Bacterial dry matter content and biomass estimations.

Approximately 20% dry-matter content appears to be an accepted standard value for bacterial cells. We have found that the dry-matter content of bacteria may be more than twice as high as generally assumed. The main reason for the low estimates seems to be that proper corrections for intercellular water have not been made when estimating the wet weight of the cells. Using three different bacterial strains, we determined a dry-matter content of cells ranging from 31 to 57%, suggesting not only that the accepted standard value is much too low but also that it is far from standard. To convert bacterial biovolume into biomass (carbon content), we suggest that 0.22 g of C cm-3 should be used as a conversion factor.     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.     

Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy.

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Identification of quantitative trait loci for dry-matter,starch, and β-carotene content in sweetpotato

Development of orange-fleshed sweetpotatoes (OFSP) is desired for the improvement of the food supply and nutritional status of millions of people in developing countries, particularly in sub-Saharan Africa. However, sweetpotato [Ipomoea batatas (L.) Lam] breeding is challenging due to its genetic complexity, and marker-assisted breeding tools are needed to facilitate crop improvement. We identified quantitative trait loci (QTL) for dry-matter, starch, and β-carotene content in a hexaploid sweetpotato mapping population derived from a cross between Tanzania, a white-fleshed, high dry-matter African landrace, and Beauregard, an orange-fleshed, low dry-matter sweetpotato cultivar popular in the USA. Two parental maps were constructed using a population of 240 clones. Strong correlations were observed between starch and dry-matter content (r > 0.8, P < 0.0001) in the storage roots, while moderate correlations (r = –0.6, P < 0.0001) were observed for β-carotene and starch content. In both parental maps, QTL analysis revealed the presence of 13 QTL for storage root dry-matter content, 12 QTL for starch content, and 8 QTL for β-carotene content. Multiple QTL regression models developed for segregation of alleles in each parent explained 15–24% of the variation in dry-matter content, 17–30% of the starch content, and 17–35% of β-carotene content. To the best of our knowledge, this research presents the only QTL mapping study published to date for dry-matter, starch, and β-carotene content in sweetpotato. This work improves our understanding of the inheritance of these important traits in sweetpotato, and represents a first step toward the long-term goal of developing marker-assisted breeding tools to facilitate sweetpotato breeding efforts.     

Physiological comparison of cells with high and low alcohol dehydrogenase activities in bacterial populations consuming ethanol

Measuring the physiological heterogeneity of natural and industrial microbial populations is essential to studying, modelling and monitoring of microbial populations. It was discovered that populations of Escherichia coli and Bacillus megaterium growing in medium with ethanol as an external source of energy have two actively respiring but physiologically different subpopulations. Cells of one subpopulation have negligibly low alcohol dehydrogenase (ADH) activity (ADH-L cells) and cells of the other have high ADH activity (ADH-H cells). The subpopulation of ADH-H bacterial cells was measured using 10 min incubations of cells in a 1% solution of allyl alcohol for fast selective killing of cells with high activity of ADH and flow cytometry detection of dead cells after this incubation. The content of ADH-H cells during exponential phase of batch culture varied from 9 % to 90 % and lowered to zero for a few hours during starvation of the population. ADH-L cells are actively respiring cells and not depolarized cells. The simultaneous presence of ADH-L and ADH-H cells growing in the medium with ethanol can be explained by the fact that ADH-H cells oxidize actively external ethanol whereas ADH-L cells oxidize only intracellular storage carbohydrates. The method for enumeration of cells with high ADH activity can be used to monitor the heterogeneity of bacterial populations consuming ethanol as a sole source of carbon and energy.     

Preferential use of an anode as an electron acceptor by an acidophilic bacterium in the presence of oxygen

Several anaerobic metal-reducing bacteria have been shown to be able to donate electrons directly to an electrode. This property is of great interest for microbial fuel cell development. To date, microbial fuel cell design requires avoiding O(2) diffusion from the cathodic compartment to the sensitive anodic compartment. Here, we show that Acidiphilium sp. strain 3.2 Sup 5 cells that were isolated from an extreme acidic environment are able to colonize graphite felt electrodes. These bacterial electrodes were able to produce high-density electrocatalytic currents, up to 3 A/m(2) at a poised potential of +0.15 V (compared to the value for the reference standard calomel electrode) in the absence of redox mediators, by oxidizing glucose even at saturating air concentrations and very low pHs.     

Protein Quality of the Bacterium Hydrogenomonas eutropha

Hydrogenomonas eutropha cells harvested from semicontinuous autotrophic culture and washed free of substrate contain about 13% of nitrogen on a dry-solids basis. Biological value and digestibility of the bacterial nitrogen were determined in the rat by use of an abbreviated Mitchell-Thomas nitrogen balance technique and casein as the standard protein. Casein nitrogen was 99% digestible, and that of both whole boiled and sonically ruptured bacterial cells was 93%. Biological value of casein and the bacterial preparations was uniformly 77%. Amino acid composition of the bacteria, as in the case of casein, indicates a first limitation of sulfur-containing amino acids. These compositional features suggest that H. eutropha may be potentially valuable as a protein supplement in animal feeds.     

Effects of an Abrupt Change in Ration from All Roughage to High Concentrate upon Rumen Microbial Numbers in Sheep

When three sheep were abruptly changed from a ration of 100% orchardgrass hay to 60% cracked corn-40% orchardgrass hay, fed at equal dry-matter intakes, significant increases in concentration were observed in the rumen microbial population. Bacterial numbers (colony counts) per gram of rumen contents did not appear to have stabilized within 21 days after the ration change; however, protozoan numbers per milliliter plateaued after 5 days. The concentration of cellulose-digesting bacteria varied considerably between animals and decreased in all animals with the change. Changes were observed in total and molar percentages of volatile fatty acids, which were typical for the two types of rations. Although the concentration of protozoa increased after the ration change, only minor differences were observed in their percent generic distribution. A significant decrease in rumen volume was measured in two of the three sheep with the change in ration; however, fluid turnover rates were not significantly affected. Rates of rumen dry-matter turnover were slower with the concentrate ration, although rumen dry-matter digestion was increased. Calculation of total bacterial numbers based on total rumen volume completely negated the effect of ration change in one animal, whereas total numbers in the other two animals were still significantly different between rations and very similar between animals. Adjustment of total protozoa numbers did not alter the trends seen previously with concentration values.     

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

The composition of processed abattoir blood and its use in finishing-pig diets

Abattoir blood with added formalin was heated by steam injection at a processing factory. The range in dry matter content of the processed blood was 7.0–13.4%, and in the crude protein content of the dry matter, 90.7–94.5%. Available lysine varied from 6.9 to 7.8% of the dry matter. Up to 4% blood in the dry matter of a diet was accepted by finishing pigs with no health problems, and when blood was substituted for 2% herring meal on a dry-matter basis, there was no significant difference in pig performance. The effect of varying concentrations of formalin on bacterial count was examined when the blood was stored at either 5°C or 16°C. A concentration of 0.1% formalin was sufficient to prevent an increase in total bacterial count at the lower temperature, but at the higher temperature, 0.3% and 0.65% were necessary to prevent increases for 2–3 and for 7–8 days respectively.     

Culturability of Stream Bacteria Assessed at the Assemblage and Population Levels

Lotic bacterial communities can be examined at multiple levels: from the assemblage level to populations of individual species. In stream environments, as in many other systems, the percentage of bacteria that are culturable is quite low. In this study, the culturability of the overall bacterial assemblage, as well as the culturability of three common species (Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida), was determined in samples collected from four streams on three dates. Colony hybridization (colonies were grown on modified nutrient agar) and fluorescent in situ hybridization were used to calculate the percentage of cells of a given species that were culturable. Approximately half of the overall assemblage was estimated to be viable but nonculturable cells (VBNC). The culturability of two of the species was low (0.29% for A. calcoaceticus and 0.46% for P. putida), whereas the value for B. cepacia (2.48%) exceeded the overall assemblage level culturability (0.90%). Overall, both bacterial assemblages and populations were dominated by VBNC. These results show quantitatively that not all members of a species that has culturable representatives are culturable when retrieved from natural populations, likely because of interspecific phenotypic and genotypic variability. Thus, the large pool of nonculturable cells includes representatives of species that are, under some circumstances, culturable.     

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X. fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.     

Characterisation of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

Cells and protoplasts isolated from three different tissues of sunflower hypocotyls and cultured either in liquid or agarose medium were compared in terms of their volume, DNA content, division potential and embryoid formation. Epidermal and external cortical cells differ from other tissue cells by their small size, their weak response to plasmolysis and their low DNA content (around 1C). They contribute only very weakly to the dividing protoplast population. In contrast, protoplasts from cortical and medullar cells both have similar division potential, reaching 50%. The nuclear DNA content of these two cell types, as well as their corresponding protoplasts, has a 2C value, taking root tip cells in G0 phase as standard. The culture conditions induce the same specific response in protoplasts isolated from both tissues: exclusively loose colony formation in liquid medium, and mainly production of embryoids in agarose medium.     

Molecular vs culture methods for the detection of bacterial faecal indicators in groundwater for human use

AIMS: The current standard culture methods are unable to detect nongrowing bacteria and, thus, might not be sufficient for precise monitoring of the microbiological quality of waters. The use of a molecular method such as PCR could be a valid alternative to detect bacterial faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and reveal the presence of culturable and nonculturable bacterial forms. METHODS AND RESULTS: The presence of E. coli and Ent. faecalis cells in 30 groundwater samples was evaluated with the standard culture method and compared with a specific PCR protocol. A substantial percentage (50%) of the samples not containing culturable cells proved positive in the search for Ent. faecalis DNA by PCR. Quantification by competitive PCR (cPCR) of the DNA detected allowed us to calculate the number of nonculturable cells present in water samples: the number varied from 2 to 120 cells ml(-1). Only four samples were positive for E. coli DNA and the corresponding nonculturable cells varied from 24 to 70 ml(-1). CONCLUSIONS: This study demonstrates that the standard culture methods in use are unable to detect a substantial proportion of the bacterial population which is nonculturable but, as previously demonstrated, potentially still viable and able to express those pathogenic factors needed for causing infections in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health it is necessary to develop and use methods which detect the nonculturable as well as culturable bacteria present in water.     

The relationship between cell size and viability of soil bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (dwarf) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4m, about 10% for cells with diameters between 0.4 and 0.6m, and 30–40% for cells with diameters above 0.6m. Only 10–20% of the viable cells with diameters <0.4m increased their diameter to >0.4m prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.     

Experimental and theoretical kinetics study of antibacterial killing mediated by human natural killer cells

We have recently shown that purified human NK cells, both resting and activated, efficiently kill gram-negative and gram-positive bacteria in vitro. To investigate the mechanism of NK cell-mediated cytotoxicity against Escherichia coli we have developed a mathematical model of the kinetics using the experimental data. The kinetics of killing are characterized by initial target bacterial multiplication, followed by rapid bacterial death. Experiments demonstrates that for each donor there is a threshold number of effector cells necessary to observe a net killing effect. Below the threshold, even use of high effector-to-target ratios lack killing activity and the bacterial growth cannot be stopped. In contrast, if the number of NK cells is larger than the threshold, complete killing is achieved, even at ratios as low as 1/1000. The threshold number varies among donors, ranging between 1200 and 12000 purified NK cells/tube, and provides a quantitative measure of antibacterial activity. Performing the assay at 4 degrees C raised the threshold number required for killing. Experiments performed in Boyden chambers confirm that NK cell-bacteria contact is not necessary for efficient killing, although the kinetics of bacterial lysis is slower. The fit between model and data supports the hypothesis that the bactericidal mechanism is extracellular and is mediated by an anti-microbial factor released from NK cells. Accumulated evidence also indicates that this factor is distinguishable from the mechanisms mediating tumor cell cytotoxicity.     

MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120

MreB is a bacterial actin that plays important roles in determination of cell shape and chromosome partitioning in Escherichia coli and Caulobacter crescentus. In this study, the mreB from the filamentous cyanobacterium Anabaena sp. PCC 7120 was inactivated. Although the mreB null mutant showed a drastic change in cell shape, its growth rate, cell division and the filament length were unaltered. Thus, MreB in Anabaena maintains cell shape but is not required for chromosome partitioning. The wild type and the mutant had eight and 10 copies of chromosomes per cell respectively. We demonstrated that DNA content in two daughter cells after cell division in both strains was not always identical. The ratios of DNA content in two daughter cells had a Gaussian distribution with a standard deviation much larger than a value expected if the DNA content in two daughter cells were identical, suggesting that chromosome partitioning is a random process. The multiple copies of chromosomes in cyanobacteria are likely required for chromosome random partitioning in cell division.     

Clinical significance of telomerase activity in peritoneal disseminated cells: gastrointestinal cancers

Early detection and accurate staging of gastrointestinal (GI) cancers are difficult. The aim of this study was to evaluate whether telomerase activity (TA) in exfoliated/disseminated epithelial cells could be used as a reliable marker for GI cancers. TA was evaluated with the real-time RTQ-TRAP in immunomagnetically sorted peritoneal epithelial cells from 60 patients undergoing surgical treatment. Thirty-two patients were clinically diagnosed with a variety of GI cancers: 1 had premalignant disease, 2 had history of GI cancers, and 25 patients were clinically negative for cancer. Here we report that all types and all cases of gastrointestinal cancers were telomerase positive, thereby demonstrating 100% sensitivity for cancer. Eighteen of 25 nonmalignant cases had undetectable levels of TA, 2 had low, and 5 of 25 expressed high TA levels. Because normal epithelial cells usually have low TA and a lesser tendency to exfoliate compared with cancer cells, it is of great importance to have close follow-up for these patients to exclude possible malignant disease. We conclude that RTQ-TRAP assessment of TA in immunomagnetically sorted peritoneal epithelial cells has 100% sensitivity and 100% negative predictive value for GI cancers, and therefore, can be considered as a valuable tool and useful addition to current standard diagnostic methods. Clinical significance of unusually high telomerase activity in some clinically negative for cancer cases requires further study.