Endosymbiotic Microbiota of the Bamboo Pseudococcid Antonina crawii (Insecta, Homoptera)

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcid Antonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned from A. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the γ subdivision of the division Proteobacteria (γ-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a β-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences of Spiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections of A. crawii and in situ hybridization with specific oligonucleotide probes. The γ- and β-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the γ- and β-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the β-Proteobacteria symbiont was detected in all five pseudococcids, the γ-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only in A. crawii.     

Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins.     

Characterization of chemoautotrophic bacterial symbionts in a gutless marine worm Oligochaeta, Annelida) by phylogenetic 16S rRNA sequence analysis and in situ hybridization.

The phylogenetic relationships of chemoautotrophic endosymbionts in the gutless marine oligochaete Inanidrilus leukodermatus to chemoautotrophic ecto- and endosymbionts from other host phyla and to free-living bacteria were determined by comparative 16S rRNA sequence analysis. Fluorescent in situ hybridization confirmed that the 16S rRNA sequence obtained from these worms originated from the symbionts. The symbiont sequence is unique to I. leukodermatus. In phylogenetic trees inferred by both distance and parsimony methods, the oligochaete symbiont is peripherally associated with one of two clusters of chemoautotrophic symbionts that belong to the gamma subdivision of the Proteobacteria. The endosymbionts of this oligochaete form a monophyletic group with chemoautotrophic ectosymbionts of a marine nematode. The oligochaete and nematode symbionts are very closely related, although their hosts belong to separate, unrelated animal phyla. Thus, cospeciation between the nematode and oligochaete hosts and their symbionts could not have occurred. Instead, the similar geographic locations and habitats of the hosts may have influenced the establishment of these symbioses.     

Endosymbioses between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea)

Siboglinid tubeworms do not have a mouth or gut and live in obligate associations with bacterial endosymbionts. Little is currently known about the phylogeny of frenulate and moniliferan siboglinids and their symbionts. In this study, we investigated the symbioses of two co-occurring siboglinid species from a methane emitting mud volcano in the Arctic Ocean (Haakon Mosby Mud Volcano, HMMV): Oligobrachia haakonmosbiensis (Frenulata) and Sclerolinum contortum (Monilifera). Comparative sequence analysis of the host-specific 18S and the symbiont-specific 16S rRNA genes of S. contortum showed that the close phylogenetic relationship of this host to vestimentiferan siboglinids was mirrored in the close relationship of its symbionts to the sulfur-oxidizing gammaproteobacterial symbionts of vestimentiferans. A similar congruence between host and symbiont phylogeny was observed in O. haakonmosbiensis: both this host and its symbionts were most closely related to the frenulate siboglinid O. mashikoi and its gammaproteobacterial symbiont. The symbiont sequences from O. haakonmosbiensis and O. mashikoi formed a clade unaffiliated with known methane- or sulfur-oxidizing bacteria. Fluorescence in situ hybridization indicated that the dominant bacterial phylotypes originated from endosymbionts residing inside the host trophosome. In both S. contortum and O. haakonmosbiensis, characteristic genes for autotrophy (cbbLM) and sulfur oxidation (aprA) were present, while genes diagnostic for methanotrophy were not detected. The molecular data suggest that both HMMV tubeworm species harbour chemoautotrophic sulfur-oxidizing symbionts. In S. contortum, average stable carbon isotope values of fatty acids and cholesterol of -43 per thousand were highly negative for a sulfur oxidizing symbiosis, but can be explained by a (13)C-depleted CO(2) source at HMMV. In O. haakonmosbiensis, stable carbon isotope values of fatty acids and cholesterol of -70 per thousand are difficult to reconcile with our current knowledge of isotope signatures for chemoautotrophic processes.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Symbiotic bacteria associated with stomach discs of human lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation "Candidatus Riesia pediculicola" for the louse symbionts.     

Two Intracellular Symbiotic Bacteria from the Mulberry Psyllid Anomoneura mori (Insecta, Homoptera)

We characterized the intracellular symbiotic bacteria of the mulberry psyllid Anomoneura mori by performing a molecular phylogenetic analysis combined with in situ hybridization. In its abdomen, the psyllid has a large, yellow, bilobed mycetome (or bacteriome) which consists of many round uninucleated mycetocytes (or bacteriocytes) enclosing syncytial tissue. The mycetocytes and syncytium harbor specific intracellular bacteria, the X-symbionts and Y-symbionts, respectively. Almost the entire length of the bacterial 16S ribosomal DNA (rDNA) was amplified and cloned from the whole DNA of A. mori, and two clones, the A-type and B-type clones, were identified by restriction fragment length polymorphism analysis. In situ hybridization with specific oligonucleotide probes demonstrated that the A-type and B-type 16S rDNAs were derived from the X-symbionts and Y-symbionts, respectively. Molecular phylogenetic analyses of the 16S rDNA sequences showed that these symbionts belong to distinct lineages in the γ subdivision of the Proteobacteria. No 16S rDNA sequences in the databases were closely related to the 16S rDNA sequences of the X- and Y-symbionts. However, the sequences that were relatively closely related to them were the sequences of endosymbionts of other insects. The nucleotide compositions of the 16S rDNAs of the X- and Y-symbionts were highly AT biased, and the sequence of the X-symbiont was the most AT-rich bacterial 16S rDNA sequence reported so far.     

Symbiotic Bacteria Associated with Stomach Discs of Human Lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “Candidatus Riesia pediculicola” for the louse symbionts.     

Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma- and epsilonproteobacteria at four Mid-Atlantic Ridge hydrothermal vent fields

The shrimp Rimicaris exoculata from hydrothermal vents on the Mid-Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free-living relative. Both the epsilon- and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma- and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host-symbiont selectivity play a role in structuring the epibiosis of R. exoculata.     

Bacterial host specificity of Lucinacea endosymbionts: Interspecific variation in 16S rRNA sequences

Abstract Three tropical lucinid clams ( Codakia orbiculata, Codakia pectinella and Lucina nassula ) from a shallow coastal environment have been studied regarding to their thioautotrophic bacterial endosymbionts. The 16S rRNA genes (rDNA) from these three endosymbionts were amplified using PCR. Phylogenetic analysis by distance matrix and parsimony methods always placed the newly examined symbionts within the monophyletic group composed of symbionts of the bivalve superfamily Lucinacea. A same single 16S rRNA sequence was found in C. orbiculata and C. pectinella and was identical to that found in C. orbicularis and Linga pensylvanica , two other lucinids living in the same type of environment. These data indicate that a same symbiont species may be associated with different host species. Lucina nassula hosts a symbiont with a distinct 16S rDNA sequence, but very closely related to the former.     

Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.     

Free-living tube worm endosymbionts found at deep-sea vents

Recent evidence suggests that deep-sea vestimentiferan tube worms acquire their endosymbiotic bacteria from the environment each generation; thus, free-living symbionts should exist. Here, free-living tube worm symbiont phylotypes were detected in vent seawater and in biofilms at multiple deep-sea vent habitats by PCR amplification, DNA sequence analysis, and fluorescence in situ hybridization. These findings support environmental transmission as a means of symbiont acquisition for deep-sea tube worms.     

Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences.

The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis [C. R. Woese, Microbiol. Rev. 51: 221-271, 1987]). Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.     

Acidovorax-like symbionts in the nephridia of earthworms

Dense accumulations of bacteria in the excretory organs, nephridia, were first described more than 75 years ago in members of the annelid family Lumbricidae (earthworms). These nephridial symbionts were assumed to play a role in the degradation of proteins in the excretory fluid for nitrogen recycling. In the present study, the phylogenetic affiliation of the nephridial bacteria of the earthworms Lumbricus terrestris, Aporrectodea tuberculata, Octolasion lacteum and Eisenia foetida was resolved. The 16S rRNA gene sequences of the symbionts formed a monophyletic cluster within the genus Acidovorax. Similarity between symbiont sequences from different host species was 95.5-97.6%, whereas similarity was> 99% between symbiont sequences from individuals of the same species. Densely packed bacteria were detected in the ampulla of the nephridia by fluorescence in situ hybridization (FISH) using Acidovorax-specific oligonucleotide probes. No other bacterial cells could be found by FISH, although a few sequences other than Acidovorax had been found by PCR and cloning. These results suggest that the Acidovorax-earthworm symbiosis is a stable, host-specific association that has evolved from a common bacterial ancestor. Given the close phylogenetic relationship of the symbionts to proteolytic, free-living Acidovorax species, they may indeed play a role in protein degradation during nitrogen excretion by earthworms.     

Dual symbiosis in a Bathymodiolus sp. mussel from a methane seep on the Gabon continental margin (Southeast Atlantic): 16S rRNA phylogeny and distribution of the symbionts in gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 microM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.     

Coupling of bacterial endosymbiont and host mitochondrial genomes in the hydrothermal vent clam Calyptogena magnifica

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Vesicomyidae) depends for its nutrition on sulfur-oxidizing symbiotic bacteria housed in its gill tissues. This symbiont is transmitted vertically between generations via the clam's eggs; however, it remains uncertain whether occasionally symbionts are horizontally transmitted or acquired from the environment. If symbionts are transmitted strictly vertically through the egg cytoplasm, inheritance of symbiont lineages should behave as if coupled to the host's maternally inherited mitochondrial DNA. This coupling would be obscured, however, with low rates of horizontal or environmental transfers, the equivalent of recombination between host lineages. Population genetic analyses of C. magnifica clams and associated symbionts from eastern Pacific hydrothermal vents clearly supported the hypothesis of strictly maternal cotransmission. Host mitochondrial and symbiont DNA sequences were coupled in a clam population that was polymorphic for both genetic markers. These markers were not similarly coupled with sequence variation at a nuclear gene locus, as expected for a randomly mating sexual population. Phylogenetic analysis of the two cytoplasmic genes also revealed no evidence for recombination. The tight association between vesicomyid clams and their vertically transmitted bacterial endosymbionts is phylogenetically very young (<50 million years) and may serve as a model for the origin and evolution of eukaryotic organelles.     

Molecular and ecological evidence for species specificity and coevolution in a group of marine algal-bacterial symbioses

The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of Prionitis, P. lanceolata (from central California) and P. decipiens (from Peru). This investigation reports gall formation on a third related host, Prionitis filiformis. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the alpha subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gall-bearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.     

Coexistence of bacterial sulfide oxidizers, sulfate reducers, and spirochetes in a gutless worm (Oligochaeta) from the Peru margin

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, approximately 300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the gamma-Proteobacteria (Gamma 1 and 2 symbionts), two members of the delta-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the delta-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.     

Bacterial endosymbionts of free-living amoebae

The occurrence of bacterial endosymbionts in free-living amoebae has been known for decades, but their obligate intracellular lifestyle hampered their identification. Application of the full cycle rRNA approach, including 16S rRNA gene sequencing and fluorescence in-situ hybridization with 16S rRNA-targeted oligonucleotide probes, assigned the symbionts of Acanthamoeba spp. and Hartmannella sp. to five different evolutionary lineages within the Proteobacteria, the Bacteroidetes, and the Chlamydiae, respectively. Some of these bacterial symbionts are most closely related to bacterial pathogens of humans, and it has been suggested that they should be considered potential emerging pathogens. Complete genome sequence analysis of a chlamydia-related symbiont of Acanthamoeba sp. showed that this endosymbiont uses similar mechanisms for interaction with its eukaryotic host cell as do the well-known bacterial pathogens of humans. Furthermore, phylogenetic analysis suggested that these mechanisms have been evolved by the ancestor of these amoeba symbionts in interplay with ancient unicellular eukaryotes.