Patterns of gene expression in Bacillus subtilis colonies.

Bacillus subtilis 5:7, a derivative of macrofiber-producing strain FJ7, carries the lacZ reporter gene within Tn917 at an unknown location in the host genome. Expression of the host gene carrying lacZ within colonies of 5:7 was observed by examining growth under different conditions in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). At a high plating density small colonies arose that expressed the host gene early and throughout the colony, whereas at a low density large colonies were produced that expressed the host gene late in development and only in cells forming a ring pattern close to the colony periphery. A highly regulated spatial and temporal gene expression pattern was observed in growth from cross-streaks, suggesting that gene expression is responsive to concentration gradient fields established by neighboring growth. Colonies cultured on agar blocks revealed that expression was governed by depletion of a medium component and also by the geometry of the substrate upon which the colonies grew. At least three factors influenced the control of expression: (i) the concentration of a diffusible component of the medium exhausted by cell growth, (ii) a spatial-temporal factor related to growth within the colony, and (iii) the geometry of the growth substrate.     

Anaerobic incubation enhances the colony formation of a polA recB strain of Escherichia coli K-12.

Escherichia coli strain E247 (polA1 recB21) has reduced colony formation (even at the permissive temperature of 30 degrees C) because of a poor suppressor mutation (sup-126). The colony formation was enhanced in the absence of oxygen about 3-fold at 30 degrees C and 10(6)-fold at 43 degrees C, suggesting that a polA recB strain was inviable due to oxygen toxicity. Colony formation was also increased by incubation in an agar medium containing the reducing agent thioglycolate and incubation in the presence of chloroform-killed Saccharomyces cerevisiae pet+ cells, but not pet cells. Since the E247 strain viability was inversely dependent on the oxygen pressure and since the strain was more sensitive to superoxide radical than either the polA or the recB mutant, it seems likely that the polA and recB genes play a role in repairing DNA damage during respiration.     

Escherichia coli K-12 cell-cell interactions seen by time-lapse video.

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.     

Oligotrophic bacterioplankton with a novel single-cell life strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4',6'-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 micro g of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.     

Oligotrophic Bacterioplankton with a Novel Single-Cell Life Strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4′,6′-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 μg of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.     

Expression of motility in strains of the non-motile species Aeromonas media

Abstract Four isolates of the non-motile species Aeromonas media , icluding the type strain, were demonstrated by transmission electronk microcopy to possess polar flagella. Motility appeared to be restricted to old, regularly subcultured strains, and coincided with an increase in colony size and a concomitant reduction in the amount of brown diffusible pigment produced by colonies on tryptone soya agar.     

Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms.

Factors governing the morphogenesis of Bacillus subtilis colonies as well as the spatial-temporal pattern of expression of a reporter gene during colony development were examined by systematically varying the initial nutrient levels and agar concentrations (wetness), the relative humidity throughout incubation, and the genotype of the inoculum. A relationship between colony form and reporter gene expression pattern was found, indicating that cells respond to local signals during colony development as well as global conditions. The most complex colony forms were produced by motile strains grown under specific conditions such that cells could swim within the colony but not swarm outward uniformly from the colony periphery. The wetness of the growth environment was found to be a critical factor. Complex colonies consisted of structures produced by growth of finger-like projections that expanded outward a finite distance before giving rise to a successive round of fingers that behaved in a similar fashion. Finger tip expansion occurred when groups of cells penetrated the peripheral boundary. Although surfactin production was found to influence similar colony forms in other B. subtilis strains, the strains used here to study reporter gene expression do not produce it. The temporal expression of a reporter gene during morphogenesis of complex colonies by motile strains such as M18 was investigated. Expression arose first in cells located at the tips of fingers that were no longer expanding. The final expression pattern obtained reflects the developmental history of the colony.     

Development of Streptococcal L-Form Colonies

The development and architecture of L-form agar colonies produced from protoplasts and L-phase bodies were studied by both light and scanning electron microscopy. Agar blocks containing L-phase microcolonies of group A Streptococcus strains ADA and GL8 and group D Streptococcus strain F24 as well as longitudinal sections of mature colonies were used as samples. Initially, granules of about 0.5 mum in diameter were produced by multiple condensation and fragmentation of protoplasts and large bodies. Surface growth by granules ensued and infiltration into agar occurred only after 10 to 11 hr of incubation at 37 C. Club-shaped granules were noted and division seemed to take place by simple fission. The configuration of large bodies and granules in mature colonies suggested budding as another means of replication. Acellular spaces inside the colonies appeared to have been formed by lysis of large bodies or by the envelopment of space by the extending growth of minute granules. Whereas no significant strain variation was noted in colonies of less than 24 hr of incubation, fully mature colonies were differentiated on uniform media.     

Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of Structural Properties and Kinetic Constraints on Bacillus cereus Growth

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter⁻¹. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.     

Ultraviolet-Sensitive Mutator mutU4 of Escherichia coli Inviable with polA

Attempts to transduce the ultraviolet-sensitive mutator lesion mutU4 into strains deficient in deoxyribonucleic acid polymerase I (polA) were unsuccessful. Mutator recombinants were found when the polA recipient had first been reverted to Pol(+) by selection for resistance to methyl methanesulfonate. The inviability of the mutU4 polA double mutant was demonstrated by a reduction in the absolute number of transductants when the recipient was polA as compared with Pol(+), and selection was made for markers very close to mutU4. Double mutants containing mutU4 and polA4, which determines a cold-sensitive polymerase, were unable to grow at 24 C, the nonpermissive temperature.     

Chromium(VI) and apparent phenotypic reversion in Salmonella TA100

After treatment with potassium chromate at concentrations causing ultramicroscopic cellular lesions, a significant proportion (up to 75%) of TA100 colonies fail to replicate on fresh minimal plates containing biotin. This suggests that chromium(VI) may not always induce his- reversion to his+ in Salmonella TA100. The terms 'false' or phenotypic reversion have been used to distinguish such instances from 'true' or genotypic reversion, where progeny his+ cells readily grow on biotin replica plates. Results of the present study indicate that the majority of chromate-exposed colonies, initially scored as his-, are identifiable as his+ after 24 h culture on nutrient agar. Moreover, chromate exerts a cytostatic effect on TA100 since early colony development is suppressed at high chromate concentrations. A gradual chemical reduction of chromium(VI) ions by normal media compounds is probably responsible for the re-emergence of colony growth during prolonged incubation of test plates. Thus, temporary growth inhibition at high chromate concentration appears to be responsible for most of the non-replicating colonies detected in mutagenicity assays of chromium(VI).     

Formation of eosinophilic-like granulocytic colonies by mouse bone marrow cells in vitro

Formation of granulocytic and macrophage colonies in agar cultures of mouse marrow or spleen cells was stimulated by the addition of medium from pokeweed mitogen-stimulated cultures of mouse spleen cells (PKW-CM). Approximately 5% of the colonies developing were large, dispersed granulocytic colonies (DG-colonies) composed of cells with eosinophilic cytoplasmic granules. The capacity to stimulate DG-colonies was shown by media conditioned by PKW-treated lymphoid and peritoneal cells but not by other cells or organ fragments. Velocity sedimentation studies indicated that cells generating DG-colonies were separable from cells generating regular granulocytic or macrophage colonies. DG-colonies did not survive if transfered to cultures containing other forms of CSF. The active colony stimulating factor in pokeweed mitogen-conditioned medium which stimulates DG-colony formation was antigenically distinct from the factor stimulating granulocytic and macrophage colony formation, was separable electrophoretically from the latter factor and on gel filtration had an apparent molecular weight of 50,000. Although the cells in DG-colonies have not been established to be eosinophils, DG-colonies represent an interesting new system for analysing further aspects of the control of growth and differentiation in hemopoietic populations.     

The requirement of IHF protein for extrachromosomal replication of the Escherichia coli oriC in a mutant deficient in DNA polymerase I activity

It is shown here that plasmids containing the replication origin of Escherichia coli (oriC) cannot replicate in an extrachromosomal state in E. coli cells with the polA1hip3 double mutation. This E. coli mutant is deficient in the polymerizing function of DNA polymerase I (Pol I) and is unable to produce functional IHF protein. The inability of the oriC minichromosomes to replicate in the absence of IHF is dependent on the absence of Pol I; cells with the polA+himA- or polA+hip- mutation, which are deficient in the alpha and beta subunits of the IHF heterodimer, respectively, can support replication of the oriC replicons. We propose that IHF-deficient cells utilize an alternative pathway of the DNA replication in which Pol I is required. In vitro DNA binding assays revealed that the IHF binding site resides between the oriC coordinates 110 and 122 and is adjacent to the DnaA "box" 1. Within the area protected by IHF we found at least 1 out of 11 GATC methylation sites present in oriC. The consequences of lack of IHF protein binding to the oriC and the indirect effects of the IHF deficiency on the oriC replication are discussed.     

Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.

Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.     

The effect of sodium chloride concentration and pH on the growth of Salmonella typhimurium colonies on solid medium

The growth of Salmonella typhimurium colonies on a model food system (agar solidified culture medium) was followed. Colony radius, determined using computer image analysis (IA) techniques, and viable cell number per colony were measured as indices of colony growth, and the effect of [NaCl] (0.5–3.5% (w/v)) and pH (7.0–5.0) on colony growth at 30°C was observed; colonies were point inoculated from serial dilutions. Colony growth (between 13 and 26 h after inoculation) was linear when expressed in terms of radius, and exponential when expressed in terms of viable cell number per colony. Overall, both increasing the [NaCl] and decreasing the pH had little effect on colony growth, other than to delay the onset of linear radial growth. Initial specific growth rate (μ) ranged from 0.73 to 0.87 h⁻¹. Thin films of agar medium on microscope slides allowed the growth of microcolonies to be observed after just 4 h incubation. A greater understanding of the growth kinetics of bacterial colonies, and the effects of environment on such data, may enable better control of foodborne bacterial pathogens, and consequently an improvement in food product safety.     

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.     

Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.