Hydrogenosome behavior during the cell cycle in Tritrichomonas foetus

The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments. The hydrogenosome behavior in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle, is reported here. The hydrogenosomes were followed by light and transmission electron microscopy during the whole cell cycle. Videomicroscopy, immunofluorescence microscopy, and immunocytochemistry were also used. It is shown that the hydrogenosomes divide at any phase of the cell cycle and that the organellar division is not synchronized. During the interphase the hydrogenosomes are distributed mainly along the axostyle and costa, and at the beginning of mitosis migrate to around the nucleus. Three forms of hydrogenosome division were seen: (1). segmentation, where elongated hydrogenosomes are further separated by external membranous profiles; (2). partition, where rounded hydrogenosomes, in a bulky form, are further separated by a membranous internal septum and, (3). a new dividing form: heart-shaped hydrogenosomes, which gradually present a membrane invagination leading to the organelle division. The hydrogenosomes divide at any phase of the cell cycle. A necklace of intramembranous particles delimiting the outer hydrogenosomal membrane in the region of organelle division was observed by freeze-etching. Similarities between hydrogenosomes and mitochondria behavior during the cell cycle are discussed.     

Doublet Formation and Cytoplasmic Symbionts in the Ciliate Urostyla grandis

The formation of homopolar doublets can be induced by the action of antibiotics which inhibit the growth of cytoplasmic bacterial symbionts whose cell cycle appears to be controlled by the host. They multiply during the macronuclear S-phase of the ciliate and become enclosed in vesicles and are largely destroyed just before cell division is completed. Since doublet formation is due to an incomplete cell division, and because experimental disturbances at the cell cortex of dividing ciliates also lead to doublets, the symbionts are thought to contribute some factor which is essential for normal cytokinesis of the host cell.     

An anaerobic protozoon, with symbiotic methanogens, living in municipal landfill material

Abstract We have established that anaerobic protozoa do live in municipal landfill material although they probably spend much of the time encysted, especially in the drier (< 40% water) site. At least eight species were observed; they were readily isolated by adding anoxic water to dry landfill samples. The ciliate Metopus palaeformis was frequently i isolated; it appears to be ubiquitous in anaerobic landfills. It has a polymorphic life cycle, it is positive for hydrogenase, each ciliate contains about 500 bromoethanesulfonate-sensitive methanogen symbionts (probably Methanobacterium formicicum ), and maximum cell densities in culture exceed 3000 per ml. The methanogens are not attached to the hydrogenosomes, neither do they undergo morphological transformation; the ciliate receives no measurable energetic advantage from its symbionts. The ciliate encysts in response to a shortage of food or water, and the methanogens remain viable within the cysts. When the protozoon excysts, the methanogens resume growth and cell division within the trophic form of the ciliate. Unlike free-living methanogens, the M. palaeformis -methanogen consortium is not particularly sensitive to oxygen; the symbiotic methanogens remain viable following exposure of the consortium to atmospheric oxygen for several days. Dispersal of methanogen-bearing protozoan cysts through oxygenated environments is a potential mechanism of transfer between landfill sites and other anaerobic environments. Anaerobic protozoan consortia are theoretically capable of making a significant contribution to methane generation from wet landfill sites.     

Synthesis of peptidoglycan and membrane during the division cycle of rod-shaped, gram-negative bacteria.

A modified procedure for determining the pattern of peptidoglycan synthesis during the division cycle has allowed the measurement of the rate of side wall synthesis during the division cycle without the contribution due to pole formation. As predicted by a model proposing that the surface growth of the cell is regulated by mass increase, we find a decrease in side wall synthesis in the latter half of the division cycle. This supports the proposal that, upon invagination, pole growth accommodates a significant proportion of the increasing cell mass and that residual side wall growth occurs in response to the residual mass increase not accommodated by pole volume. The observed side wall synthesis patterns support the proposal that mass increase is a major, and possibly sole, regulator of bacterial surface increase. Membrane synthesis during the division cycle of the gram-negative, rod-shaped bacteria Escherichia coli and Salmonella typhimurium has also been measured with similar methods. The rate of membrane synthesis--measured by incorporation of radioactive glycerol or palmitate relative to simultaneous labeling with radioactive leucine--exhibits the same pattern as peptidoglycan synthesis. The results are compatible with a model of cell surface growth containing the following elements. (i) During the period of the division cycle prior to invagination, growth of the cell occurs predominantly in the side wall and the cell grows only in length. (ii) When invagination begins, pole growth accommodates some cytoplasmic increase, leading to a concomitant decrease in side wall synthesis. (iii) Surface synthesis increases relative to mass synthesis during the last part of the division cycle because of pole formation. It is proposed here that membrane synthesis passively follows the pattern of peptidoglycan synthesis during the division cycle.     

Hydrogenosome-Methanogen Assemblages in the Echinoid Endocommensal Plagiopylid Ciliates,Lechriopyla mystax Lynch, 1930 and Plagiopyla minuta Powers, 19331

ABSTRACT. Lechriopyla mystax Lynch, 1930 and Plagiopyla minula Powers, 1933 contain hydrogenosome-methanogen assemblages similar to those reported for other plagiopylid ciliates. These assemblages are stacks of elongate ovoid hydrogenosomes alternating with methanogens; these stacks are surrounded by cisternae of the rough endoplasmic reticulum that are often accompanied by Golgi complexes. The individual methanogens in the larger ciliate, L. mystax, are about four times the volume of those in the smaller ciliate, P. minuta, but both ciliates appear to contain Gram-negative methanococcoid bacteria, possibly Methanoplanus sp. The endoplasmic reticulum-Golgi complex probably plays a significant role in exploitation of the methanogens by the host ciliate.     

Modeling the Individual Growth of Tetrahymena Sp. and its Population Consequences

ABSTRACT. Three types of mathematical growth models are presented to describe the individual growth of the ciliate Tetrahymena sp. feeding on the bacterium Pseudomonas fluorescens . Both organisms were isolated from a domestic waste-water treatment plant. Growth of individual ciliates and the consequences for the whole population are considered. Experimental data, obtained by following the individual ciliate during its lifespan from cell division to cell division, are used for parameter estimations. Differences between growth models for individuals turn out to have little effect on the specific population growth rate and the mean cell volume. In case of exponential growth of individuals the unstructured and structured population models are equivalent, even in time-variant environments. This knowledge can be applied in the stability analysis of food chains or forced systems. The results obtained facilitates quantification of protozoa biomass as a function of bacterial biomass in chemostats. More specifically, it highlights the dynamic behaviour of bacteria and protozoa in waste-water treatment plants.     

Division Cycle of Myxococcus xanthus III. Kinetics of Cell Growth and Protein Synthesis

The kinetics of cell growth and protein synthesis during the division cycle of Myxococcus xanthus was determined. The distribution of cell size for both septated and nonseptated bacteria was obtained by direct measurement of the lengths of 8,000 cells. The Collins-Richmond equation was modified to consider bacterial growth in two phases: growth and division. From the derived equation, the growth rate of individual cells was computed as a function of size. Nondividing cells (growth phase) comprised 91% of the population and took up 87% of the time of the division cycle. The absolute and specific growth rates of nondividing cells were observed to increase continually throughout the growth phase; the growth rate of dividing cells could not be determined accurately by this technique because of changes in the geometry of cells between the time of septation and physical separation. The rate of protein synthesis during the division cycle was measured by pulselabeling an exponential-phase culture with radio-active valine or arginine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of protein synthesis as a function of cell size was obtained. Nondividing cells showed an increase in both the absolute and specific rates of protein synthesis throughout the growth phase; the specific rate of protein synthesis for dividing cells was low when compared to growthphase cells. Cell growth and protein synthesis are compared to the previously reported kinetics of deoxyribonucleic acid and ribonucleic acid synthesis during the division cycle.     

Monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors

Continuous growth of individual bacteria has been previously studied by direct observation using optical imaging. However, optical microscopy studies are inherently diffraction limited and limited in the number of individual cells that can be continuously monitored. Here we report on the use of the asynchronous magnetic bead rotation (AMBR) sensor, which is not diffraction limited. The AMBR sensor allows for the measurement of nanoscale growth dynamics of individual bacterial cells, over multiple generations. This torque-based magnetic bead sensor monitors variations in drag caused by the attachment and growth of a single bacterial cell. In this manner, we observed the growth and division of individual Escherichia coli, with 80-nm sensitivity to the cell length. Over the life cycle of a cell, we observed up to a 300% increase in the rotational period of the biosensor due to increased cell volume. In addition, we observed single bacterial cell growth response to antibiotics. This work demonstrates the non-microscopy limited AMBR biosensor for monitoring individual cell growth dynamics, including cell elongation, generation time, lag time, and division, as well as their sensitivity to antibiotics.     

Hydrogenosomes under microscopy

A hydrogenosome is a hydrogen-producing organelle, evolutionary related to mitochondria and is found in Parabasalia protozoa, certain chytrid fungi and certain ciliates. It displays similarities to and differences from mitochondria. Hydrogenosomes are spherical or slightly elongated organelles, although very elongated hydrogenosomes are also found. They measure from 200 nm to 1 μm, but under stress conditions can reach up to 2 μm. Hydrogenosomes are surrounded by two closely apposed membranes and present a granular matrix. Cardiolipin has been detected in their membranes, and frataxin, which is a conserved mitochondrial protein involved in iron metabolism, was also recently found. Hydrogenosomes have one or multiple peripheral vesicles, which incorporate calcium. The peripheral vesicle can be isolated from the hydrogenosomal matrix and can be considered as a distinct hydrogenosomal compartment. Dysfunctional hydrogenosomes can be removed by an autophagic process and further digested by lysosomes. Hydrogenosomes divide in three different ways, like mitochondria, by segmentation, partition and the heart form. They may divide at any phase of the cell cycle. Nucleoid or electron dense deposits found in hydrogenosomes can be considered artifacts or dysfunctional hydrogenosomes. The hydrogenosome does not contain a genome, although DNA has already been detected in one anaerobic ciliate. Hydrogenosomes can be considered as good drug targets since their metabolism is distinct from mitochondria.     

测定绿脓杆菌分裂指数反映生长势头的初步研究

本文以细胞生物学理论为基础,细菌细胞发育周期形态和群体细菌分裂相数量的变化为依据,研究了测定绿脓杆菌DI方法。建立了数学模型,分析了生长过程中DI动态。结果表明,DI可较准确地反映细菌生长势头。实验采用同龄细菌选择和同步培养技术,免疫荧光染色,显微镜下直接观察分裂相细菌,并计算其占总菌数的比例。在对数期前DI已上升,开始后迅速达高峰,接近稳定期时已下降到较低水平。与生长速度比较,两者动态基本一致,但DI高峰较早出现77分钟。认为DI与生长速度关系密切,是与细菌生命活动物质交换的三流学说相伴随的一种特殊信息流。     

Phylogenetic Analysis and Fluorescence <Emphasis Type="Italic">In Situ</Emphasis> Hybridization Detection of Archaeal and Bacterial Endosymbionts in the Anaerobic Ciliate <Emphasis Type="Italic">Trimyema Compressum</Emphasis>

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 μg ml⁻¹), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host’s fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.     

Using single cell cultivation system for on-chip monitoring of the interdivision timer in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cell cycle

Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.     

Evolution of Amitosis of the Ciliate Macronucleus: Gain of the Capacity to Divide

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.     

Evolution of amitosis of the ciliate macronucleus: gain of the capacity to divide

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.     

Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division

The budding yeast, Saccharomyces cerevisiae, was grown exponentially at different rates in the presence of growth rate-limiting concentrations of a protein synthesis inhibitor, cycloheximide. The volumes of the parent cell and the bud were determined as were the intervals of the cell cycle devoted to the unbudded and budded periods. We found that S. cerevisiae cells divide unequally. The daughter cell (the cell produced at division by the bud of the previous cycle) is smaller and has a longer subsequent cell cycle than the parent cell which produced it. During the budded period most of the volume increase occurs in the bud and very little in the parent cell, while during the unbudded period both the daughter and the parent cell increase significantly in volume. The length of the budded interval of the cell cycle varies little as a function of population doubling time; the unbudded interval of the parent cell varies moderately; and the unbudded interval for the daughter cell varies greatly (in the latter case an increase of 100 min in population doubling time results in an increase of 124 min in the daughter cell's unbudded interval). All of the increase in the unbudded period occurs in that interval of G1 that precedes the point of cell cycle arrest by the S. cerevisiae alpha-mating factor. These results are qualitatively consistent with and support the model for the coordination of growth and division (Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Exp. Cell. Res. 105:79-98.) This model states that growth and not the events of the DNA division cycle are rate limiting for cellular proliferation and that the attainment of a critical cell size is a necessary prerequisite for the "start" event in the DNA-division cycle, the event that requires the cdc 28 gene product, is inhibited by mating factor and results in duplication of the spindle pole body.     

Reactivation of stationary Tetrahymena : A contribution to the question of G0 state

Stationary cells of Tetrahymena were reactivated to exponential growth phase by transfer to fresh medium. The sequence of resuming cell cycle events was analysed by scoring the division index, the labelling index for macro- and micronuclei and the increase in cell number. By long-term labelling it was found that all cells replicate in stationary phase cultures. They also divide eventually. Upon transfer to fresh medium a small fraction of cells (about 3%) divide immediately, whereas the rest divide 3 h later after having replicated their macronuclear DNA. The kinetics of entry into the S phase indicates that these cells have a lag period of about 2 h before they resume progress through the cell cycle. It takes more than 1 h until all cells have begun replication. These data show that in stationary cultures all cells proceed through the events of the cell cycle. The cell cycle phases are extended differentially, G1 taking the largest part. During G2 cells pass very slowly through a certain stage close to division. Under the present conditions there is no indication for cells being in a resting state that is not part of the cell cycle, from which they can be restimulated and which has been called the G0 state. The criteria to demonstrate a resting state of this nature are discussed.     

Ecological Traits of the Algae‐Bearing Tetrahymena utriculariae (Ciliophora) from Traps of the Aquatic Carnivorous Plant Utricularia reflexa

Trap fluid of aquatic carnivorous plants of the genus Utricularia hosts specific microbiomes consisting of commensal pro‐ and eukaryotes of largely unknown ecology. We examined the characteristics and dynamics of bacteria and the three dominant eukaryotes, i.e. the algae‐bearing ciliate Tetrahymena utriculariae (Ciliophora), a green flagellate Euglena agilis (Euglenophyta), and the alga Scenedesmus alternans (Chlorophyta), associated with the traps of Utricularia reflexa. Our study focused on ecological traits and life strategies of the highly abundant ciliate whose biomass by far exceeds that of other eukaryotes and bacteria independent of the trap age. The ciliate was the only bacterivore in the traps, driving rapid turnover of bacterial standing stock. However, given the large size of the ciliate and the cell‐specific uptake rates of bacteria we estimated that bacterivory alone would likely be insufficient to support its apparent rapid growth in traps. We suggest that mixotrophy based on algal symbionts contributes significantly to the diet and survival strategy of the ciliate in the extreme (anaerobic, low pH) trap‐fluid environment. We propose a revised concept of major microbial interactions in the trap fluid where ciliate bacterivory plays a central role in regeneration of nutrients bound in rapidly growing bacterial biomass.     

Surface-limited growth: a model for the synchronization of a growing bacterial culture through periodic starvation

This article analyses the Surface-Limited Growth Model put forward to explain the very tight synchrony, over more than ten division cycles, obtained experimentally by subjecting a growing bacterial culture to alternating periods of starvation and dilution, using inorganic phosphate as the limiting substrate. The Model states that when an essential nutrient is in limited supply, the rate of growth of an individual cell will be proportional to its surface area (and the current concentration of the limiting substance) rather than to its volume. This decrease in dimensionality from volume to surface is expected to favor the smaller cells and so result ultimately in a narrower size distribution. The Surface-Limited Growth Model deals with cell growth under unusual nutritional conditions, and its predictions depend on how the cell replication cycle is assumed to behave under these same circumstances. Two alternatives are considered: the volume at which cells divide is the same during the starvation phase as during steady-state exponential growth, and the cells adjust immediately to the changing growth rate. In the latter case, we have tested both C + D constant with time and C + D variable (where C + D is the time between initiation of chromosome replication and the corresponding cell division), the incremental value at any instant being computed separately for each individual cell from its current effective growth rate. The simulation results are of two sorts depending on the auxiliary assumptions used. Either the dilution-starvation cycles have no effect whatsoever on the cell volume distribution, or the width of the distribution decreases gradually with time, approaching zero slowly and asymptotically, but the mean cell volume decreases as well--directly contradicting experimental observations. We conclude that the Surface-Limited Growth Model is incapable of explaining the synchronization of cells by periodic starvation of a growing bacterial culture.