The Biometric Analysis of Bacterial Cells in Soil

The biometric analysis of bacterial cells in soil by light, fluorescence, and scanning electron microscopy showed that their average size is 0.8 m in diameter, 1.4 m in length, and 0.7 m³ in volume. In soil loci with enhanced microbiological activity (the rhizoplane of plants and the intestinal tract of soil invertebrates), the average size of bacterial cells was found to be 40% smaller than that of cells occurring in other parts of soil. This is the first experimental evidence showing that the metabolic activity of soil bacteria, their concentration, and allometric parameters are related.     

Morphometric analysis of bacteria associated with soil Myriapoda

Scanning electron microscopy revealed that the average cell size of bacteria associated with the digestive tract of soil Myriapoda was 0.65 microm in diameter, 1.36 microm in length, and 0.60 microm3 in volume. An example of Myriapoda illustrated that the intestinal tract bacteria of soil invertebrates shared the following features: (1) a high density level in this habitat; (2) existence mostly in the form of vegetative cells; (3) a cell size significantly smaller than that of bacteria functioning in soil; (4) a cell size closer to the lower limits of the size range characteristic for bacterial cultures grown in laboratory media. All this suggests that the bacterial community of the digestive tract differs from the typical soil community not only in composition, but also in a higher level of physiological activity.     

Morphometric analysis of bacteria associated with soil millipedes

Scanning electron microscopy revealed that the average cell size of bacteria associated with the digestive tract of soil millipedes was 0.65 μm in diameter, 1.36 μm in length, and 0.60 μm³ in volume. An example of millipedes illustrated that the intestinal tract bacteria of soil invertebrates share the following features: (1) a high density level in this habitat; (2) existence mostly in the form of vegetative cells; (3) a cell size significantly smaller than that of bacteria functioning in soil; (4) a cell size closer to the lower limits of the size range characteristic for bacterial cultures grown in laboratory media. All this suggests that the bacterial community of the digestive tract differs from the typical soil community not only in composition but also in a higher level of physiological activity.     

Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method.

A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrdUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, Lobs, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of X-rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions Lobs continues to increase only until operating replicons have completed their replication. This value for Lobs then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before X-irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 micron, whereas for Chinese hamster ovary cells, the average replicon size is about 42 micron.     

Cell proliferation and cortical cell production in relation to wool growth

The relationship of wool growth to cell proliferation in the follicle bulb and to the subsequent migration and growth of the fibre cortical cells was investigated in 10 Peppin Merino sheep. These sheep had been maintained on a low, medium or high level of nutrient intake to ensure a wide range in wool growth. The number and mitotic activity of the germinal cells in the follicle bulb were determined after administration of colchicine. Cortical cell size was measured following isolation of the fibre cells by acid-treatment of wool. The average fibre production of the follicle varied from 4.1 x 10(4) to 13.2 x 10(4) micron3/day in these sheep. There were also substantial differences between sheep in the mitotic activity of the germinal cells in the bulb, the rate of cell proliferation being highly correlated with the average daily fibre production of the follicle (r = + 0.88, n = 10). However, the size of the germinal cell population differed from sheep to sheep and was not closely related to the level of fibre production (r = + 0.48, n = 10). The average turnover time of these cells was inversely related to fibre production and varied from 41.6 to 19.4 h (r = -0.82, n = 10). Multiple regression analysis of the data showed that the average daily fibre production of the follicle was largely determined by the number of germinal cells present in the bulb and their rate of proliferation (R = +0.95, n = 10). Variations in cell turnover time and in cortical cell size were not significant in influencing the rate of fibre production. In these sheep, the average cortical cell varied in size from 658 to 1279 micron 3 and the positive correlation (r = + 0.83, n = 10) found between cell size and fibre production is considered to merely reflect an allometric relationship. The proportion of germinal cells contributing to the fibre cortex was found to be small and variable, ranging from 9.4 to 17.8%. Furthermore, this proportion was not related to the nutritional level of these sheep, and it is thought that the variability in the distribution of cells to the fibre may be attributed to genetic differences between sheep.     

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry

Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm³). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm. Received: 21 January 1999; Accepted: 12 April 1999     

An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction

Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.     

Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon

Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mr values (high Mr TMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mr values (low Mr TMs) have no significant protection even when the actin filaments have been fully saturated with low Mr TMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol. 106, 1973-1983). While nonmuscle caldesmon alone or low Mr TMs alone show no significant protection against fragmentation by gelsolin, the low Mr TMs coupled with 83-kDa protein are able to protect actin filaments. Further, high Mr TMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 +/- microns, and gelsolin shortens the average length to 0.084 +/- 0.039 micron. Similar short average lengths are obtained when gelsolin severs actin complexed with low Mr TMs (0.080 +/- 0.045 micron) or with nonmuscle caldesmon (0.11 +/- 0.072 micron) while longer average length (0.22 +/- 0.18 micron) is measured in the presence of high Mr TMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e. 0.61 +/- 0.37 micron in the presence of low Mr TMs and 1.57 +/- 0.97 micron in the presence of high Mr TMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high Mr TMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.     

Effect of Metal Oxide Nanoparticles on Microbial Community Structure and Function in Two Different Soil Types

Increased availability of nanoparticle-based products will, inevitably, expose the environment to these materials. Engineered nanoparticles (ENPs) may thus find their way into the soil environment via wastewater, dumpsters and other anthropogenic sources; metallic oxide nanoparticles comprise one group of ENPs that could potentially be hazardous for the environment. Because the soil bacterial community is a major service provider for the ecosystem and humankind, it is critical to study the effects of ENP exposure on soil bacteria. These effects were evaluated by measuring bacterial community activity, composition and size following exposure to copper oxide (CuO) and magnetite (Fe₃O₄) nanosized (<50 nm) particles. Two different soil types were examined: a sandy loam (Bet-Dagan) and a sandy clay loam (Yatir), under two ENP concentrations (1%, 0.1%). Results indicate that the bacterial community in Bet-Dagan soil was more susceptible to change due to exposure to these ENPs, relative to Yatir soil. More specifically, CuO had a strong effect on bacterial hydrolytic activity, oxidative potential, community composition and size in Bet-Dagan soil. Few effects were noted in the Yatir soil, although 1% CuO exposure did cause a significant decreased oxidative potential and changes to community composition. Fe₃O₄ changed the hydrolytic activity and bacterial community composition in Bet-Dagan soil but did not affect the Yatir soil bacterial community. Furthermore, in Bet-Dagan soil, abundance of bacteria annotated to OTUs from the Bacilli class decreased after addition of 0.1% CuO but increased with 1% CuO, while in Yatir soil their abundance was reduced with 1% CuO. Other important soil bacterial groups, including Rhizobiales and Sphingobacteriaceae, were negatively affected by CuO addition to soil. These results indicate that both ENPs are potentially harmful to soil environments. Furthermore, it is suggested that the clay fraction and organic matter in different soils interact with the ENPs and reduce their toxicity.     

Resuscitation of Vibrio vulnificus from the viable but nonculturable state.

Stationary-phase-grown cells of the estuarine bacterium Vibrio vulnificus became nonculturable in nutrient-limited artificial seawater microcosms after 27 days at 5 degrees C. When the nonculturable cells were subjected to temperature upshift by being placed at room temperature, the original bacterial numbers were detectable by plate counts after 3 days, with a corresponding increase in the direct viable counts from 3% to over 80% of the total cell count. No increase in the total cell count was observed during resuscitation, indicating that the plate count increases were not due to growth of a few culturable cells. Chloramphenicol and ampicillin totally inhibited resuscitation of the nonculturable cells when added to samples that had been at room temperature for up to 24 h. After 72 h of resuscitation, the inhibitors had an easily detectable but reduced effect on the resuscitated cells, indicating that protein and peptidoglycan synthesis were still ongoing. Major changes in the morphology of the cells were discovered. Nonculturable cells of V. vulnificus were small cocci (approximately 1.0 micron in diameter). Upon resuscitation, the cells became large rods with a size of mid-log-phase cells (3.0 microns in length). Four days after the cells had become fully resuscitated, the cell size had decreased to approximately 1.5 micron in length and 0.7 micron in width. The cells were able to go through at least two cycles of nonculturability and subsequent resuscitation without changes in the total cell count. This is the first report of resuscitation, without the addition of nutrient, of nonculturable cells, and it is suggested that temperature may be the determining factor in the resuscitation from this survival, or adaptation, state of certain species in estuarine environments.     

Spatial Ecology of Bacteria at the Microscale in Soil

Despite an exceptional number of bacterial cells and species in soils, bacterial diversity seems to have little effect on soil processes, such as respiration or nitrification, that can be affected by interactions between bacterial cells. The aim of this study is to understand how bacterial cells are distributed in soil to better understand the scaling between cell-to-cell interactions and what can be measured in a few milligrams, or more, of soil. Based on the analysis of 744 images of observed bacterial distributions in soil thin sections taken at different depths, we found that the inter-cell distance was, on average 12.46 µm and that these inter-cell distances were shorter near the soil surface (10.38 µm) than at depth (>18 µm), due to changes in cell densities. These images were also used to develop a spatial statistical model, based on Log Gaussian Cox Processes, to analyse the 2D distribution of cells and construct realistic 3D bacterial distributions. Our analyses suggest that despite the very high number of cells and species in soil, bacteria only interact with a few other individuals. For example, at bacterial densities commonly found in bulk soil (10⁸ cells g⁻¹ soil), the number of neighbours a single bacterium has within an interaction distance of ca. 20 µm is relatively limited (120 cells on average). Making conservative assumptions about the distribution of species, we show that such neighbourhoods contain less than 100 species. This value did not change appreciably as a function of the overall diversity in soil, suggesting that the diversity of soil bacterial communities may be species-saturated. All in all, this work provides precise data on bacterial distributions, a novel way to model them at the micrometer scale as well as some new insights on the degree of interactions between individual bacterial cells in soils.     

Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages.

We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average decrease in cell capacitance of 8 pF was seen after exposure of the cells to immune complexes. Cells in which particle uptake was inhibited by cytochalasin B treatment before exposure to immune complexes showed an average increase of 0.5 pF. The decrease in membrane capacitance after exposure of cells to particulate stimuli was absent with the soluble stimulus, platelet-activating factor, further confirming that decreases in membrane capacitance were due to particle uptake. Exposure of cells to sized latex particles resulted in a graded, stepwise decrease in membrane capacitance. The average step size for 0.8-micron particles was 250 fF, and the average step change for the larger 3.2-micron particles was 480 fF, as calculated from Gaussian fits to the step size amplitude histograms. The predicted step size for the individual particles based upon the minimum amount of membrane required to enclose a particle and a specific capacitance of 10 fF/micron2 was 20 and 320 fF, respectively. The step size for the smaller particles deviates significantly from the predicted size distribution, indicating either a possible lower limit to the size of the phagocytic vacuole or multiple particles taken up within a single phagosome. Dynamic interaction between phagocytosis and exocytosis was observed in a number of cells as a biphasic response consisting of an initial rapid increase in capacitance, consistent with cellular exocytosis, followed by stepwise decreases in capacitance.     

Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC)

The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor. Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150–180 micron) to a mean diameter of 42.5 micron (size fraction: 25–45 micron) enhanced the bioleaching rate from 0.05 kg m⁻³ h⁻¹ to 0.098 kg m⁻³ h⁻¹. The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells. The values of specific growth rate (μ) and yield factor (Y) were 0.018–0.025 h⁻¹ and 0.67 × 10¹¹–1.45 × 10¹¹ cells (g iron)⁻¹, respectively. Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells. The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite. Received: 11 December 1998 / Accepted: 9 April 1999     

Unique ultrastructure in the elementary body of Chlamydia sp. strain TWAR.

The ultrastructure of two prototype strains (TW-183 and AR-39) of Chlamydia sp. strain TWAR was described. The TWAR elementary body (EB) demonstrated a unique morphology and structure distinct from those of other chlamydial organisms. It was pleomorphic but typically pear shaped. The average size was 0.38 micron, with a long axis of 0.44 micron, a short axis of 0.31 micron, and a ratio of the long to the short axes of 1.42. The cytoplasmic mass was round, with an average diameter of 0.24 micron. There was a large periplasmic space. Small, round electron-dense bodies (0.05 micron in diameter), which were attached to the cytoplasm by a stringlike structure, were seen in the periplasmic space. These features are in contrast to those of other chlamydiae, which are typically round with a narrow or barely discernible periplasmic space. The TWAR reticulate body (RB) was morphologically and structurally similar to those of other Chlamydia species, having an average diameter of 0.51 micron and being circular in shape. The ultrastructural observations of the intracellular growth of TWAR in HeLa cells revealed that TWAR underwent the same developmental cycle as do other chlamydiae, i.e., transformation of EB to RB, multiplication by binary fission, and maturation by transformation of RB to EB via the intermediate-form stage.     

Patterns in Abundance,Cell Size and Pigment Content of Aerobic Anoxygenic Phototrophic Bacteria along Environmental Gradients in Northern Lakes

There is now evidence that aerobic anoxygenic phototrophic (AAP) bacteria are widespread across aquatic systems, yet the factors that determine their abundance and activity are still not well understood, particularly in freshwaters. Here we describe the patterns in AAP abundance, cell size and pigment content across wide environmental gradients in 43 temperate and boreal lakes of Québec. AAP bacterial abundance varied from 1.51 to 5.49 x 10⁵ cells mL^-1, representing <1 to 37% of total bacterial abundance. AAP bacteria were present year-round, including the ice-cover period, but their abundance relative to total bacterial abundance was significantly lower in winter than in summer (2.6% and 7.7%, respectively). AAP bacterial cells were on average two-fold larger than the average bacterial cell size, thus AAP cells made a greater relative contribution to biomass than to abundance. Bacteriochlorophyll a (BChla) concentration varied widely across lakes, and was not related to AAP bacterial abundance, suggesting a large intrinsic variability in the cellular pigment content. Absolute and relative AAP bacterial abundance increased with dissolved organic carbon (DOC), whereas cell-specific BChla content was negatively related to chlorophyll a (Chla). As a result, both the contribution of AAP bacteria to total prokaryotic abundance, and the cell-specific BChla pigment content were positively correlated with the DOC:Chla ratio, both peaking in highly colored, low-chlorophyll lakes. Our results suggest that photoheterotrophy might represent a significant ecological advantage in highly colored, low-chlorophyll lakes, where DOC pool is chemically and structurally more complex.     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

X-ray microanalytic method for measurement of dry matter and elemental content of individual bacteria.

A method for the determination of dry matter and elemental content of individual bacterial cells is described. The method is based on energy-dispersive X-ray microanalysis in a transmission electron microscope. A theory for area correction of intensity is developed. Escherichia coli in the late exponential phase of growth and early stationary phase (glucose limited) had an average dry matter content of 278 and 154 fg/cell, respectively. Of the elements detected, sodium, magnesium, phosphorus, sulphur, chlorine, potassium, and calcium together made up 15 to 17% of the dry matter content. A phosphorus content of 4.2 to 5.4% of the dry matter was found in these cells. Volume measurements of air-dried cells gave an average of 1.20 to 1.25 micron3. These results emphasize that dry matter content and elemental composition can be measured directly on single cells from complex microbial communities.     

X-ray microanalytic method for measurement of dry matter and elemental content of individual bacteria

A method for the determination of dry matter and elemental content of individual bacterial cells is described. The method is based on energy-dispersive X-ray microanalysis in a transmission electron microscope. A theory for area correction of intensity is developed. Escherichia coli in the late exponential phase of growth and early stationary phase (glucose limited) had an average dry matter content of 278 and 154 fg/cell, respectively. Of the elements detected, sodium, magnesium, phosphorus, sulphur, chlorine, potassium, and calcium together made up 15 to 17% of the dry matter content. A phosphorus content of 4.2 to 5.4% of the dry matter was found in these cells. Volume measurements of air-dried cells gave an average of 1.20 to 1.25 micron3. These results emphasize that dry matter content and elemental composition can be measured directly on single cells from complex microbial communities.     

DNA replication fork progression rate and temporal organization of S phase in normal epidermis and in basal cell carcinoma

The double-pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S-phase duration in BCC, compared with normal skin. The mean RU size was 76 +/- 4 micron in BCC, not significantly different from the 68 +/- 6 micron value found in normal skin, so the mean of those two values (i.e., 72 micron), was used in further calculations. The rate of replication fork progression was 0.59 +/- 0.005 micron/min in the normal epidermis and 0.33 +/- 0.03 micron/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8-fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8-fold increase in S-phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined "sets". These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S-phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10.