Rate Of Colony Expansion Research Articles

The Loricrin-Like Protein (LLP) of Phytophthora infestans Is Required for Oospore Formation and Plant Infection.

Loricrin-like protein (LLP) is characterized by a high content of glycine residues and is a major component of plant cell wall. Here, we identified a Phytophthora infestans ortholog of plant LLP, named PiLLP. In P. infestans, PiLLP is strongly expressed in asexual and sexual developmental stages, including in sporangia, zoospores and germinating cysts, and during oospore formation, as well as in the early stages of infection and during hydrogen peroxide stress. Compared with the wild type, the PiLLP-silenced transformants were defective in oospore formation, had slower colony expansion rates, produced less sporangia with lower germination and zoospore-release rates, and were more sensitive to hydrogen peroxide. Moreover, Nile red staining, and PiLLP-red fluorescent protein fusions indicated that PiLLP is involved in oogonia formation. The silenced transformants also had severely diminished virulence levels that could be partially restored with diphenyleneiodium treatments. The analysis of catalase activity showed a decrease of catalase activity in silenced transformants. Thus, PiLLP is important for sexual and asexual reproduction, and is required for oxidative stress tolerance and plant infection.

Innovative High Gas Pressure Microscopy Chamber Designed for Biological Cell Observation.

An original high-pressure microscopy chamber has been designed for real-time visualization of biological cell growth during high isostatic (gas or liquid) pressure treatments up to 200 MPa. This new system is highly flexible allowing cell visualization under a wide range of pressure levels as the thickness and the material of the observation window can be easily adapted. Moreover, the design of the observation area allows different microscope objectives to be used as close as possible to the observation window. This chamber can also be temperature controlled. In this study, the resistance and optical properties of this new high-pressure chamber have been tested and characterized. The use of this new chamber was illustrated by a real-time study of the growth of two different yeast strains - Saccharomyces cerevisiae and Candida viswanathii - under high isostatic gas pressure (30 or 20 MPa, respectively). Using image analysis software, we determined the evolution of the area of colonies as a function of time, and thus calculated colony expansion rates.

From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate.

The organization of cells, emerging from cell–cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called “van Gogh bundles”) of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

Hypoxic Culture of Human Pluripotent Stem Cell Lines is Permissible Using Mouse Embryonic Fibroblasts

Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate. Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A. Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers. The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.

Aqueous films limit bacterial cell motility and colony expansion on partially saturated rough surfaces

Bacterial motility is a key mechanism for survival in a patchy environment and is important for ecosystem biodiversity maintenance. Quantitative description of bacterial motility in soils is hindered by inherent heterogeneity, pore-space complexity and dynamics of microhydrological conditions. Unsaturated conditions result in fragmented aquatic habitats often too small to support full bacterial immersion thereby forcing strong interactions with mineral and air interfaces that significantly restrict motility. A new hybrid model was developed to study hydration effects on bacterial motility. Simulation results using literature parameter values illustrate sensitivity of colony expansion rates to hydration conditions and are in general agreement with measured values. Under matric potentials greater than -0.5 kPa (wet), bacterial colonies grew fast at colony expansion rates exceeding 421 +/- 94 microm h(-1); rates dropped significantly to 31 +/- 10 microm h(-1) at -2 kPa; as expected, no significant colony expansion was observed at -5 kPa because of the dominance of capillary pinning forces in the submicrometric water film. Quantification of hydration-related constraints on bacterial motion provides insights into optimal conditions for bacterial dispersion and spatial ranges of resource accessibility important for bioremediation and biogeochemical cycles. Results define surprisingly narrow range of hydration conditions where motility confers ecological advantage on natural surfaces.

The Porous Surface Model, a Novel Experimental System for Online Quantitative Observation of Microbial Processes under Unsaturated Conditions

Water is arguably the most important constituent of microbial microhabitats due to its control of physical and physiological processes critical to microbial activity. In natural environments, bacteria often live on unsaturated surfaces, in thin (micrometric) liquid films. Nevertheless, no experimental systems are available that allow real-time observation of bacterial processes in liquid films of controlled thickness. We propose a novel, inexpensive, easily operated experimental platform, termed the porous surface model (PSM) that enables quantitative real-time microscopic observations of bacterial growth and activity under controlled unsaturated conditions. Bacteria are inoculated on a porous ceramic plate, wetted by a liquid medium. The thickness of the liquid film at the surface of the plate is set by imposing suction, corresponding to soil matric potential, to the liquid medium. The utility of the PSM was demonstrated using Pseudomonas putida KT2440 tagged with gfp as a model bacterium. Single cells were inoculated at the surface of the PSM, and the rate at which colonies expanded laterally was measured for three matric potentials (-0.5, -1.2, and -3.6 kPa). The matric potential exerted significant influence on colony expansion rates, with a faster rate of spreading at -0.5 than at -1.2 or -3.6 kPa (diameter increase rate, ca. 1,000, 200, and 17 microm h(-1), respectively). These differences can be attributed to cell motility, strongly limited under the most negative matric potential. The PSM constitutes a tool uniquely adapted to study the influence of liquid film geometry on microbial processes. It should therefore contribute to uncovering mechanisms of microbial adaptation to unsaturated environments.

A re-examination of twitching motility in Pseudomonas aeruginosa.

Twitching motility is a form of solid surface translocation which occurs in a wide range of bacteria and which is dependent on the presence of functional type IV fimbriae or pili. A detailed examination of twitching motility in Pseudomonas aeruginosa under optimal conditions in vitro was carried out. Under these conditions (at the smooth surface formed between semi-solid growth media and plastic or glass surfaces) twitching motility is extremely rapid, leading to an overall radial rate of colony expansion of 0.6 mm h(-1) or greater. The zones of colony expansion due to twitching motility are very thin and are best visualized by staining. These zones exhibit concentric rings in which there is a high density of microcolonies, which may reflect periods of expansion and consolidation/cell division. Video microscopic analysis showed that twitching motility involves the initial formation of large projections or rafts of aggregated cells which move away from the colony edge. Behind the rafts, individual cells move rapidly up and down trails which thin and branch out, ultimately forming a fine lattice-like network of cells. The bacteria in the lattice network then appear to settle and divide to fill out the colonized space. Our observations redefine twitching motility as a rapid, highly organized mechanism of bacterial translocation by which P. aeruginosa can disperse itself over large areas to colonize new territories. It is also now clear, both morphologically and genetically, that twitching motility and social gliding motility, such as occurs in Myxococcus xanthus, are essentially the same process.

Mesoscale and Microscale Observations of Biological Growth in a Silicon Pore Imaging Element

Better physical understanding is needed of the processes affecting biological growth in aquifers, filtration beds, and recharge basins. Toward this end, a two-dimensional random width network pore model etched in a silicon wafer was developed to simulate microbial growth in porous media representative of fine sand. This Silicon Pore Imaging Element (SPIE) was seeded with a mixed culture and fed with 0.34 mM acetate under aerobic conditions and at fixed flow rate. Twelve filamentous colonies grew in a dense manner in the upgradient and lateral directions and at low density in the downgradient direction. Heterogeneous colonization led to empty zones. Particle tracking suggested rerouting of flow due to biomass growth. Microscale time-lapse measured filamentous growth rates (0.5 to 1.6 μm/min) were in good agreement with measured mesoscale colony expansion rates. Rather than the microscale concept of biomass developing at the surface of soil grains, filamentous growth may be better represented as mesoscale colonies spanning over several pores and separated from each other by open flow channels. Biological clogging might be prevented if such flow channels could be kept open in some manner.

Effect of physical conditions on the spatial and temporal dynamics of the soil‐borne fungal pathogen Rhizoctonia solani

The transmission of infection by many soil‐borne fungal parasites of plants depends on the ability of the fungus to grow on or through soil. Progress in analysing the effects of soil physical factors on the temporal and spatial dynamics of fungal growth has been hindered by technical difficulties of quantifying fungal biomass in soil and heterogeneity in soil properties. In this paper we use a combination of a monoclonal antibody‐based immunosorbent assay and microscopy to analyse the effects of soil physical properties on the spatial and temporal dynamics of colonies of the economically important fungus Rhizoctonia solani Kühn growing in two dimensions and three dimensions in a sand. Combinations of different particle‐size distributions and matric potential are used to manipulate the air‐filled pore volume and pore‐size distribution independently of each other. Temporal dynamics are measured by the change in fungal biomass over time whereas spatial dynamics relate to fungal spread and are measured by the colony size, the rate of colony expansion and the biomass distribution within colonies. We show that the fungus spreads more than three times further over surfaces than through sand, even though the same amount of biomass is produced in each case. Pore‐size distribution and air‐filled pore space both affected the extent and rate of fungal spread in three dimensions within sand, with more rapid and extensive spread in a coarse sand compared with a fine sand at identical air‐filled pore volume. The spread of fungal hyphae along surfaces was affected neither by differences in surface texture nor by air‐filled volume, and was substantially more homogeneous than for three‐dimensional spread. We argue that the relative impermeability of sand surfaces to penetration by hyphae might be influenced by the ability of the fungus to branch within a confined space rather than simply to penetrate the pores. The broader epidemiological and ecological consequences of preferential spread by parasitic and saprophytic fungi along surfaces rather than through the dense soil volume are discussed.

Effect of validamycin A and l-sorbose on the growth and morphology of Rhizoctonia cerealis and Rhizoctonia solani

Validamycin A had no effect on the doubling timo of Rhizoctonia cerealis in liquid medium. However, when R. cerealis was grown on solid medium, the antibiotic caused a decrease in the rate of colony expansion and decreases in the compartment length and the radius of leading hyphae. In contrast the antibiotic caused an increase in hyphal density, and this increase was correlated with the observed reduction in compartment length and with the reduction in colony radial growth rate. The interval between inoculation and the onset of inhibition in colony radial growth rate was inversely related to antibiotic concentration, but, at least over the range 0.2 to 1.0 μ M, the final rate of expansion attained by validamycin-inhibited colonies grown on medium containing 5 m M glucose was independent of antibiotic concentration. The inhibitory effect of validamycin A on colony expansion was antagonized by glucose. R. cerealis and R. solani appeared to respond to validamycin A in a similar manner. l-Sorbose caused a decrease in the rate of expansion of colonies of R. cerealis and a decrease in the intercalary compartment length of leading hyphae, but the paramorphogen did not cause a decrease in hyphal radius.

The use of Diethystilbestrol as a Temporary Chemosterilant in Black-Tailed Prarie Dogs (Cynomys ludovicianus)

The following is an abstract of a manuscript submitted to the Journal of Range Management - 25 September 1982. Abstract. The effectiveness of diethylstilbestrol (DES), a synthetic estrogen, as a reproductive inhibitor in black-tailed prairie dogs (Cynomys ludovicianus) was examined in a 4-year study at Wind Cave National Park, South Dakota. In 1979 and 1980, a study colony was monitored to determine age structure, reproductive success of individual animals, and rate of colony expansion. In 1981, the colony was divided into control and experimental areas (Fig. 1). Treatment with DES during the breeding season resulted in complete curtailment of reproduction on the experimental side while reproduction of the control group was similar to that of previous years. Results were identical in 1982 when treatment was reversed (Fig. 2). There were no obvious effects of DES treatment on the subsequent reproductive capability of study animals. In 1981, surface expansion of the study colony was significantly less on the DES-treated side compared with previous years (Fig. 3). Prospects of using DES for managing prairie dog populations are discussed. The authors visualize an integrated approach to prairie dog management; that is, use of rodenticide for initial reduction and irregular DES treatments thereafter to maintain the population at low levels.

The role of regulation of cell speed in the behavior of Physarum polycephalum amoebae

Studies on the behavior of wild-type and mutant Physarum polycephalum amoebae have revealed that regulation of cell speed results in different patterns of cell dispersion in different environments and have shown that P. polycephalum can be used for genetic studies of the mechanisms responsible for this element of cell behavior. Colonies generated by clonal populations of amoebae growing on E. coli display alternate colony morphologies depending on the pH of the culture medium and the presence of live E. coli as a nutrient. In the larger ‘spreading colonies’ cells at the outside of a colony are dispersed over a wide band of bacteria while in the smaller ‘aggregate ring colonies’ most cells moving on bacteria are aggregated in a regularly shaped ring on a narrow band of bacteria at the border of the bacterial lawn created when amoebae completely consume the bacteria available in the colony center. Measurements of cell growth, the rate of colony expansion, and the rate of single cell movement show that cells in contact with bacteria move more slowly in aggregate ring than in spreading colonies. Moreover, since in aggregate ring colonies the rate of movement of cells in contact with bacteria is also reduced relative to that of cells moving on adjacent regions of the agar surface, inhibition of cell speed appears to be at least partially responsible for generating the aggregate ring morphology. Characterization of the behavior of a single locus mutant which generates spreading colonies under conditions where aggregate ring colonies are normally formed has provided additional evidence that a specific mechanism is involved in controlling the distribution of amoebae through regulation of cell speed. Furthermore, the studies of this mutant have shown that aberrant colony morphology can be used as an easily recognized phenotype for identifying and studying mutants with defects which affect the regulation of cell speed.

Effects of continuous competition in two species of cellular slime mold: Dictyostelium discoideum and Polysphondylium pallidum.

Competitively induced population changes were studied in the laboratory. The experimental organisms chosen for the work were two cellular slime mold species: Dictyostelium discoideum and Polysphondylium pallidum. Culturing these organisms on a temperature gradient of 15 °C to 30 °C induces long-term continuous competition for food and space. The organisms respond by changing their rates of resource exploitation and their susceptibility to interference.Before competition, D. discoideum interfered with P. pallidum fruiting, body formation. This effect was not observed after competition. Specifically, when grown alone before competition, P. pallidum fruited from 18° to 37 °C and D. discoideum from 9° to 27 °C. In mixed cultures, before competition. P. pallidum fruited from about 24° to 37 °C and D. discoideum from about 9° to 27 °C. In mixed cultures, after continued competition, P. pallidum fruited from about 20° to 37 °C and D. discoideum from about 9° to 24 °C. There is evidence to suggest that the change is genetic and related to parasexuality in P. pallidum.Rates of resource exploitation (which depend upon spore germination times and amoeba colony expansion rates) also changed. Germination times were unaltered but amoeba colony expansion rates increased.Apparently continued competition resulted in convergence and divergence with respect to resource use. Because P. pallidum gained the ability to fruit in the presence of D. discoideum it converged with respect to resource use. At the same time the two species diverged by increasing rates of resource use over different segments of the temperature range.