The dps gene of symbiotic "Candidatus Legionella jeonii" in Amoeba proteus responds to hydrogen peroxide and phagocytosis

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.     

Purification and characterization of the GroESLx chaperonins from the symbiotic X-bacteria in Amoeba proteus.

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized sigma(70)-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37 degrees C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37 degrees C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37 degrees C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx(7) and GroELx(14) in two stacks of GroELx(7). In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.     

Amoeba proteus displays a walking form of locomotion

This report deals with observations on the directional locomotion of amoeba before and after fixation and scanning electron microscopy. The study was aimed at visualization of the stepwise events of directional movements. After the analysis of the data it is proposed that the amoeba undergoes a sequence of movement events that can be defined as a walking form of locomotion.     

Dynamics of the cytoskeleton in Amoeba proteus

Fluorescein-labeled muscle actin was microinjected into Amoeba proteus and followed during intracellular redistribution by means of the image-intensification technique. The fully polymerization-competent protein becomes part of the endogenous actomyosin system undergoing dynamic changes over time periods of several hours. Single-frame analysis of long-term sequences enabled the direct demonstration of both the contractile activities and morphological transformations of microfilaments in normally locomoting, immobilized and phagocytozing specimens. In normally locomoting cells the filament layer undergoes continuous changes in spatial distribution depending on the actual pattern of cytoplasmic streaming and cell shape. The highest degree of differentiation is always maintained in the intermediate region between the front and the uroid, thus indicating this segment of the cortex to be the most important site in generating motive force for pseudopodium formation and ameboid movement. In immobilized cells contracted by the application of ruthenium red or relaxed by different anesthetics, the filament layer forms a continuous thick sheath beneath the cell surface or becomes completely disintegrated. In phagocytozing cells the local polymerization of actin at the tip of pseudopodia forming the food-cup and around the nascent phagosome points to a significant participation of the actomyosin system in the process of capturing and constricting prey organisms. Although our results provide clear evidence for the overall importance of motive force generation according to the hydraulic pressure theory, some motile phenomena exist in Amoeba proteus that cannot exclusively be explained by this mechanism.     

The Contractile Vacuole in Amoeba proteus: Temperature Effects

The influence of temperature on the various aspects of the contractile vacuole cycle of Amoeba proteus has been established. In the upper temperature range (20, 25 and 30 C) an increase in temperature results in shorter vacuolar cycles with greater systolic (final) volumes. The systole is rapid and always complete. At 35 C the vacuole shows the effect of heat stress, cycles are irregular in volume and duration with only partial systoles. In the lower temperature range (15, 10 and 5 C), a new phenomenon has been observed, the plateau. Instead of undergoing systole, after reaching a certain critical volume the vacuole abruptly ceases to grow in size and remains in a state of pause for a well defined period of time, ending at a comparatively slow but complete systole. The duration of this plateau as well as its inception and termination seem quite precisely controlled. Its effect, a decrease in the fluid output by the vacuole, is such as to adjust vacuolar output to near constant Q₁₀ kinetics over our temperature range. This is correlated with a single straight line fit in an Arrhenius plot. Available data do not permit a complete explanation of the nature of the plateau. It could represent a steady state between 2 opposing phenomena: active fluid influx into the vacuole and osmotic losses from the vacuole into the relatively hypertonic cytoplasm.     

The Chromatin Extrusion Phenomenon in Amoeba proteus Cell Cycle

Amoeba proteus is possibly the best known of all unicellular eukaryotes. At the same time, several quintessential issues of its biology, including some aspects of the cell cycle, remain unsolved. Here, we show that this obligate agamic amoebae and related species have a special type of cyclic polyploidy. Their nucleus has an euploid status only for a small fraction of the cell cycle, during metaphase and telophase. The rest of the time it has an aneuploid status, which is a consequence of polyploidization. Extrusion of “excess” chromatin from the nucleus in late interphase and during prophase results in depolyploidization. Such a strategy of life cycle in unicellular eukaryotes is thought to be the main mechanism of “resetting” the Muller's ratchet and is a satisfactory alternative to the meiotic recombination for agamic protists.     

Peptide stimulation of phagocytosis in Amoeba proteus

Summary Normal articular cartilages from the weightbearing areas of the femoral condyles of the knee joints of 11 patients (3–20 years old) and of 35 Schwarzkopf sheep (3 months to 2 years old) were studied using the electron microscope. The study has shown that the matrix of normal articular cartilage is not only composed of collagen fibrils and proteoglycans, but also contains two types of elastic system fibres. Small elastic fibres can be identified in the superficial and lower radiate zones of cartilage of man and sheep. Similar to elastic fibres in other tissues, they consist of a central amorphous core and are surrounded by aggregates of 10 nm microfibrils. Another type of elastic system fibres, oxytalan fibres, are found in the intermediate and upper radiate zones of the articular cartilage.     

Lysolecithin as a modifier of induced pinocytosis in Amoeba proteus

Lysolecithin was found to modify cation-induced pinocytosis in Amoeba proteus. It is shown here that lysolecithin (LPC) in the concentration range of 10(-15) to 10(-10) g/ml has the same effect on Na+ -induced pinocytosis as cAMP and a pinocytosis regulating factor (PRF) which is secreted by the amoeba. Thus, LPC activated Na+-induced pinocytosis in starved amoebae and decreased the sensitivity to the inducer in normal cells. Pinocytosis depressed by treatment with EGTA or dibucaine became normal upon addition of LPC to the inducer. These effects were also obtained with lysolecithin isolated from the amoeba. It is suggested that PRF and amoeba LPC may be closely related and that phospholipase activity of the amoeba may regulate its capacity for pinocytosis.     

The Feeding of Amoeba proteus on Paramecium aurelia*

SYNOPSIS. Density of prey (Paramecium aurelia) and predator (Amoeba proteus) were varied while volume of inorganic medium was kept constant. Variations in density of prey had little effect on the rates of feeding and reproduction of the amoebae; but with increasing predator density the amoebae captured the paramecia less rapidly and ingested fewer before dividing, altho division size did not change appreciably. Therefore, amoebae of a low density population with a constant food supply carry more nutritional reserves from generation to generation than do those in a denser population.     

Cytoplasmic Incorporation of a Ribonucleic Acid Precursor in Amoeba proteus

The question of RNA synthesis in enucleate cytoplasm of Amoeba has been approached experimentally by incubating enucleate amoebae in a labelled RNA precursor and determining the incorporation into RNA autoradiographically. The results indicate that there is a cytoplasmic incorporation mechanism which can operate in the absence of the nucleus. A comparison is made between Acetabularia and Amoeba with respect to the origins of cytoplasmic RNA. It is concluded that the existing data are consistent with the assumption that some cytoplasmic RNA is of nuclear origin in both organisms.     

The ecological energetics of Amoeba proteus (Protozoa)

The consumption, production and respiration of Amoeba proteus were measured in the laboratory for cells cultured over a range of Tetrahymena pyriformis concentrations (125–4 000 cells/0.5 ml) at 10, 15 and 20 °C. Differences were attributed to both temperature and prey availability. A series of generation energy budgets were constructed for amoebae grown under the above conditions. The biological efficiencies linking the parameters of the budget equation were calculated. Assimilation efficiencies ranged from 22–59% regardless of temperature. Net production efficiencies were high at 15 and 20 °C (65–82%) but low at 10 °C (11–49%). Gross production efficiencies were also higher at 15 and 20 °C (16–47%) than at 10 °C (4–29%). The ecological implications of this investigation are discussed.