Colony Formers Research Articles

Better together? Lessons on sociality from Trichodesmium.

The N2-fixing cyanobacterium Trichodesmium is an important player in the oceanic nitrogen and carbon cycles. Trichodesmium occurs both as single trichomes and as colonies containing hundreds of trichomes. In this review, we explore the benefits and disadvantages of colony formation, considering physical, chemical, and biological effects from nanometer to kilometer scale. Showing that all major life challenges are affected by colony formation, we claim that Trichodesmium's ecological success is tightly linked to its colonial lifestyle. Microbial interactions in the microbiome, chemical gradients within the colony, interactions with particles, and elevated mobility in the water column shape a highly dynamic microenvironment. We postulate that these dynamics are key to the resilience of Trichodesmium and other colony formers in our changing environment.

Morphological and taxonomic implications of the formation of colonies in Cyclostephanos and Stephanodiscus (Bacillariophyta)

ABSTRACT The importance of diatoms in bioindication and palaeolimnology is significant. For these applications, and species identification in general, a sound understanding of morphological variability is essential. Some species of the centric diatom genera Cyclostephanos and Stephanodiscus can form long, chain-like colonies. In these genera colony formation is associated with the morphology of the spines, which hold the cells together. One objective of this study was to investigate other possible differences in the valve morphology of solitary and colonial cultures. In addition, all strains were sequenced to account for the possibility that solitary and colonial forms may be different species, using three different loci (LSU, rbcL, cox1). The results show that colony formation not only affects the spine morphology but also other morphological features of the valve. The current interpretation that C. invisitatus and C. delicatus are facultative colony formers is supported by the results of this study. The evidence furthermore suggests that C. incognitus is a morphotype of C. invisitatus. A similar morphological pattern leads to the hypothesis that S. hantzschii and S. binderanus could possibly be solitary and colonial forms of the same species, which is also in agreement with the molecular analysis. These findings have implications for the taxonomy of the group and its application and open up a way for future investigations of morphological changes due to colony formation in other groups and taxa.

Isolation, Screening and Characterization of Exopolysaccharide Producing Bacteria

Exopolysaccharides(EPSs) are essential metabolites synthesized and excreted by certain microorganisms in response to extreme condition of pH, temperature, salinity, osmotic stress and other contaminants for survival in such adverse environment. The present study focuses on isolation of exopolysaccharide producing bacteria from extreme environment of oil polluted soil of Ogoni land and marine water of Bonny Island. Screening of EPS producing abilities of the selected isolates were estimated using two approaches viz gravimetric analysis of EPS dry weight and quantification assay for total carbohydrate content by phenol sulphuric acid method. Result revealed that a total of forty (40) different colonies were suspected to produce exopolysaccharide after preliminary screening by selecting thick ropy-like colony formers on agar medium. Secondary screening indicated that twelve (12) isolates produced precipitates above 1000mg/l of total dry weight and eight (8) isolates produced highest EPS yield above 1000mg/l of culture media with carbohydrate content determination. Five isolates with the code WAS1, WAS11, SC6, SOS7 and SOS10 produced significantly higher EPS compared to other bacterial colonies isolated and were termed as most potent EPS producers. These isolates were identified based on 16S rDNA sequence as Providencia stuarti, Escherichia coli, Shewanella chilikensis and Bacillus nealsonii. The result of the present study indicated that these strains have the potentials of producing high exopolysaccharides and can be explored in biotechnological industries.

CURRENT KNOWLEDGE OF THE LIFE CYCLES OF PHAEOCYSTIS GLOBOSA AND PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE)(1).

Despite continuous efforts since the 1950s and more recent advances in culturing flagellates and nonflagellate cells of the prymnesiophyte Phaeocystis, a number of different life-cycle models exist today that appear to apply for P. globosa Scherff. and P. antarctica G. Karst., both spherical colony formers. In one such model, this life cycle consists of three different flagellates and one nonmotile cell stage that is embedded in carbohydrate matrix-forming colonies of different sizes and forms. Recently, noncolonial aggregates of diploid nonmotile cells attached to surfaces of diatoms were put forward as a new stage in the sexual life cycle of P. antarctica. However, it can be discussed that these "attached aggregates" (AAs) are an intermediate between motile diploid flagellates, with their well-known tendency to adhere to surfaces, and the young spherical colony with its diploid nonmotile cells, which in nature is commonly found attached to diatoms. A life-cycle model pertaining to both P. globosa and P. antarctica is presented.

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 μm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

Protection of bacterial spores in space, a contribution to the discussion on Panspermia.

Spores of Bacillus subtilis were exposed to space in the BIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed with clay, red sandstone, Martian analogue soil or meteorite powder, in dry layers as well as in so-called 'artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases < or = 10(-6)). The same low survival was obtained behind a thin layer of clay acting as an optical filter. The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay, rock or meteorites, and up to 100% survival was reached in soil mixtures with spores comparable to the natural soil to spore ratio. These data confirm the deleterious effects of extraterrestrial solar UV radiation. Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are in direct contact with the spores. The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta of a few cm in diameter could be sufficiently large to protect bacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may not provide sufficient protection for spores to survive. The data are also pertinent to search for life on Mars and planetary protection considerations for future missions to Mars.

Response of primary colon cancer cells in hybrid spheroids to 5-fluorouracil.

We have measured the clonogenic survival of cells isolated directly from colon cancer surgical specimens and treated with 5-fluorouracil (5-FU). Enzymatically disassociated cells were incorporated into hybrid spheroids, consisting predominantly of nonproliferating HeLa feeder cells. Aliquots were exposed for 1.5 hr to a range of concentrations of 5-FU. From the decrease in clonogenic survival, as deduced from the frequency of colony formers among hybrid spheroids after chemical treatment, we were able to construct survival curves in 50% of the surgical specimens tested. A striking revelation was the presence of a resistant plateau in the survival curves, reminiscent of the solid tumor response to treatment with 5-FU. This resistance was absent in monolayer cultures. Evidence is presented that this resistance is due to the absence of, or delay in, cell cycle progression of cells residing in hybrid spheroids.

The Role of Ice Nucleation-active Bacteria on Frost Damage of Tea Plants.

Buds and folded terminal leaves on young shoots of tea plant (Camellia sinensis (L.) Kuntze) carried gemmisphere bacteria at the median population of 2.8×106 cfu/bud or 4.8×107 cfu/g. The bacteria were detected in a log normal distribution and characterized with high population of ice nucleation-activity negative, irregularly-shaped, Gram-positive and orange colored colony formers. The ice nucleation-active (INA) bacteria, Xanthomanas campestris and Erwinia ananas, were the components of these gemmisphere bacteria. X. campestris was predominant all year round, the median population being 5.5×104 cfu/bud in contrast to 3.1×102 cfu/bud in E. ananas, although the populations varied depending on the localities, fields, growth stages of tea plants and/or neighboring plant species. No relationship was confirmed between the distribution frequency and/or population of these INA bacteria and frost damage induced under either natural conditions or artificial ones as well as freeze injuries inflicted on aseptically cultured shoots. Young leaves growing in field suffered freeze injuries at -2 to -3°C irrespective of the degree of leaf expansion, suggesting the involvement of plant intrinsic ice nuclei rather than INA bacteria in the frost damage of tea plant.

A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression

We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.

Effect of oxolinic acid on fecal microflora of goldfish ( Carassius auratus)

The present study was performed to investigate the effect of oxolinic acid (OA) on the intestinal microflora of goldfish ( Carassius auratus). OA was orally administrated to three goldfish at 20 mg/kg body weight for 7 days, and fecal microflora was successively determined. All three fish had Aeromonas hydrophila as a dominant component while pin-hole colony formers and Bacteroides type A were the dominant organisms in two out of three goldfish. Plesiomonas shigelloides and Pseudomonas spp. predominated in one fish. The pin-hole colony formers was suggested to be A. hydrophila which was sensitive to OA. The ability of fecal microflora to resist OA did not significantly change during the experimental period of 10 days. These results strongly suggested that the intestinal microflora of goldfish was not affected markedly by the oral administration of OA at the dosage of 20 mg OA/kg fish for 7 days.

RECOVERY OF VIRAL CAPACITY IN IRRADIATED EXPONENTIALLY GROWING CELLS

Exponentially growing cells of the PtK-2 line (ATCC No. CCL56, from the marsupial Potorous tridactylus) require protein and RNA synthesis in a limited period following UV-radiation damage for optimal recovery as colony formers [Overberg et al. (1988) Mutat. Res. 194, 83-92]. Overall behavior suggests the operation of damage-induced recovery processes. The capacity of confluent cell monolayers for infection with unirradiated herpes simplex virus 1 (HSV-1) is sharply reduced by UV-irradiation. We have followed capacity changes in exponentially growing cells after irradiation and varying amounts of photoreactivation by means of an infectious center assay. These changes closely parallel changes of colony formation. Spontaneous recovery of capacity in the dark occurs over approximately the same time period that the UV sensitivity of colony formation depends on macromolecular synthesis. The effect of photoreactivation is complementary rather than additive to this recovery, suggesting that the dark recovery in this period concerns pyrimidine dimers in cell DNA.

Fucose Binding Lectin for Characterizing Acute Myeloid Leukemia Progenitor Cells

The reactivity of acute myeloid leukemia cells (AML) was determined in 29 patients using the fucose binding lectin Ulex europaeus agglutinin (UEA) as surface marker. We show a marked heterogeneity in the UEA-binding abilities of the cells in these patients as determined by fluorescence analysis of the blasts labeled with the UEA coupled to the fluorescent molecule FITC. The results suggest a correlation between the capability of AML blast cells to bind UEA and cytologic maturation, because in 1 of 10 M1, 3 of 8 M2, 6 of 8 M4, and 1 of 3 M5 cytology types UEA binding to the leukemic cells was apparent. In 13 cases, the cells gave rise to colonies in vitro. The amount of UEA binding to AML colony-forming cells (AML-CFU) was determined by cell sorting and subsequent colony culture of UEA-negative, intermediately positive, and highly fluorescent cells. AML-CFU from none of the four patients with M1 cytology were UEA positive, whereas they showed intense reactivity with the lectin in 1 of 4 cases with M2 cytology and in all 4 cases of M4. In these five cases with strongly UEA positive AML-CFU, the fluorescence distribution of the colony formers differed from that of the total leukemia population, indicating that AML-CFU represent a subpopulation of AML cells with specific UEA-binding properties. Normal bone marrow myeloid and multipotential colony-forming cells (CFU-GM, CFU-GEMM) showed low or no binding of UEA. UEA-FITC appears a useful reagent for membrane analysis of AML-CFU. In certain cases, UEA-FITC labeling may be applied to discriminate AML-CFU from normal hematopoietic progenitors.

Fucose binding lectin for characterizing acute myeloid leukemia progenitor cells

The reactivity of acute myeloid leukemia cells (AML) was determined in 29 patients using the fucose binding lectin Ulex europaeus agglutinin (UEA) as surface marker. We show a marked heterogeneity in the UEA- binding abilities of the cells in these patients as determined by fluorescence analysis of the blasts labeled with the UEA coupled to the fluorescent molecule FITC. The results suggest a correlation between the capability of AML blast cells to bind UEA and cytologic maturation, because in 1 of 10 M1, 3 of 8 M2, 6 of 8 M4, and 1 of 3 M5 cytology types UEA binding to the leukemic cells was apparent. In 13 cases, the cells gave rise to colonies in vitro. The amount of UEA binding to AML colony-forming cells (AML-CFU) was determined by cell sorting and subsequent colony culture of UEA-negative, intermediately positive, and highly fluorescent cells. AML-CFU from none of the four patients with M1 cytology were UEA positive, whereas they showed intense reactivity with the lectin in 1 of 4 cases with M2 cytology and in all 4 cases of M4. In these five cases with strongly UEA positive AML-CFU, the fluorescence distribution of the colony formers differed from that of the total leukemia population, indicating that AML-CFU represent a subpopulation of AML cells with specific UEA-binding properties. Normal bone marrow myeloid and multipotential colony-forming cells (CFU-GM, CFU-GEMM) showed low or no binding of UEA. UEA-FITC appears a useful reagent for membrane analysis of AML-CFU. In certain cases, UEA-FITC labeling may be applied to discriminate AML-CFU from normal hematopoietic progenitors.

Defective T-cell differentiation in acquired immune deficiency syndrome (AIDS).

A decline in T-cell lymphocyte number is the central characteristic of acquired immune deficiency syndrome (AIDS). The reason for the loss of these cells is not well understood. We investigated the hypothesis that defects in T-cell differentiation contributed to T-cell loss using an in vitro colony assay that measures T-cell precursor (CFU-T) frequency. The results indicate a substantial generalized decrease in CFU-T in people with AIDS (P less than 0.01), most of whom have Kaposi's sarcoma, and an occasionally severe decrease in CFU-T in people with ARC. Some of the cells from low colony formers suppressed colony formation by control cells. In addition, plasma from people with AIDS was less supportive of colony growth than control plasma. Decreased Ia expression on adherent mononuclear cells did not correlate with colony formation. A defect in T-cell repopulation can help explain the loss of T cells associated with AIDS.

The intestinal microflora of bullfrog Rana catesbeiana at different stages of its development.

Microflora in the contents of intestinal tracts of Bullfrog at different stages of its development was investigated using 7 different agar media, and major bacterial components were as follows: Tadepoles, Bacteroides A-like, other Bacteroidaceae, helically coiled and filamentous rods, Pseudomonas, A. hydrophila and pin-hole colony formers; metamorphosing tadpoles with hind legs, Bacteroides A-like, other Bacteroidaceae, Clostridium and pin-hole colony formers; frogs, Bacteroidaceae, Clostridium, C. freundii, C. diversus, E. coli, E. hafniae, K. pneumoniae, A. hinshawii and P. shigelloides. The differences of the intestinal microflora may depend on the changes of physiological conditions of host animals, and it is found that frogs is an important experimental animal to study the interaction between the intestinal microflora and host animals.

Antigen-initiated B lymphocyte differentiation. XX. Colony-forming B lymphocytes are not identical with the intermediate, "pre-progenitor" subset of primary or secondary B cells.

The surface immunoglobulin isotype and the cell cycle status of B lymphocyte agar-colony-forming cells (BL-CFC) were studied in order to test a hypothesis, based on their culture behavior, that they represent a mixture of virgin and memory "intermediate" or "pre-progenitor" B cells. If so, BL-CFC would be equivalent to the minor subset of B cells initiating adoptive immune responses. Most BL-CFC were found to be s-IgD+, whereas most progenitors of primary or secondary adoptive responses were s-IgD-. An intense nonspecific stimulus, in the form of horse erythrocytes injected i.p., failed to throw BL-CFC into cell cycle, as judged by hydroxyurea suicide experiments, whereas the progenitors of adoptive responses were thrown into cell cycle. It was concluded that BL-CFC as a whole were not "pre-progenitor" B cells, but more closely resembled the typical "direct progenitor" B cell. Some data derived from unprimed animals, namely, the level of dividing BL-CFC as assessed by hydroxyurea killing and sedimentation velocity distribution, together with the special sensitivity of even nondividing BL-CFC to killing by 3H-TdR, suggested that the agar culture system might select more activated B cells as colony formers.

Three Phases in Transformation of Human Lymphocytes by Epstein‐Barr Virus, as Revealed by Colony Formation in Soft Agar

Human cord lymphocytes were examined for colony formation in soft agar medium at various times after infection with Epstein-Barr virus (EBV). The transformation process could be divided into the following three sequential phases: (1) A pre-early phase, lasting about 10 hr post infection (pi), during which the number of transformed colonies was virus dose dependent. (2) An early phase, between about 10 and 60 hr pi, during which cloning efficiency was markedly lower than that of the pre-early phase. (3) A later phase, starting at about 60 hr pi, during which cloning efficiency was restored and the number of colony formers increased in an exponential fashion. EBV-associated nuclear antigen-positive cells first appeared at the beginning (12 hr pi) of the early phase and increased continuously thereafter. Increased DNA synthesis began at about 48 hr pi. These results suggest that the cloning efficiency of the infected cells changes during the transformation process, possibly reflecting altered cell sensitivity to agar.

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function.

Escherichia coli mutants, called groNB, which block the growth of bacteriophage lambda at the level of action of the gene N product, have been isolated as survivors at 42 degrees C of bacteria carrying a) the defective prophage lambda bio11 i lambda cI857 delta H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage lambda cI857. In addition, groNB bacterial mutants have been isolated at 37 degrees C, as large colony formers in the presence of lambda i lambda cI h434, lambda i lambda cI h lambda, and lambda i lambda cI h80 phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42 degrees C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of lambda growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro+ for lambda growth. Lambda transducing phages carrying the groNB+ bacterial gene have been isolated. GroNB mutant bacteria lysogenized by the transducing phage acquire the Gro+ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.

Synthesis of HbA and HbF in Erythroid Colonies Cultured From Human Fetal Liver and Umbilical Cord Blood

AbstractErythroid colonies were grown in vitro in 0.1-ml plasma clots from 12 to 17 wk human fetal liver tissue obtained from prostaglandin-induced abortuses, and from full-term umbilical cord bloods. Synthesis of α, β, and γ globin chains in the colonies was measured after varying periods of culture and after short-term incubation of the tissue source from which the colonies were derived. In suspensions of fetal liver cells, the ß/γ synthesis ratio averaged 0.12 (range 0.10–0.15). In the erythroid colonies grown from the liver tissue, the average ß/γ ratio after 5–14 days of culture was 0.16 (range 0.09–0.24). In cord blood suspensions, the average ß/γ ratio was 0.60 (range 0.39–0.74), and in the erythroid colonies, after 9–14 days of culture, it was 0.74 (range 0.42–1.17). A tendency towards an increased ratio was noted in the later clot cultures from both sources. The relative synthesis of β and γ globin in erythroid colonies grown in plasma clots from these “fetal” sources reflected the period of human development from which the colony progenitors were obtained. It would therefore seem that the proportion of HbA and HbF synthesized by developing human erythropoietic tissues is already determined by the stage of erythroid maturation of the precursor cells that function as in vitro colony formers.

Variations in granulocyte colony forming cell numbers in adult blood.

Blood granulocyte colony forming units (CFU-C) were studied in normal adults to establish: (i) a normal range; (ii) variability due to the culture technique; (iii) variability of blood CFU-C within individuals. Thirty men studied had 98 (range 8--300) CFU-C X 10(3)/1, and 28 women studied had 44 (range 0--260) CFU-C X 10(3)/1. This difference was significant (P less than 0.001). There was also a significant sex difference in the total number of cells forming colonies and clusters per litre; and in the incidence of colony formers and of cluster formers in buffy coat and mononuclear cell blood fractions. CFU-C were assayed in four subjects over a 10 week period. When buffy coat cells were used as a source of colony stimulation the week to week variation in the combined growth of the four subjects was wide (+/- 36%) but with conditioned medium the variation was smaller (+/- 14%). In all subjects colony and cluster growth varied in the same way (r = 0.77, P = .001) but there was no correlation with the total leucocyte count. A 3--4 week cyclical change in CFU-C/1 was found which was independent of the variation inherent in the technique. The physiological significance of the sex difference and the apparent cyclical changes in blood CFU-C are not explained, but the results emphasize the wide fluctuations in CFU-C that may occur in normal individuals.