Abundance and distribution of anaerobic protozoa and their contribution to methane production in Lake Cadagno (Switzerland)

Abstract In the uupermost layers of the anoxic sediment in Lake Cadagno, 9 different species of anaerobic protozoa were identified. The total number of these organisms was about 580 cells·ml⁻¹ sediment. Most pf these protozoa contained endosymbiotic methanogenic bacteria which in total amounted to 10⁶ methanogens·ml⁻¹ sediment. In addition to the methanogenic endosymbionts, cells of Metopus setosus and Caenomorpha lata also contained a non-fluorescent bacterial rod inside the cytoplasm. In some individual cells of C. lata this second type of endosymbiotic bacterium was sometimes the only endosymbiont observed. Contrary to earlier suggestions, anaerobic protozoa do not seem to play a major role in methane production at least in Lake Cadagno. No significant methane production due to the anaerobic protozoa and their methanogenic endosymbionts was found in situ. Isolated ciliates and amoebae produced methane at 12°C, but not at 6°C, probably as a result of temperature limitation. In the sediment of Lake Cadagno sulfate reduction seemed to be the dominant terminal degradation process.     

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Endosymbiotic interactions in anaerobic protozoa

Several aspects of the endosymbiosis of methanogenic archaea with anaerobic protozoa are reviewed. Special attention is payed to the role of hydrogenosomes and plastid-like organelles that seem to provide the substrates for the methanogenic endosymbionts. Evidence is presented that hydrogenosomes evolved several times in the various protoctistan taxa. Hydrogenosomes are seemingly different, and their common denominator is the production of hydrogen. The absence of nucleic acids and a protein-synthesizing machineny hampers the analysis of their divergent evolutionary history, and molecular genetic data argue not only for different but even a chimeric origin of the hydrogenosomes.     

Endosymbiosis and the design of eukaryotic electron transport

The bioenergetic organelles of eukaryotic cells, mitochondria and chloroplasts, are derived from endosymbiotic bacteria. Their electron transport chains (ETCs) resemble those of free-living bacteria, but were tailored for energy transformation within the host cell. Parallel evolutionary processes in mitochondria and chloroplasts include reductive as well as expansive events: On one hand, bacterial complexes were lost in eukaryotes with a concomitant loss of metabolic flexibility. On the other hand, new subunits have been added to the remaining bacterial complexes, new complexes have been introduced, and elaborate folding patterns of the thylakoid and mitochondrial inner membranes have emerged. Some bacterial pathways were reinvented independently by eukaryotes, such as parallel routes for quinol oxidation or the use of various anaerobic electron acceptors. Multicellular organization and ontogenetic cycles in eukaryotes gave rise to further modifications of the bioenergetic organelles. Besides mitochondria and chloroplasts, eukaryotes have ETCs in other membranes, such as the plasma membrane (PM) redox system, or the cytochrome P450 (CYP) system. These systems have fewer complexes and simpler branching patterns than those in energy-transforming organelles, and they are often adapted to non-bioenergetic functions such as detoxification or cellular defense.     

Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester

After immobilization of anaerobes on polyurethane foam in a thermophilic, fixed-bed, anaerobic digester supplied with acetate, the results of real-time PCR analysis indicated that the major immobilized methanogenic archaea were Methanosarcina spp., and that the major free-living methanogenic archaea were Methanosarcina and Methanobacterium spp. 16S rRNA gene densities of Methanosarcina spp. and Methanobacterium spp. immobilized on the polyurethane foam were 7.6x10(9) and 2.6x10(8) copies/cm3, respectively. Immobilized methanogenic archaea could be concentrated 1000 times relative to those in the original anaerobically digested sludge from a completely mixed thermophilic digester supplied with cattle waste. On the other hand, immobilized bacteria could be concentrated only 10 times. The cell densities of the immobilized methanogenic archaea and bacteria were higher than those of the free-living methanogenic archaea and bacteria in the reactor. The results of clone analysis indicate that the major methanogenic archaea of the original thermophilic sludge are members of the order Methanomicrobiales, and that the major methanogenic archaea immobilized on the polyurethane foam are Methanosarcina spp., and those of the liquid phase are Methanobacterium spp. The results of the real time PCR analysis approximately agree with those of the clone analysis. These results indicate that real-time PCR analysis is useful for quantitatively describing methanogenic communities.     

Endosymbiosis,cell evolution,and speciation

In 1905, the Russian biologist C. Mereschkowsky postulated that plastids (e.g., chloroplasts) are the evolutionary descendants of endosymbiotic cyanobacteria-like organisms. In 1927, I. Wallin explicitly postulated that mitochondria likewise evolved from once free-living bacteria. Here, we summarize the history of these endosymbiotic concepts to their modern-day derivative, the “serial endosymbiosis theory”, which collectively expound on the origin of eukaryotic cell organelles (plastids, mitochondria) and subsequent endosymbiotic events. Additionally, we review recent hypotheses about the origin of the nucleus. Model systems for the study of “endosymbiosis in action” are also described, and the hypothesis that symbiogenesis may contribute to the generation of new species is critically assessed with special reference to the secondary and tertiary endosymbiosis (macroevolution) of unicellular eukaryotic algae.     

A briefly argued case that mitochondria and plastids are descendants of endosymbionts,but that the nuclear compartment is not

Recent findings are summarized in support of the view that mitochondria (including hydrogenosomes) and plastids (including complex ones) descend from symbiotic associations of once free-living organisms. The reasoning behind endosymbiotic hypotheses stems from a comparison of biochemistry and physiology in organelles with that in free-living cells; their strength is shown to lie in the specific testable predictions they generate about expected similarity patterns among genes. Although disdained for many decades, endosymbiotic hypotheses have gradually become very popular. In the wake of that popularity, endosymbiotic hypotheses have been formulated to explain the origins of eukaryotic cell compartments and structures that have no biochemical similarity to free-living cells. In particular, it has become fashionable in recent years to entertain the century-old notion that the nucleus might also descend from an endosymbiotic bacterium. A critique of that hypothesis is formulated and a simple alternative to it is outlined, which derives the nuclear compartment in a mitochondrion-bearing cell.     

Mutation rate and genome reduction in endosymbiotic and free-living bacteria

Genome reduction has been considered the hallmark of endosymbiotic bacteria, such as endocellular mutualists or obligatory pathogens until it was found exactly the same in several free-living bacteria. In endosymbiotic bacteria genome reduction is mainly attributed to degenerative processes due to small population size. These cannot affect the free-living bacteria with reduced genomes because they are known to have very large population sizes. It has been proposed that selection for simplification drove genome reduction in these free-living bacteria. For at least one of them (Prochlorococcus), genome reduction is associated with accelerated evolution and we suggest an alternative hypothesis based on increase in mutation rate as the primary cause of genome reduction in free-living bacteria.     

Anaerobiosis and Symbiosis with Bacteria in Free-living Ciliates

SYNOPSIS. Marine, sediment-dwelling ciliates were examined for cytochrome oxidase activity by a cytochemical method and for fine structural details. Species of Plagiopylidae (Trichostomatida), i.e. Plagiopyla frontata, Sonderia vorax and Sonderia sp., and of Heterotrichida, i.e., Parablepharisma pellitum, Parablepharisma sp., Metopus contortus, Metopus vestitus and Caenomorpha capucina ; previously considered to be obligate anaerobes because of their sulfide-containing habitat, do not have cytochrome oxidase activity or mitochondria with cristae or tubuli. The evolutionary origin and significance of anaerobic ciliates is discussed. Most of the anaerobic ciliates harbor a flora of ecto- and endosymbiotic bacteria as demonstrated by transmission and scanning electron micrographs. It is speculated that the bacteria may utilize the metabolic end products of the protozoa for growth and energy yielding processes. These associations are also compared with other, previously described cases of symbiosis involving prokaryotes and protozoa.     

Anaerobiosis and symbiosis with bacteria in free-living ciliates.

Marine, sediment-dwelling ciliates were examined for cytochrome oxidase activity by a cytochemical method and for fine structural details. Species of Plagiopylidae (Trichostomatida), i.e. Plagiopyla frontata, Sonderia vorax and Sonderia sp., and of Heterotrichda, i.e., Parablepharisma pellitum, Parablepharisma sp., Metopus contortus, Metopus vestitus and Caenomorpha capucina; previously considered to be obligate anaerobes because of their sulfide-containing habitat, do not have cytochrome oxidase activity or mitochondria with cristae or tubuli. The evolutionary origin and significance of anaerobic ciliates is discussed. Most of the anaerobic ciliates harbor a flora of ecto- and endosymbiotic bacteria as demonstrated by transmission and scanning electron micrographs. It is speculated that the bacteria may utilize the metabolic end products of the protozoa for growth and energy yielding processes. These associations are also compared with other, previously described cases of symbiosis involving prokaryotes and protozoa.     

Hydrogenosomes in some anaerobic protozoa resemble mitochondria

Abstract Microbodies in six anaerobic ciliated protozoa ( Metopus, Brachonella, Plagiopyla, Parablepharisma, Sonderia, Saprodinium ) were found to be enclosed by two membranes. The inner membrane showed extensive infolding, division stages were observed, and in all genera apart from Sonderia and Parablepharisma , the microbodies contained an hydrogenase and were attached to methanogenic bacteria. Some of these ciliates are related to aerobic species with mitochondria. We believe that the microbodies are hydrogenosomes, that they are derived from mitochondria, and that their biochemical modification has incurred little change in the original mitochondrial ultrastructure. These observations weaken the case for the independent origin of hydrogenosomes from anaerobic prokaryotes.     

Synchronous Division of an Endosymbiotic Methanogenic Bacterium In the Anaerobic Ciliate Plagiopyla Frontata Kahl

ABSTRACT The life cycle of a methanogenic bacterium, symbiotic within the marine, free-living anaerobic ciliate Plagiopyla frontata , was studied using light microscopy and transmission electron microscopy (TEM). the bacteria are disc-shaped. During the growth phase of the host, bacteria and hydrogenosomes (organelles which ferment pyruvate into acetate and hydrogen) are arranged in conglomerates resembling stacks of coins in which bacteria and hydrogenosomes alternate; hydrogenosomes always cap the ends of the stacks. During the growth phase, numbers of hydrogenosomes and bacteria remain constant (about 5,000 and 3,500 per cell, respectively). Hydrogenosomes increase in volume shortly after cell division. Methanogens increase in volume slowly during the growth phase of the ciliate and rapidly when the ciliate begins to divide. the hydrogenosomes divide mainly during the initial phases of cell division while the methanogens divide synchronously during the last phase of ciliate division. the timing of reproduction of the symbionts is controlled by the host-cell cycle. the ciliate is known to receive an energetic advantage from its symbionts. the suppression of continuous bacterial reproduction may trigger the secretion of excess bacterial production as soluble organic compounds, for use by the ciliate.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.     

Association of methanogenic bacteria with rumen protozoa

Methanogenic bacteria superficially associated with rumen entodiniomorphid protozoa were observed by fluorescence microscopy. A protozoal suspension separated from strained rumen fluid (SRF) by gravity sedimentation exhibited a rate of methane production six times greater (per millilitre) than SRF. The number of protozoa (per millilitre) in the protozoal suspension was three times greater than that of SRF; however, the urease activity of this fraction was half that of SRF. The methanogenic activity of SRF and the discrete fractions obtained by sedimentation of protozoa correlated with the numbers of protozoa per millilitre in each fraction. Gravity-sedimented protozoa, washed four times with cell-free rumen fluid, retained 67-71% of the recoverable methanogenic activity. Thus it is evident from our observations that many methanogens adhere to protozoa and that the protozoa support methanogenic activity of the attached methanogens. When protozoa-free sheep were inoculated with rumen contents containing a complex population of protozoa, methanogenic activity of the microflora in SRF samples was not significantly enhanced.     

An overview of endosymbiotic models for the origins of eukaryotes, their ATP-producing organelles (mitochondria and hydrogenosomes), and their heterotrophic lifestyle.

The evolutionary processes underlying the differentness of prokaryotic and eukaryotic cells and the origin of the latter's organelles are still poorly understood. For about 100 years, the principle of endosymbiosis has figured into thoughts as to how these processes might have occurred. A number of models that have been discussed in the literature and that are designed to explain this difference are summarized. The evolutionary histories of the enzymes of anaerobic energy metabolism (oxygen-independent ATP synthesis) in the three basic types of heterotrophic eukaryotes those that lack organelles of ATP synthesis, those that possess mitochondria and those that possess hydrogenosomes--play an important role in this issue. Traditional endosymbiotic models generally do not address the origin of the heterotrophic lifestyle and anaerobic energy metabolism in eukaryotes. Rather they take it as a given, a direct inheritance from the host that acquired mitochondria. Traditional models are contrasted to an alternative endosymbiotic model (the hydrogen hypothesis), which addresses the origin of heterotrophy and the origin of compartmentalized energy metabolism in eukaryotes.     

Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria)

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approximately 98.5%, have been reported to occur as obligate endosymbionts of ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S-23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex life cycle. These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and obligately free-living bacteria ever revealed.     

Suggested mitochondrial ancestry of nonmitochondrial ATP/ADP carrier

One of the major evolutionary events that transformed an endosymbiotic bacterium into a mitochondrion was the acquisition of the ATP/ADP carrier (AAC) in order to supply the host with respiration-derived ATP. Along with the mitochondrial carrier, an unrelated carrier is known, which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic α-proteobacteria. This nonmitochondrial carrier was recently described in rickettsia-like endosymbionts (RLE), a group of obligate intracellular bacteria classified with the order Rickettsiales, which have diverged after free-living α-proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on nonmitochondrial AAC were re-analyzed in the present work, using both DNA and protein sequences and various methods including Bayesian analysis. The data presented are consistent with the classic endosymbiont theory for the origin of mitochondria and suggest that even the last but one common ancestor of rickettsiae and organelles was an endosymbiotic bacterium, in which AAC first originated.     

Strain differentiation of epidemic typhus rickettsiae (Rickettsia prowazekii) by DNA restriction endonuclease analysis

We have investigated the number of methanogenic bacteria in different samples of fresh feces and effluents from the anaerobic digestion of cattle and swine waste and municipal sludges. We have further compared conventional anaerobic techniques with the rigorous, oxygen-free, anaerobic, experimental conditions obtained in a controlled glove cabinet atmosphere. It was found that the total number of oxygen-intolerant methanogenic bacteria was about 10–100 times higher than that of anaerobic oxygen-tolerant methanogenic bacteria. We have also developed a simple technique for the growth of methanogenic bacteria that permits the realization of rigorous anaerobic conditions without the use of a glove cabinet.     

Association of methanogenic bacteria with flagellated protozoa from a termite hindgut

Epifluorescence microscopy was used to examine hindgut contents ofZootermopsis angusticollis (Hagen) termites for the presence of methanogenic bacteria, which can be identified on the basis of the fluorescence of the novel cofactors F₄₂₀ and F₃₅₀. Small, autofluorescent, rod-shaped bacteria of theMethanobrevibacter sp. morphotype were observed associated with three flagellates tentatively identified asTrichomitopsis termopsidis (Cleveland),Tricercomitus termopsidis Kirby andHexamastix termopsidis Kirby. Methanogens were not observed associated with any other protozoal morphotypes and were not numerous in the free-living state inZ. angusticollis hindgut fluid. Electron micrographs of thin sections of hindgut protozoa suggest methanogens are endosymbionts in the small trichomonad protozoa. Our observations are consistent with the finding of Odelson and Breznak that methane is a minor endproduct of the metabolism of termite gut microbiota.Deceased.