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.     

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.     

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.     

Systematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoaMetopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. andPelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells ofMetopus palaeformis, Metopus contortus, Trimyema sp. andCyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera;Methanobacterium, Methanocorpusculum andMethanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. andMetopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis andM. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses inMetopus must postdate the speciation ofM. palaeformis andM. contortus. Despite this,Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.     

Development of Respiration and Mitochondria in Mucor genevensis After Anaerobic Growth: Absence of Glucose Repression

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO(4) fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.     

Isolation and culturing of a most common anaerobic ciliate, Metopus sp

The study includes isolation of anaerobic ciliate, Metopus sp. from an anaerobic reactor and development of its monoculture under laboratory conditions. Separation by centrifugation followed by micromanipulatory isolation resulted in obtaining pure Metopus culture with less bacterial contamination. The isolated Metopus sp. had the mean dimensions of 32 x 123 microm with the generation time of 53 h. Among the different basal media tried, the ciliate mineral medium (CMV) with 1% wheat powder suspension was the most suitable one for Metopus growth. The temperature and pH ranges, for the best growth of Metopus, were 30-35 degrees C and 6-7, respectively. Higher concentrations of volatile fatty acids (VFA) such as acetate, butyrate and propionate had adverse effect on Metopus growth and prevented its growth beyond 0.05 M concentration. Maximum COD removal was in CMV medium by the growth of anaerobic Metopus sp.     

Studies on the uptake and metabolism of adenosine 3':5'-cyclic monophosphate and N6,O2-dibutyryl 3':5'-cyclic adenosine monophosphate in sea urchin eggs

Structurally abnormal mitochondria were found in Candida utilis cells grown in the presence of either high copper concentrations or copper deficiency as compared with cells grown in standard media (copper 60 μg/l). In cells grown under conditions of copper deficiency the average size of the mitochondria is 0.6 μm compared with 0.2 μm of the normal cell, and the cristae have an abnormal appearance. In cells grown in the presence of high copper concentrations the mitochondrial size is up to 2–3 μm with poorly developed cristae and abnormal appearance of the matrix area.     

The Endocommensal Ciliates of Venezuelan Sea Urchins

SYNOPSIS. Two hundred and fifty-nine specimens of the sea urchins Echinometra lucunter (201), Diadema antillarum (52), Tripneustes ventricosus (5) and Eucidaris tribuloides (1), collected on the littoral of the Federal District of Venezuela, were examined for intestinal ciliates.
Iron hematoxylin and silver-impregnation staining technics permitted determination of eight of the nine ciliates found: Anophrys aglycus, A. elongata, Cohnilembus caeci, Biggaria bermudensis, B. echinometris, Cyclidium rhabdotectum, Metopus circumlabens and M. rotundus . An unidentified species of Euplotes was also found.     

Ultrastructural changes of organelles in coleoptile cells during anaerobic germination of rice seeds

Summary Ultrastructural changes of organelles, especially those of mitochondria in rice seedlings germinated under strictly anaerobic conditions were investigated.The embryos of dry seeds had slightly modified mitochondria, characterized by an electron transparent matrix with few cristae and electron opaque patches. These mitochondria developed normally for 48 hours irrespective of whether oxygen was present or not. However, after 72 hours' germination under anaerobic conditions vesiculation of the cristae developed and progressed greatly for the subsequent 24 hours and most of the spaces in the mitochondrion became occupied with vacuolated cristae. Vesiculation seemed to be the effect of cessation of energy supply from the mitochondria themselves.Ultrastructural changes of other organelles such as the plastids and endoplasmic reticulum were also observed after 96 hours under anaerobic conditions.     

MEMBRANOUS STRUCTURES IN YEASTS

1. Most yeast cells carrying out active respiration have spherical or ellipsoidal mitochondria, with plate-like cristae. 2. Cytoplasmic petite strains of Saccharomyces cerevisiae have aberrant mitochondria, often containing whorled membranes. Mutants with deficiencies in the tricarboxylic acid cycle have mitochondria which appear normal when the cells are grown in low levels of glucose. 3. Cells of normal and petite S. cerevisiae grown strictly anaerobically show no recognizable mitochondrial profiles. 4. Carbon substrates which can only be respired promote the development of well-defined mitochondria. In certain facultatively anaerobic yeasts respiration is suppressed by glucose and the mitochondria under these conditions are large, pleomorphic and few in number. Other fermentable carbohydrates do not give this repression. 5. A number of antibacterial antibiotics, which inhibit mitochondrial protein synthesis, cause a disorganization of the mitochondrial cristae. 6. In yeast cells adapting from anaerobic to aerobic conditions mitochondria appear to develop from proliferations of the endoplasmic reticulum, which become progressively more organized. 7. Vacuoles often contain granular material, but in S. cerevisiae the vacuole, which has been described as a lysosome, frequently contains myelin-like lipid inclusions. The material in these inclusions is apparently derived from spherosomes. 8. Endoplasmic reticulum, orientated parallel to the plasmalemma, may be associated with fermentative ability in certain facultatively anaerobic yeasts. Endoplasmic reticulum is also actively involved in the budding process. 9. Normally the yeast-cell plasmalemma shows only minor convolutions, but in chloramphenicol-grown Rhodotorula glutinis the plasmalemma produces vesicular structures termed ‘paramural bodies’. 10. The yeast nuclear membrane has about 200 pores occupying 6–8 % of the total surface area. The nuclear membrane remains intact during mitotic division in yeasts until the daughter nuclei separate.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles.

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48-144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual "noncristate" mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of "noncristate" mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inhibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48–144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual “noncristate” mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of “noncristate” mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Paragonimus westermani possesses aerobic and anaerobic mitochondria in different tissues,adapting to fluctuating oxygen tension in microaerobic habitats

We previously showed that adult Paragonimus westermani, the causative agent of paragonimiasis and whose habitat is the host lung, possesses both aerobic and anaerobic respiratory chains, i.e., cyanide-sensitive succinate oxidase and NADH-fumarate reductase systems, in isolated mitochondria (Takamiya et al., 1994). This finding raises the intriguing question as to whether adult Paragonimus worms possess two different populations of mitochondria, one having an aerobic succinate oxidase system and the other an anaerobic fumarate reductase system, or whether the worms possess a single population of mitochondria possessing both respiratory chains (i.e., mixed-functional mitochondria). Staining of trematode tissues for cytochrome c oxidase activity showed three types of mitochondrial populations: small, strongly stained mitochondria with many cristae, localised in the tegument and tegumental cells; and two larger parenchymal cell mitochondria, one with developed cristae and the other with few cristae. The tegumental and parenchymal mitochondria could be separated by isopycnic density-gradient centrifugation and showed different morphological characteristics and respiratory activities, with low-density tegumental mitochondria having cytochrome c oxidase activity and high-density parenchymal mitochondria having fumarate reductase activity. These results indicate that Paragonimus worms possess three different populations of mitochondria, which are distributed throughout trematode tissues and function facultatively, rather than having mixed-functional mitochondria.     

The resistance to anoxia and the mitochondrial fine structure of rice seedlings

Summary Under anoxia, rice coleoptiles have a remarkable capacity to grow and to preserve undamaged mitochondrial structure and functions. The transfer of aerobically grown intact seedlings to anaerobic conditions resulted in the appearance of unusual mitochondria in coleoptiles as well as in leaves and roots. These mitochondria become filled with stacks of extended cristae, but, obviously, are not affected structurally (only in the root cortex the cells are damaged after a longer period without oxygen). On the contrary, the mitochondria and other organelles of excised coleoptiles, roots and leaves disintegrate after a relatively short exposure to an oxygen-free environment. The degeneration can be avoided if the excised organs are supplied with glucose. Then the mitochondrial fine structure resembles that of intact plants kept under anaerobic conditions.The observations suggest that the capacity of rice coleoptiles to grow under anoxia and to preserve undamaged mitochondria and other organelles is not caused by the resistance of the cell organelles to oxygen deficiency, but rather by the ability of the seedling to transport organic compounds easily, even under the exclusion of oxygen, from the grain to the coleoptile where they can be utilized by glycolysis.     

Glutathione amide and its perthiol in anaerobic sulfur bacteria.

Chromatium species produced the novel biological thiol glutathione amide, gamma-L-glutamyl-L-cysteinylglycine amide (GASH), when grown photoheterotrophically. GASH was largely converted to the corresponding perthiol during photoautotrophic growth on sulfide, suggesting that GASH may have a function in anaerobic sulfide metabolism. This unprecedented form of glutathione metabolism was probably present in anaerobic ancestors of modern cyanobacteria and purple bacteria.     

Mitochondrial biogenesis in Neurospora crassa. I. An ultrastructural and biochemical investigation of the effects of anaerobiosis and chloramphenicol inhibition

The isolation of a new class of mutants permitting facultative anaerobiosis in Neurospora crassa is described. Backcross analyses to the obligate aerobe prototroph (An ^-) indicate single nuclear gene inheritance (An ^-/An ⁺). An ⁺ and An ^- are indistinguishable in morphology and growth rates under aerobic conditions. Anaerobic growth requires nutritional supplements that are dispensable for aerobic growth. Conidiogenesis, conidial germination, and vegetative growth rate are suppressed by anaerobiosis. An ⁺ mutants produce substantial quantities of ethanol under anaerobic conditions. Anaerobiosis and chloramphenicol both affect mitochondrial enzyme activity and morphology. Chloramphenicol inhibition leads to reduction in cytochrome oxidase and swollen mitochondria with few cristae. Anaerobiosis leads to reduction in both cytochrome oxidase and malate dehydrogenase activities, enlarged mitochondria with fewer cristae, enlarged nuclei, and other alterations in cellular morphology. The fine structure of anaerobically grown cells changes with the time of anaerobic growth. We conclude that either inhibition of mitochondrial membrane synthesis or inhibition of respiration might lead to the observed alterations in mitochondria.     

Electromigration, a tool for studies on anaerobic ciliates

Abstract By applying electric current, anaerobic ciliates could be extracted from sludge samples. All the freshwater ciliates tested had one point of optimal current and voltage where they reached the highest net speed of migration in the electric field. The swimming behavior of Metopus es was tested under different current and voltage conditions. During electromigration the freshwater, marine and rumen ciliates moved from the anode to the cathode. In the anaerobic freshwater ciliates high numbersof methanogenic bacteria of different size were present. At the depth in the sludge with the highest number of Metopus minimus (660 cells ·ml⁻¹, a peak of methane production also occurred.     

Two Anaerobic Ciliates (Ciliophora,Armophorea) from China: Morphology and SSU rDNA Sequence,with Report of a New Species,Metopus paravestitus nov. spec

The morphology and phylogeny of two metopid ciliates, collected from anaerobic habitats in China, were investigated using live observation, protargol staining method, and SSU rDNA sequencing. The new species Metopus paravestitus nov. spec. can be distinguished by a combination of the following features: oblong cell with densely arranged ectobiotic prokaryotes perpendicular to cell surface, filiform intracytoplasmic structures packed in the anterior portion of the cell. Our work also demonstrates the wide geographical distribution of Metopus es (Müller, 1776) Lauterborn, 1916. The order Metopida is consistently depicted as a paraphylum in SSU rDNA phylogeny. Metopus paravestitus nov. spec. is closely related to its marine congeners than to freshwater forms. The present study confirms once again the non-monophyly of the genus Metopus and genus Metopidae.     

Ultrastructural Changes during Swelling and Contraction of Mitochondria from Cold-hardened and Non-hardened Winter Wheat

Mitochondria isolated from both 2 and 24 C grown winter wheat (Triticum aestivum L.) undergo spontaneous swelling in isomolar KCI solutions, but only 24 C mitochondria exhibit a substrate-induced contraction response. Electron microscopic examination revealed that 24 C mitochondria have more clearly defined cristae, less matrix material, and are generally more electron-dense than 2 C mitochondria. During swelling, the matrix material of both 2 and 24 C mitochondria expands and the mitochondria become less electron-dense. After partial swelling, 24 C mitochondria contract upon addition of succinate, and regain structural characteristics similar to those of untreated mitochondria. In contrast, mitochondria from 2 C seedlings continue to swell after addition of substrate, and many of the mitochondria become irregular in shape and lose much of their matrix material. A comparison of results obtained from absorbancy measurements, electron microscopy, and a Coulter Counter indicate that swelling and contraction involve changes both in over-all volume, and internal structural characteristics of mitochondria from 2 and 24 C grown seedlings. Electron microscopic examination of shoot cells showed that mitochondria in 24 C grown seedlings possessed more recognizable cristae and greater internal organization than mitochondria in 2 C seedlings.     

Ciliates from a fresh water sulfuretum

Ciliates were collected from a freshwater sulfuretum, Lake Cisó, which is part of a gypsum karstic area whose main feature is Lake Banyoles (Girona, Spain). Chromatium, Lamprocystis and Chlorobium are the major phototrophic sulfur bacteria in Lake Cisó. Blooms of a photosynthetic cryptomonad (up to 5 X 10(5) ind ml-1) were found at the metalimnion. The community of ciliates could be divided in three groups: aerobic, cosmopolitan, genera such as Stentor and Vorticella, in the epilimnion; a large population (up to 10(4) ind ml-1) of Coleps, adapted to low concentrations of both oxygen and sulfide, together with a few individuals of the equally sulfide-tolerant genus Paramecium, in the metalimnion, and anaerobic, true sulfide-loving genera such as Plagiopyla and Metopus, in the hypolimnion, where sulfide concentration was between 0.6 and 1.2 mM.