Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

Synchronous plasma membrane electrochemical potential oscillations during yeast colony development and aging

Microorganisms that survive in natural environments form organized multicellular communities, biofilms and colonies with specific properties. During stress and nutrient limitation, slow growing and senescent cells in such communities retain vital processes by maintaining plasma membrane integrity and retaining the ability to generate transmembrane electrochemical gradients. We report the use of a Saccharomyces cerevisiae colonial model to show that population growth in a multicellular community depends on nutrient diffusion and that resting cells start to accumulate from the beginning of the second acidic phase of colony development. Despite differentiation of colony members, synchronous transmembrane potential oscillation was detected in the organized colony. The electrochemical membrane potential periodically oscillated at frequencies between those for circadian to infradian rhythms during colony aging and transiently decreased at time points previously linked with rebuilding of yeast metabolism. Despite extensive decreases in the intracellular ATP concentration and in the amount and activity of the plasma membrane proton pump during nutrient limited growth and colony aging, the transmembrane electrochemical potential appeared to be maintained above a level critical for population survival.     

Growth rates of yeast colonies on solid media

Previous measurements of growth rates of giant yeast colonies on solid media are shown to be unreliable as they depend strongly on extraneous factors such as the proximity of other colonies and the dimensions of the apparatus used. The hitherto unexplained dependence of the growth rate on the square root of the growth limiting nutrient concentration is explained by constructing a theory based on the diffusion of nutrient towards the colony which makes use of many ideas used in the theory of flame propagation. The theory also explains why the temperature dependence of the homogeneous growth constant is different from that observed in the surface colony, and it requires the existence of a lag phase in the homogeneous culture kinetics if the velocity of propagation of the culture is to be independent of inoculum size and shape. Both phenomena are known to occur.     

Aging and longevity of yeast colony populations: metabolic adaptation and differentiation

Yeast multicellular colonies possess several traits that are absent from individual yeasts. These include the ability to synchronize colony population development and adapt its metabolism to different environmental changes, such as nutrient depletion. This, together with cell diversification to cell variants with distinct metabolic and other properties, contributes to the main goal of the colony population: to achieve longevity. In this respect, a benefit to individual cells is subordinated to the benefit to the whole population, exhibiting a kind of altruistic behaviour. For example, some colony cells located at particular positions undergo regulated cell dying and provide components to other cells located in more propitious areas. The enhancement of techniques that enable the in vivo investigation of three-dimensional spatiotemporal colony development may lead to new discoveries on metabolic differentiation and regulation in the near future.     

WL营养琼脂对葡萄酒相关酵母的鉴定效果验证

利用WL营养琼脂对采自葡萄园和葡萄汁发酵过程中的35株酵母菌进行了分类鉴定,同时进行了5.8S-ITS和26S rDNA D1/D2区的扩增与测序。结果表明利用WL营养琼脂的鉴定结果与测序结果基本符合。WL营养琼脂是一种较为有效的葡萄酒相关酵母菌的分类鉴定培养基。     

Role of distinct dimorphic transitions in territory colonizing and formation of yeast colony architecture

Microbial populations in nature often form organized multicellular structures (biofilms, colonies) occupying different surfaces including host tissues and medical devices. How yeast cells within such populations cooperate and how their dimorphic switch to filamentous growth is regulated are therefore important questions. Studying population development, we discovered that Saccharomyces cerevisiae microcolonies early after their origination from one cell successfully occupy the territory via dimorphic transition, which is induced by ammonia and other volatile amines independently on cell ploidy and nutrients. It results in oriented pseudohyphal cell expansion in the direction of ammonia source, which consequently leads to unification of adjacent microcolonies to one more numerous entity. The further population development is accompanied by another dimorphic switch, which is strictly dependent on Flo11p adhesin and is indispensable for proper formation of biofilm-like aerial 3-D colony architecture. In this, Flo11p is required for both elongation of cells organized to radial clusters (formed earlier within the colony) and their subsequent pseudohyphal expansion. Just before this expansion, Flo11p relocalizes from the bud-neck of radial cell clusters also to the tip of elongated cells.     

Flo11p, drug efflux pumps, and the extracellular matrix cooperate to form biofilm yeast colonies

Much like other microorganisms, wild yeasts preferentially form surface-associated communities, such as biofilms and colonies, that are well protected against hostile environments and, when growing as pathogens, against the host immune system. However, the molecular mechanisms underlying the spatiotemporal development and environmental resistance of biofilms and colonies remain largely unknown. In this paper, we show that a biofilm yeast colony is a finely tuned, complex multicellular organism in which specialized cells jointly execute multiple protection strategies. These include a Pdr1p-regulated mechanism whereby multidrug resistance transporters Pdr5p and Snq2p expel external compounds solely within the surface cell layers as well as developmentally regulated production by internal cells of a selectively permeable extracellular matrix. The two mechanisms act in concert during colony development, allowing growth of new cell generations in a well-protected internal cavity of the colony. Colony architecture is strengthened by intercellular fiber connections.     

Continuous cultivation of fission yeast: Analysis of single-cell protein synthesis kinetics

A fundamental problem in microbial reactor analysis is identification of the relationship between environment and individual cell metabolic activity. Population balance equations provide a link between experimental measurements of composition frequency functions in microbial populations on the one hand and macromolecular synthesis kinetics and cell division control parameters for single cells on the other. Flow microfluorometry measurements of frequency functions for single-cell protein content in Schizosaccharomyces pombe in balanced exponential growth have been analyzed by two different methods. One approach utilizes the integrated form of the population balance equation known as the Collins-Richmond equation, and the other method involves optimization of parameters in assumed kinetic and cell division functional forms in order to best fit measured frequency functions with corresponding model solutions. Both data interpretation techniques indicate that rates of protein synthesis increase most in small protein content cells as the population specific growth rate increases, leading to parabolic single-cell protein synthesis kinetics at large specific growth rates. Utilization of frequency function data for an asynchronous population is shown in this case to be a far more sensitive method for determination of single-cell kinetics than is monitoring the metabolic dynamics of a single cell or, equivalently, synchronous culture analyses.     

Cell differentiation within a yeast colony: metabolic and regulatory parallels with a tumor-affected organism

Nutrient sensing and metabolic reprogramming are crucial for metazoan cell aging and tumor growth. Here, we identify metabolic and regulatory parallels between a layered, multicellular yeast colony and a tumor-affected organism. During development, a yeast colony stratifies into U and L cells occupying the upper and lower colony regions, respectively. U cells activate a unique metabolism controlled by the glutamine-induced TOR pathway, amino acid-sensing systems (SPS and Gcn4p) and signaling from mitochondria with lowered respiration. These systems jointly modulate U cell physiology, which adapts to nutrient limitations and utilize the nutrients released from L cells. Stress-resistant U cells share metabolic pathways and other similar characteristics with tumor cells, including the ability to proliferate. L cells behave similarly to stressed and starving cells, which activate degradative mechanisms to provide nutrients to U cells. Our data suggest a nutrient flow between both cell types, resembling the Cori cycle and glutamine-NH(4)(+) shuttle between tumor and healthy metazoan cells.     

Cell division in the volume of Flavobacterium sp.22 colonies

Six-day-old colonies of Flavobacterium sp. 22 were studied by electron microscopy. Direct evidence was obtained of bacterial cell division across the entire colony volume, indicating that the colony growth of Flavobacterium sp. 22 is not purely peripheral. It is argued that the colony shape is determined not only by peripheral growth but also by physical forces acting upon a droplet of liquid on the surface. For bacterial colonies developing on solid nutrient media, the intercellular matrix plays the role of such a liquid.     

Colony pattering ofAspergillus oryzae on agar media

To clarity the effects of nutrient concentration and diffusion on the pattern formation of fungal colonies, the colony patterning ofAspergillus oryzae at various nutrient and agar levels was studied experimentally and was summarized in a colony morphology diagram. Roles of the nutrient content and the relaxation of nutrient distribution on the colony patterning were discussed based on a computer model of the mycelial growth. The colony morphology changed from compact to ramified as the nutrient and agar levels were lowered. No clear boundary was found between these two morphologies. The deterioration of substrate around the growing colony was detected when the morphic switching from homogeneous into splitting patterns emerged in the growth of ramified colonies. In the mycelial growth model, dense compact colonies developed at low growth rates and high nutrient influx into the colonized area. Under low nutrient levels, splitting colonies appeared at high growth rates as compared with the nutrient influx.     

ARRESTED GROWTH OF CHINESE HAMSTER OVARY (CHO) CELL COLONIES ON AGAR

Chinese hamster ovary (CHO) cells plated on agar form two classes of colonies; those which increase continuously in diameter and those which become arrested in outward growth. All colonies continue to increase in mass and thickness as demonstrated by computer-assisted analysis of time sequence photographs of several thousand colonies and by examination of histological sections. Colonies which shift from a monolayer to a mounded morphology at fairly large colony diameter (?1 mm) continue to increase in diameter. Colonies in which mounding occurs at smaller colony diameter (?1 mm) cease to increase in diameter but continue to increase in thickness, as demonstrated by histological examination and by computer-assisted analysis. Rapid cell division occurs at the edge of all colony classes, as shown by the distribution of mitotic figures. In arrested colonies these dividing cells must move toward the colony core to compensate for dying cells. Necrotic cells are found as a discrete zone at the air-colony interface in all cases for CHO cell colonies growing on agar. As such, necrosis is probably due to limitations in nutrient diffusion upward from the agar rather than oxygen diffusion downward.     

Self-similar colony morphogenesis by gram-negative rods as the experimental model of fractal growth by a cell population.

The ability to form a fractal colony was shown to be common among several species of the family Enterobacteriaceae. Bacterial spreading growth in a two-dimensional field of nutrient concentration was indicated to be important for this experimental self-similar morphogenesis. As a basic analogy, the diffusion-limited aggregation model was suggested. Fractal dimensions of colonies were mostly in the range of values from 1.7 to 1.8, similar to those of the two-dimensional diffusion-limited aggregation model. Bacterial characteristics and culture conditions inducing changes in fractal patterns and growth rates were identified. The contribution of the bacterial multicellular nature to fractal morphogenesis is discussed.     

Self-similar colony morphogenesis by gram-negative rods as the experimental model of fractal growth by a cell population.

The ability to form a fractal colony was shown to be common among several species of the family Enterobacteriaceae. Bacterial spreading growth in a two-dimensional field of nutrient concentration was indicated to be important for this experimental self-similar morphogenesis. As a basic analogy, the diffusion-limited aggregation model was suggested. Fractal dimensions of colonies were mostly in the range of values from 1.7 to 1.8, similar to those of the two-dimensional diffusion-limited aggregation model. Bacterial characteristics and culture conditions inducing changes in fractal patterns and growth rates were identified. The contribution of the bacterial multicellular nature to fractal morphogenesis is discussed.     

Cell division across the volume of colonies of flavobacterium sp. 22

Six-day-old colonies ofFlavobacterium sp. 22 were studied by electron microscopy. Direct evidence was obtained of bacterial cell division across the entire colony volume, indicating that the colony growth ofFlavobacterium sp. 22 is not purely peripheral. It is argued that the colony shape is determined not only by peripheral growth but also by physical forces acting upon a droplet of liquid on the surface. For bacterial colonies developing on solid nutrient media, the intercellular matrix plays the role of such a liquid.     

Unequal cell division, growth regulation and colony size of mammalian cells: a mathematical model and analysis of experimental data.

This work describes mathematically the dynamics of expansion of cell populations from the initial division of single cells to colonies of several hundred cells. This stage of population growth is strongly influenced by stochastic (random) elements including, among others, cell death and quiescence. This results in a wide distribution of colony sizes. Experimental observations of the NIH3T3 cell line as well as for the NIH3T3 cell line transformed with the ras oncogene were obtained for this study. They include the number of cells in 4-day-old colonies initiated from single cells and measurements of sizes of sister cells after division, recorded in the 4-day-old colonies. The sister cell sizes were recorded in a way which enabled investigation of their interdependence. We developed a mathematical model which includes cell growth and unequal cell division, with three possible outcomes of each cell division: continued cell growth and division, quiescence, and cell death. The model is successful in reproducing experimental observations. It provides good fits to colony size distributions for both NIH3T3 mouse fibroblast cells and the same cells transformed with the rasEJ human cancer gene. The difference in colony size distributions could be fitted by assuming similar cell lifetimes (12-13 hr) and similar probabilities of cell death (q = 0.15), but using different probabilities of quiescence, r = 0 for the ras oncogene transformed cells and r = 0.1 for the non-transformed cells. The model also reproduces the evolution of distributions of sizes of cells in colonies, from a single founder cell of any specified size to the stable limit distribution after eight to ten cell divisions. Application of the model explains in what way both random events and deterministic control mechanisms strongly influence cell proliferation at early stages in the expansion of colonies.