of
organelle protein import mechanisms. Trends Cell Biol 11, 358–361.
LaGier, M. J., Tachezy, J., Stejskal, F., Kutisova, K. & Keithly, J. S.
(2003). Mitochondrial-type iron–sulphur cluster biosynthesis genes
(IscS and IscU ) in the apicomplexan Cryptosporidium parvum.
Microbiology 149, 3519–3530.
Luna-Arias, J. P., Hernandez-Rivas, R., Dios-Bravo, G., Garcia, J.,
Mendoza, L. & Orozco, E. (1999). The TATA-box binding protein of
Entamoeba histolytica: cloning of the gene and location of the protein
by immunofluorescence and confocal microscopy. Microbiology 145,
33–40.
Mai, Z., Ghosh, S., Frisardi, M., Rosenthal, B., Rogers, R. &
Samuelson, J. (1999). Hsp60 is targeted to a cryptic mitochondrion-
derived organelle (‘‘crypton’’) in the microaerophilic protozoan
parasite Entamoeba histolytica. Mol Cell Biol 19, 2198–2205.
Marchat, L. A., Pezet-Valdez, M., Lopez-Camarillo, C. & Orozco, E.
(2003). Entamoeba histolytica: expression and DNA binding of
1250
Tovar, J., Fischer, A. & Clark, C. G. (1999). The mitosome, a novel
organelle related to mitochondria in the amitochondrial parasite
Entamoeba histolytica. Mol Microbiol 32, 1013–1021.
Tovar, J., León-Avila, G., Sánchez, L. B., Sutak, R., Tachezy, J.,
van der Giezen, M., Hernández, M., Müller, M. & Lucocq, J. M.
(2003). Mitochondrial remnant organelles of Giardia function in
iron-sulphur protein maturation. Nature 426, 172–176.
van der Giezen, M. & Tovar, J. (2004). Mitosomes, hydrogenosomes
and mitochondria; variations on a theme? In Organelles, Genomes
and Eukaryotic Phylogeny: an Evolutionary Synthesis in the Age of
Genomics. Edited by R. P. Hirt & D. S. Horner. Boca Raton, FL: CRC
Press (in press).
Williams, B. A. P., Hirt, R. P., Lucocq, J. M. & Embley, T. M. (2002).
A mitochondrial remnant in the microsporidian Trachipleistophora
hominis. Nature 418, 865–869.
Zhu, G. & Keithly, J. S. (2002). Alpha-proteobacterial relationship
of apicomplexan lactate and malate dehydrogenases. J Eukaryot
Microbiol 49, 255–261.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 00:18:41
Microbiology 150