Pili-Induced Clustering of N. gonorrhoeae Bacteria

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.     

Inhibitory activity of interleukin 2-activated lymphocytes on human granulocyte precursors (CFU-g)

Interleukin 2-activated lymphocytes (lymphokine-activated killer [LAK] cells) cultured from 2 to 14 days were added to the cultures of granulocyte precursors (CFU-g). The LAK cells inhibited colony formation of granulocyte precursors; LAK cells cultured for five days showed the strongest inhibitory activity on colony formation. The presence of cell-to-cell interaction between LAK cells and bone marrow mononuclear cells (BMNC) in CFU-g assays emphasized the LAK cell-derived colony inhibitory activity (LAK-CIA), but cell-to-cell interaction was not always a requirement for LAK-CIA, since LAK cells were also found to inhibit colony formation without such interaction. This report shows that LAK cells can inhibit in vitro colony formation of granulocyte precursors. We therefore concluded that the observed CIA is caused by soluble factor(s) derived from LAK cells, and that E-rosette-forming cells are manifesting LAK-CIA.     

Mechanism of accessory cell requirement in inducing IL 2 responsiveness by human T4 lymphocytes that generate colonies under PHA stimulation

PHA-driven monoclonal colony formation by low concentrations of resting T4 lymphocytes in agar culture requires the presence of interleukin 2 (IL 2) and accessory cells. Using recombinant IL 2 and anti-Tac monoclonal antibody as a probe for the IL 2 receptor, we demonstrate that the requirement of accessory cells (here an irradiated B cell line) in inducing IL 2 responsiveness relies on their enhancing effect in functional IL 2 receptor expression by the T colony progenitors. Furthermore, it is shown that cell to cell interaction between accessory cells and colony progenitors results in IL 2 response, i.e., colony formation, when the IL 2 receptor density reaches a critical threshold. The asynchronism in IL 2 responsiveness expression by the T colony progenitors upon activation and the short-lived T cell-accessory cell interaction, due to accessory cell death, determine the 10% colony efficiency of the culture system. In addition, we demonstrate that the accessory function in IL 2 receptor and IL 2 responsiveness expression by the T colony progenitors can be supported by irradiated T lymphocytes as well as B cells. The absence of lineage restriction of the signal delivered by accessory cells, and the requirement of physical interaction between T colony progenitors and accessory cells, emphasize the necessity of cross-linking the activation-signal receptors in inducing IL 2 responsiveness by resting T4 cells.     

Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms

In this study, we report the isolation of colony morphology variants from Streptococcus pneumoniae serotype 3 biofilms. The colony variants differed in colony size (large, medium, and small) and their mucoid appearance on blood agar. The small nonmucoid variant (SCV) emerged during the initial attachment stage of S. pneumoniae biofilm formation and dominated over the course of biofilm growth. Mucoid variants appeared at later biofilm developmental stages. The reduction in colony size/mucoidy correlated with a decrease in capsule production and an increase in initial attachment. The large mucoid variant formed flat unstructured biofilms, failed to aggregate in liquid culture, and adhered poorly to solid surfaces. In contrast, SCVs autoaggregated in liquid culture, hyperadhered to solid surfaces, and formed biofilms with significant three-dimensional structure, mainly in the form of microcolonies. The variants showed similar antibiotic resistance/susceptibility based on a modified Kirby-Bauer test and when grown as biofilms. However, antimicrobial treatment of S. pneumoniae biofilms altered the colony variant's distribution and mainly affected the most interior areas of biofilm microcolonies. To further explore the nature of the variants, the capsule biosynthetic operon (cps3DSUM) was explored in greater detail. The genetic analysis indicated that the emergence of nonmucoid variants was due to a deletion comprising cps3DSU as well as additional genes upstream of the cps3 operon. Overall, our findings suggest that in vitro biofilm formation of S. pneumoniae serotype 3 coincides with the emergence of colony variants with distinct genotypic and phenotypic characteristics.     

Bacterial colony as a complex community

Information on the adaptive behavior of cells in the process of the formation of a colony by bacteria Shigella flexneri Rd, used as a model, was obtained with the use of light, transmission and scanning microscopy. The process of the formation of a microcolony was demonstrated; at the initial stages (8 hours) it included 3 groups of cells, and 24 hours later it exhibited sharply defined structurization. The conclusion was made on the possibility of the joint use of different methods of microscopy for the study of the development of microcolonies and colonies of bacteria.     

Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique

Abstract Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. Thus, after 40 days only 0.02–0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non-culturable bacteria in soil.     

Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter

Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.     

Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development

Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas. Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies. In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony. Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract. In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell.     

T-lymphocyte colony formation by lymphocytes from patients with aplastic anaemia

T-lymphocyte colonies were cultured using lymphocytes from patients with aplastic anaemia and normal donors to assess their respective proliferative activities. Colony numbers from aplastic patient's cells were lower than from normal donors', though this was not significant. When lymphocytes from patients were co-cultured with normal lymphocytes, inhibition of T-colony formation was observed in 8 out of 12 experiments. As the degree of inhibition was greater than if patient cells grew no colonies, then, clearly, normal T-colony formation was inhibited. This ability of patients' lymphocytes to suppress lymphopoiesis might account for the low levels of patient T-colony formation, as well as low in vivo numbers of lymphocytes found in patients with aplastic anaemia. The role of patients' lymphocytes in causing marrow aplasia was investigated. Although the incorporation of patients' lymphocytes in normal granulocyte-macrophage (GM) colony-forming systems inhibited colony growth, in only 1 out of 8 patients was this inhibition significantly greater than that caused by the addition of normal lymphocytes to GM colony systems. Therefore, lymphocytes may not be the primary cause of aplastic anaemia, except for a few rare cases.     

The power and efficiency of brood incubation in queenless microcolonies of bumble bees (Bombus terrestris L.)

1. Ground‐nesting colonies of bumble bees incubate their brood at > 30 °C if floral forage provides sufficient energy and the thermogenic power of the colony can counteract cool soil conditions. To explore the basis of incubation, the thermogenic power and sugar consumption of orphaned nests of bumble bee workers (microcolonies) were investigated under laboratory conditions. 2. This study tested experimentally the effect of variation in worker number (ranging from four to 12 adults) on a microcolony's capacity to regulate brood temperature and recover from acute cold exposure. Microcolonies were provided with ad libitum sugar syrup and minimal insulation and maintained at an ambient temperature of c. 25 °C. Energy conversion efficiency was estimated by comparing sugar consumption with the power required for artificial incubation. The joint energetics of foraging and incubation were modelled in wild colonies to explore the effect of colony size and landscape quality on thermoregulation. 3. The results showed that all sizes of microcolonies regulated brood temperature at c. 31 °C under laboratory conditions, which required 96 mW of thermogenic power. It was estimated that individual workers of B. terrestris generated an incubatory power of 35 mW. The smallest microcolonies had the highest conversion efficiency (57%), apparently because few workers were required for incubation. 4. Modelling indicated that small microcolonies of three to seven adult workers have the capacity for normal brood incubation in the wild, but that the minimum viable colony size increases as floral forage becomes poorer or more distant. 5. These preliminary findings suggest the feasibility of identifying the minimum conditions (forage quality, soil temperature, and colony size) necessary for brood incubation by queenright colonies in the wild.     

The type IV bundle-forming pilus of enteropathogenic Escherichia coli undergoes dramatic alterations in structure associated with bacterial adherence, aggregation and dispersal

BFP, a plasmid-encoded type IV bundle-forming pilus produced by enteropathogenic Escherichia coli (EPEC), has recently been shown to be associated with the aggregation of bacteria and dispersal of bacteria from bacterial microcolonies. In standard 3 h HEp-2 cell assays, EPEC adhere in localized microcolonies; after 6 h, bacterial microcolonies are no longer present, indicating that bacterial aggregation and dispersal occurs in vitro during EPEC adhesion to cultured epithelial cells. To examine the role of BFP in EPEC aggregation and dispersal, we examined HEp-2 cell adhesion of strain E2348/69 and defined E2348/69 mutants by immunofluorescence and immunoelectron microscopy. BFP was expressed initially as approximately 40 nm diameter pilus bundles that promoted bacteria-bacteria interaction and microcolony formation. BFP subsequently underwent a striking alteration in structural organization with the formation of much longer and thicker ( approximately 100 nm diameter) pilus bundles, which frequently aggregated laterally to form even thicker bundles often arranged in a loose three-dimensional network; EPEC dispersal from bacterial microcolonies was associated with this transformation of BFP from thin to thick bundles. Bacterial dispersal and transformation of BFP from thin to thick bundles did not occur with a bfpF mutant of strain E2348/69. It is concluded that BFP promotes both the formation and the dispersal of EPEC microcolonies, that the dispersal phase requires BfpF and that dispersal is associated with dramatic alterations in the structure of BFP bundles.     

The influence of T lymphocyte subsets and humoral factors on colony formation by human bone marrow and blood megakaryocyte progenitor cells in vitro

Cellular and humoral influences of T lymphocytes on human megakaryocyte colony formation in vitro were assessed by using a microagar system. Megakaryocyte colony formation from nonadherent low density T lymphocyte-depleted (NALDT-) bone marrow cells was increased significantly after the addition of aplastic anemia serum (AAS) or purified megakaryocyte colony-stimulating factor (Meg-CSF). The addition of conditioned medium obtained from phytohemagglutinin-stimulated T lymphocytes replaced, at least partially, the requirement for AAS or purified Meg-CSF for the growth of megakaryocyte colonies. The cellular influence of T lymphocytes and T lymphocyte subsets on megakaryocyte colony formation was assessed by removing either T cells from nonadherent peripheral blood mononuclear cells with monoclonal OKT4, OKT8, or OKT3 antibodies plus complement, or by adding back populations of bone marrow or blood T4+ or T8+ lymphocytes, isolated by means of fluorescence-activated cell sorting, respectively, to NALDT--bone marrow or -blood cells. When sorted T cell subpopulations were added to a fixed number of NALDT--bone marrow or -peripheral blood cells in the presence of AAS or Meg-CSF, T4+ cells enhanced megakaryocyte colony formation and T8+ cells decreased it. These studies demonstrate that although the stimulation of megakaryocytic progenitor cells by Meg-CSF may not require the presence of monocytes or T lymphocytes, T4+ lymphocytes enhance and T8+ lymphocytes down-regulate megakaryocyte colony formation induced by Meg-CSF. These observations suggest that the immune system is capable of modulating the proliferative response of human megakaryocytic progenitor cells to Meg-CSF.     

Investigating microbial (micro)colony heterogeneity by vibrational spectroscopy

Fourier transform infrared and Raman microspectroscopy are currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as 6 h, followed by the nonsubjective identification of microorganisms through the use of multivariate statistical analyses. To examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12- and 24-h cultures exhibited a significant amount of heterogeneity. Hierarchical cluster analysis of the spectra from the various positions and depths reveals the presence of different layers in the colonies. Further analysis indicates that spectra acquired from the surface of the colonies exhibit higher levels of glycogen than do the deeper layers of the colony. Additionally, the spectra from the deeper layers present with higher RNA levels than the surface layers. Therefore, the 6-h colonies with their limited heterogeneity are more suitable for inclusion in a spectral database to be used for classification purposes. These results also demonstrate that vibrational spectroscopic techniques can be useful tools for studying the nature of colony development and biofilm formation.     

Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.     

Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue

Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.     

Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms

Aims: To investigate the structural organization and dynamics of exopolysaccharides (EPS) matrix and microcolonies formation by Streptococcus mutans during the biofilm development process. Methods and Results: Biofilms of Strep. mutans were formed on saliva‐coated hydroxyapatite (sHA) discs in the presence of glucose or sucrose (alone or mixed with starch). At specific time points, biofilms were subjected to confocal fluorescence imaging and computational analysis. EPS matrix was steadily formed on sHA surface in the presence of sucrose during the first 8 h followed by a threefold biomass increase between 8 and 30 h of biofilm development. The initial formation and further development of three‐dimensional microcolony structure occurred concomitantly with EPS matrix synthesis. Tridimensional renderings showed EPS closely associated with microcolonies throughout the biofilm development process forming four distinct domains (i) between sHA surface and microcolonies, (ii) within, (iii) covering and (iv) filling the spaces between microcolonies. The combination of starch and sucrose resulted in rapid formation of elevated amounts of EPS matrix and faster assembly of microcolonies by Strep. mutans, which altered their structural organization and susceptibility of the biofilm to acid killing (vs sucrose‐grown biofilms; P < 0·05). Conclusions: Our data indicate that EPS modulate the development, sequence of assembly and spatial distribution of microcolonies by Strep. mutans. Significance and Impact of the Study: Simultaneous visualization and analysis of EPS matrix and microcolonies provide a more precise examination of the structural organization of biofilms than labelling bacteria alone, which could be a useful approach to elucidate the exact mechanisms by which Strep. mutans influences oral biofilm formation and possibly identify novel targets for effective antibiofilm therapies.     

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.     

Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3H-thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.     

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.