A Mitochondrial Ancestry of the Hydrogenosomes of Nyctotherus ovalis

Angela H. A. M. van Hoek1,*, Anna S. Akhmanova2,*, Martijn A. Huynen{dagger} and Johannes H. P. Hackstein4,*

*Department of Microbiology and Evolutionary Biology, Faculty of Science, University of Nijmegen, Nijmegen, The Netherlands; and
{dagger}Biocomputing EMBL, Heidelberg, Germany, Max-Delbrück-Centrum for Molecular Medicine, Berlin-Buch, Germany, and Bioinformatics Group, University of Utrecht, Utrecht, The Netherlands

Hydrogenosomes are membrane-bounded organelles about 1 µm in size that, like mitochondria, produce ATP (Müller 1993Citation ). They compartmentalize terminal steps of anaerobic energy metabolism but, unlike mitochondria, hydrogenosomes cannot use oxygen as an electron acceptor; they reduce protons to molecular hydrogen (Embley and Martin 1998Citation ; Martin and Müller 1998Citation ; Müller 1998Citation ). Hydrogenosomes have been found only in anaerobic protists and fungi, for example, the parabasalian flagellate Trichomonas vaginalis, the amoeboflagellate Psalteriomonas lanterna, the anaerobic ciliate Nyctotherus ovalis, and the anaerobic chytridiomycete fungi Neocallimastix frontalis and Piromyces sp. (Müller 1993Citation ).

A wealth of biochemical and molecular genetic evidence argues that hydrogenosomes share a common ancestor with mitochondria (Biagini, Finlay, and Lloyd 1997Citation ; Embley, Horner, and Hirt 1997Citation ; Sogin 1997Citation ; Martin and Müller 1998Citation ). However, since—with one notable exception—all hydrogenosomes studied so far lack a genome, a direct proof remained difficult. Recently, we provided evidence for the presence of a genome in the hydrogenosomes of the gut-dwelling anaerobic ciliate N. ovalis (Akhmanova et al. 1998Citation ). Immunogold labeling allowed colocalization of hydrogenase and double-stranded DNA in the hydrogenosomes of the ciliate. These organelles look like mitochondria, but they are surrounded by endosymbiotic methanogenic archaea that indicate (1) anoxic conditions in the immediate vicinity of the mitochondria-like organelles and (2) the presence of intracellular hydrogen (cf. Fenchel and Finlay 1995Citation ). Using reverse transcription–polymerase chain reaction (RT-PCR) and PCR approaches, we succeeded in isolating and sequencing a complete SSU rRNA that is abundantly expressed in the ciliates (Akhmanova et al. 1998Citation ). This putative hydrogenosomal SSU rRNA (accession number Y16670) proved to be greatly different with regard to nucleotide sequence and the lengths of variable regions from the respective SSU rRNA genes of the ciliate and its methanogenic endosymbionts (vanHoek et al. 1998, 1999, 2000Citation ). Notwithstanding a high degree of sequence divergence, secondary-structure analysis clearly revealed that all stem/loop structures characteristic of the "structural cores" of the SSU rRNAs (Stiegler et al. 1981Citation ; Gray, Sankoff, and Cedergren 1984Citation ) were conserved. Phylogenetic analysis based on evolutionary conserved rDNA sequence blocks (universally conserved regions U1—U8; Gray, Sankoff, and Cedergren 1984Citation ) revealed that the isolated sequence clustered together with mitochondrial SSU rRNA genes of aerobic ciliates (Akhmanova et al. 1998Citation ).

We showed earlier that the ciliates hosted by different cockroach strains exhibit substantial sequence divergence of their nuclear ribosomal genes. The degree of this sequence divergence (up to 9%), the presence of host-specific methanogenic endosymbionts, and differences in morphology and swimming behavior suggest that different cockroach strains host different (sub)species of N. ovalis (van Hoek et al. 1998, 1999, 2000Citation ). Here, we report the isolation of additional hydrogenosomal SSU rRNA genes of different N. ovalis isolates from several cockroach strains. In addition to the previously described complete hydrogenosomal SSU rRNA sequence of N. ovalis from Periplaneta americana var. Nijmegen, we isolated partial SSU rRNA sequences from ciliates hosted by the strains P. americana var. Bayer and P. americana var. Amsterdam. These sequences display substantial DNA sequence divergence, and ciliates from the host strain P. americana var. Amsterdam exhibit heteroplasmy (cf. Lightowlers et al. 1997Citation ). The SSU rRNA sequences Amsterdam 1 and Amsterdam 2 differ in the nonconserved regions of the fragment for several nucleotides. However, an alignment of conserved blocks of hydrogenosomal, mitochondrial, and bacterial SSU rRNAs is possible (fig. 1 ). The alignment allows a phylogenetic reconstruction that is supported by several methods of data analysis. Notably, phylogenetic analysis reveals monophyly of the SSU rRNAs from hydrogenosomes and mitochondria from ciliates (fig. 2a ). This would be expected if hydrogenosomes of anaerobic ciliates evolved from the mitochondria of their aerobic relatives.



View larger version (118K):
[in this window]
[in a new window]
 
Fig. 1.—An alignment of hydrogenosomal (Nyctotherus ovalis Amsterdam, N. ovalis Bayer, N. ovalis Nijmegen), mitochondrial (Schizosaccharomyces pombe, Chondrus crispus, Paramecium tetraurelia, Tetrahymena pyriformis, Oenothera sp., Reclinomonas americana, Pylaiella littoralis, Prototheca wickerhamii, Dictyostelium discoideum), and bacterial (Thermotoga maritima, Escherichia coli, Rickettsia prowazekii) SSU rRNAs. The alignment was created using the CLUSTAL X package (Jeanmougin et al. 1998Citation ). Only blocks that are well conserved across all sequences (J. Castresana, personal communication) are indicated and used for phylogenetic reconstruction. The partial sequences of the hydrogenosomal SSU rRNAs from N. ovalis from Periplaneta americana var. Amsterdam (accession numbers AJ237908 and AJ237909) and from P. americana var. Bayer (accession number AJ237907) were obtained by PCR using primers derived from conserved regions of the mitochondrial SSU rRNA of Paramecium tetraurelia (accession number K01751): 5'-TGTGCCAGCAGCCGCGGTAA-3' (positions 625–644) and 5'-CCC(AC)TACC(AG)GTACCTTGTGT-3' (positions 1637–1657). The sequence from N. ovalis from P. americana var. Amsterdam 2 is for the positions used in the alignment and phylogenetic analysis identical to that for N. ovalis var. Amsterdam 1. The numbers in parentheses are the numbers of nucleotides that have been omitted from a particular sequence.

 


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2.—a, A neighbor-joining tree based on the alignment displayed in figure 1. Bootstrap values larger than 700 are indicated. The tree was created using CLUSTAL X (Jeanmougin et al. 1998Citation ), which uses the Kimura (1980)Citation model to estimate evolutionary distances. The monophyly of the hydrogenosomal SSU rRNAs with the mitochondrial SSU rRNAs of aerobic ciliates was supported by a maximum-likelihood analysis with MOLPHY (Adachi and Hasegawa 1996Citation ). With approximate likelihood, the 1,000 best trees were calculated using a semiconstrained tree in which the Nyctotherus ovalis cluster, the Tetrahymena pyriformis + Paramecium tetraurelia cluster, and the Schizosaccharomyces pombe + Dictyostelium discoideum cluster were fixed, and using the HKY85 model (Hasegawa, Kishino, and Yano 1985Citation ) to account for biases in substitution rates and nucleotide composition. Among the 1,000 trees, one in which the ciliate mitochondria and hydrogenosomes were monophyletic had the highest probability in a full likelihood analysis, although the probability was not significantly higher than that of the best tree that did not support this specific monophyly. Furthermore, quartet puzzling (Strimmer and von Haeseler 1996Citation ) using the HKY85 model and a uniform model of rate heterogeneity, or, alternatively, eight gamma-distributed rate categories, supports the monophyly of the ciliate mitochondria and hydrogenosomes with reliability values of 99 and 95, respectively. Finally, a neighbor-joining tree in which variation among nucleotide frequencies of the various sequences was taken into account using log-determinant distances (Steel 1994Citation ) supported the monophyly of the ciliate mitochondria and hydrogenosomes with a bootstrap value of 96. Increasing the number of positions included from the alignment did not alter the clustering of the hydrogenosomal SSU rRNAs with the mitochondrial SSU rRNAs of aerobic ciliates in the neighbor-joining tree. b, Southern blot of undigested genomic DNA from N. ovalis from the various cockroach strains (from the left: Periplaneta americana var. Amsterdam, P. americana var. Bayer, P. americana var. Nijmegen, Blaberus sp. var. Amsterdam) probed with 32P-labeled DNA from the hydrogenosomal SSU rRNA gene of N. ovalis from P. americana var. Nijmegen. The probe labels a fragment with an apparent size of approximately 11 kb. The wash consisted of 70 mM Na2PO4, pH 7.3, at 65°C; exposure occurred overnight. The marker was 1–10 kb. Under these conditions, the probe does not hybridize with the 18S rDNA from N. ovalis or the SSU rDNAs from the anerobic chytrids Piromyces sp. L2 and Neocallimastix sp. E2, the methanogenic archaeon Methanomicrococcus blatticolus, or the {alpha} proteobacterium Echerichia coli. Nyctotherus ovalis cells were isolated from dissected cockroach hindguts using their characteristic galvanotactic swimming toward the anode over a distance of about 5 cm (van Hoek et al. 1999Citation ). Since all other ciliates besides the cockroach-dwelling N. ovalis species swim to the cathode under the influence of a constant DC-field (Machemer and de Peyer 1977Citation ; Wagener, Stumm, and Vogels 1986Citation ; Machemer-Röhnisch, Machemer, and Bräucker 1996Citation ; van Hoek et al. 1999Citation ), a contamination by other ciliates—whether aerobic or anaerobic—can be excluded. Moreover, it has been shown by both DNA sequencing and ribotyping (ARDRA analysis) of the SSU rDNA genes of individual ciliates that the hindgut of a given cockroach strain hosts only one host-specific Nyctotherus (sub)species (van Hoek et al. 1998Citation ).

 
Northern blotting has revealed that the hydrogenosomal SSU rRNA genes are abundantly expressed, excluding the possibility that the isolated genes are inactive or a PCR artifact (Akhmanova et al. 1998Citation ). In addition, Southern blotting strongly suggests that the SSU rRNA genes described here are regular constituents of an organelle genome: SSU rDNA probes hybridize to a band of an approximate size of 11 kb of undigested genomic DNA from the various ciliate isolates (fig. 2b ). This DNA fragment is substantially larger than the rRNA gene itself (and also a putative association of a SSU and LSU gene). Since there is also no indication for a cross-hybridization with both the ciliate’s and the endosymbiont’s SSU rDNAs (cf. also Akhmanova et al. 1998Citation ), it is likely that the DNA fragment with an apparent size of approximately 11 kb represents the hydrogenosomal genome.

We do not yet know whether the putative genome is circular or linear. Assuming a genome of 11 kb or larger (if circular), then the hydrogenosomal genome might be well within the size range of mitochondrial genomes (Gray, Burger, and Lang 1999Citation ). The most related (linear) mitochondrial genomes of the aerobic ciliates Paramecium and Tetrahymena measure approximately 40–50 kb (Nosek et al. 1998Citation ; Gray, Burger, and Lang 1999Citation ). We have not yet proven that the putative hydrogenosomal SSU rRNA genes are transcribed inside the hydrogenosomes or that the corresponding rRNAs are actually used for hydrogenosomal protein synthesis. However, to our knowledge, there is not a single report on the presence of (transcribed) mitochondrial ribosomal genes outside a mitochondrion in any eukaryote whatsoever. Moreover, immunogold labeling using antibodies against double-stranded DNA has revealed the presence of DNA in the macronucleus and the micronucleus of the ciliate, the methanogenic endosymbionts, and the mitochondria-like hydrogenosomes (Akhmanova et al. 1998Citation ; unpublished data). Since the cytoplasm is virtually free of label, it is reasonable to assume that the "mitochondrial" SSU rDNA is located in the mitochondria-like hydrogenosomes. Therefore, we have to conclude that the hydrogenosomes of N. ovalis evolved from mitochondria—most likely in a process that involved adaptation of the ciliates to anaerobic environments (Embley et al. 1995Citation ; Hirt, Wilkinson, and Embley 1998Citation ).


    Acknowledgements
 TOP
 Acknowledgements
 References
 
M.A.H. thanks Jose Castresana for assistance in the phylogenetic analyses. The research of M.A.H. has been made possible in part by a grant from the Royal Netherlands Academy of Arts and Sciences.


    Footnotes
 
William Martin, Reviewing Editor

1 Keywords: hydrogenosomes, mitochondria, evolution, anaerobic ciliates, ribosomal small-subunit RNA (SSU rRNA). Back

2 Present address: DLO State Institute for Quality Control of Agricultural Products (RIKILT), Wageningen, The Netherlands. Back

3 Present address: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands. Back

4 Address for correspondence and reprints: J. H. P. Hackstein, Department of Microbiology and Evolutionary Biology, Faculty of Science, University of Nijmegen, Toernooiveld, NL-6525 ED Nijmegen, The Netherlands. E-mail: hack{at}sci.kun.nl Back


    References
 TOP
 Acknowledgements
 References
 

    Adachi, K., and M. Hasegawa. 1996. MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Comput. Sci. Monogr. 28:1–150.

    Akhmanova, A., F. Voncken, T. van Alen, A. van Hoek, B. Boxma, G. Vogels, M. Veenhuis, and J. H. P. Hackstein. 1998. A hydrogenosome with a genome. Nature 396:527–528.

    Biagini, G. A., B. J. Finlay, and D. Lloyd. 1997. Evolution of the hydrogenosome. FEMS Microbiol. Lett. 155:133–140.[ISI][Medline]

    Embley, T. M., B. J. Finlay, P. L. Dyal, R. P. Hirt, M. Wilkinson, and A. G. Williams. 1995. Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates. Proc. R. Soc. Lond. B Biol. Sci. 262:87–93.[ISI][Medline]

    Embley, T. M., D. A. Horner, and R. P. Hirt. 1997. Anaerobic eukaryote evolution: hydrogenosomes as biochemically modified mitochondria? Trends Ecol. Evol. 12:437–441.

    Embley, T. M., and W. Martin. 1998. A hydrogen-producing mitochondrion. Nature 396:517–519.

    Fenchel, T. and B. J. Finlay. 1995. Ecology and evolution in anoxic worlds. Oxford University Press, Oxford, England.

    Gray, M. W., G. Burger, and B. F. Lang. 1999. Mitochondrial evolution. Science 283:1476–1481.

    Gray, M. W., D. Sankoff, and R. J. Cedergren. 1984. On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA. Nucleic Acids Res. 12:5837–5852.[Abstract]

    Hasegawa, M., H. Kishino, and T. Yano. 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22:160–174.[ISI][Medline]

    Hirt, R. P., M. Wilkinson, and T. M. Embley. 1998. Molecular and cellular evolution of ciliates: a phylogenetic perspective. Pp. 327–340 in G. H. Coombs, K. Vickerman, M. A. Sleigh, and A. Warren, eds. Evolutionary relationships among protozoa. The Systematics Association, Special Volume Series 56. Kluwer Academic Publishers, Dordrecht, Boston, London.

    Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins, and T. J. Gibson. 1998. Multiple sequence alignment with Clustal X. Trends. Biochem. Sci. 23:403–405.[ISI][Medline]

    Kimura, M. 1980. A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111–120.[ISI][Medline]

    Lightowlers, R. N., P. F. Chinnery, D. M. Turnbull, and N. Howell. 1997. Mammalian mitochondrial genetics: heredity, heteroplasmy and disease. Trends Genet. 13:450–455.[ISI][Medline]

    Machemer, H., and J. de Peyer. 1977. Swimming sensory cells: electrical membrane parameters receptor properties and motor control in ciliated protozoa. Verh. Dtsch. Zool. Ges. 70:86–110.

    Machemer-Röhnisch, S., H. Machemer, and R. Bräucker. 1996. Electric-field effects on gravikinesis in Paramecium. J. Comp. Physiol. [A] 179:213–226.

    Martin, W., and M. Müller. 1998. The hydrogen hypothesis for the first eukaryote. Nature 392:37–41.

    Müller, M. 1993. The hydrogenosome. J. Gen. Microbiol. 139:2879–2889.[ISI][Medline]

    ———. 1998. Enzymes and compartmentation of core energy metabolism of anaerobic protists—a special case in eukaryotic evolution? Pp. 109–132 in G. H. Coombs, K. Vickerman, M. A. Sleigh, and A. Warren, eds. Evolutionary relationships among protozoa. The Systematics Association, Special Volume Series 56. Kluwer Academic Publishers, Dordrecht, Boston, London.

    Nosek, J., L. Tomaska, H. Fukuhara, Y. Suyama, and L. Kovac. 1998. Linear mitochondrial genomes: 30 years down the line. Trends Genet. 14:184–188.[ISI][Medline]

    Sogin, M. L. 1997. Organelle origins: energy-producing symbionts in early eukaryotes? Curr. Biol. 7:R315–R317.

    Steel, M. 1994. Recovering a tree from the Markov leaf colourations it generates under a Markov model. Appl. Math. Lett. 7:19–23.

    Stiegler, P., P. Carbon, J.-P. Ebel, and C. Ehresmann. 1981. A general secondary-structure model for prokaryotic and eukaryotic RNAs of the small ribosomal subunits. Eur. J. Biochem. 120:487–495.[Abstract]

    Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964–969.[Free Full Text]

    van Hoek, A. H. A. M., V. S. I. Sprakel, T. A. van Alen, T. Brigge, G. D. Vogels, and J. H. P. Hackstein. 2000. Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates. Mol. Biol. Evol. (in press).

    van Hoek, A. H. A. M., T. A. van Alen, V. S. I. Sprakel, J. H. P. Hackstein, and G. D. Vogels. 1998. Evolution of anaerobic ciliates from the gastro-intestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives. Mol. Biol. Evol. 15:1195–1206.[Abstract]

    van Hoek, A. H. A. M., T. A. van Alen, V. S. I. Sprakel, G. D. Vogels, and J. H. P. Hackstein. 1999. Voltage-dependent reversal of galvanotaxis in Nyctotherus ovalis. J. Eukaryot. Microbiol. 46:427–433.

    Wagener, S., C. K. Stumm, and G. D. Vogels. 1986. Electromigration, a tool for studies on anaerobic ciliates. FEMS Microbiol. Ecol. 38:197–203.[ISI]

Accepted for publication September 9, 1999.