Uptake of Glucose and Phosphorus by Growing Colonies of Fusarium oxysporum as Quantified by Image Analysis

Olsson, S. 1994. Uptake of glucose and phosphorus by growing colonies of Fusarium oxysporum as quantified by image analysis. Experimental Mycology 18, 33-47. The simplest of all heterogeneous environments for fungal colony growth is the petri dish with an agar medium. As the colony grows there will be a depression of nutrient concentrations under the colony caused by the uptake of nutrients by the growing colony. Image analysis methods have been developed for measuring medium concentrations of glucose and phosphorus with simultaneous biomass density determinations in agar systems. Maps of the concentrations in the agar medium under the colony and of colony biomass density were produced. A new method for weighing fungal colonies grown on agar is also presented. For Fusarium oxysporum phosphorus and glucose uptake from the medium was the same irrespective of the C/mineral ratios in the medium within the measured range of ratios. Even the concentration profiles of the nutrients under the colony were the same irrespective of nutrient ratios. Distribution of biomass density was affected by differences in glucose concentrations, being highest at the colony margin at the lower concentrations. The results indicate that the fungal colony is able to take up nutrients at the margin in excess of the local needs.     

Kinetics and morphology of glucose-limited cultures of moulds grown in a chemostat and on solid media

The affinity (K _s value) of Geotrichum candidum for glucose determined from chemostat cultures was ca. 1 mg/l. K _s values for glucose were also estimated from the radial growth rates of colonies of G. candidum and Neurospora crassa grown on media solidified with agar or silica gel. An assessment is made of the use of colony radial growth rate to determine substrate affinities. The length of apical and intercalary hyphal comparte ments, internode length and the diameter of leading hyphaat the margin of colonies grown on solid media were all reduced at low glucose concentrations.     

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.     

Collective colony growth is optimized by branching pattern formation in Pseudomonas aeruginosa

Branching pattern formation is common in many microbes. Extensive studies have focused on addressing how such patterns emerge from local cell–cell and cell–environment interactions. However, little is known about whether and to what extent these patterns play a physiological role. Here, we consider the colonization of bacteria as an optimization problem to find the colony patterns that maximize colony growth efficiency under different environmental conditions. We demonstrate that Pseudomonas aeruginosa colonies develop branching patterns with characteristics comparable to the prediction of modeling; for example, colonies form thin branches in a nutrient‐poor environment. Hence, the formation of branching patterns represents an optimal strategy for the growth of Pseudomonas aeruginosa colonies. The quantitative relationship between colony patterns and growth conditions enables us to develop a coarse‐grained model to predict diverse colony patterns under more complex conditions, which we validated experimentally. Our results offer new insights into branching pattern formation as a problem‐solving social behavior in microbes and enable fast and accurate predictions of complex spatial patterns in branching colonies.     

Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.     

Colony pattering ofAspergillus oryzae on agar media

To clarity the effects of nutrient concentration and diffusion on the pattern formation of fungal colonies, the colony patterning ofAspergillus oryzae at various nutrient and agar levels was studied experimentally and was summarized in a colony morphology diagram. Roles of the nutrient content and the relaxation of nutrient distribution on the colony patterning were discussed based on a computer model of the mycelial growth. The colony morphology changed from compact to ramified as the nutrient and agar levels were lowered. No clear boundary was found between these two morphologies. The deterioration of substrate around the growing colony was detected when the morphic switching from homogeneous into splitting patterns emerged in the growth of ramified colonies. In the mycelial growth model, dense compact colonies developed at low growth rates and high nutrient influx into the colonized area. Under low nutrient levels, splitting colonies appeared at high growth rates as compared with the nutrient influx.     

Compatibility of Bacillus thuringiensis and granulosis virus treatments of stored grain with four grain fumigants

Wheat was fumigated with phosphine, methyl bromide, CCl₄-carbon bisulfide (80:20 by volume), or ethylene dichloride-CCl₄ (75:25 by volume) after it had been treated with either of two formulations of Bacillus thuringiensis or with a granulosis virus of the Indian meal moth, Plodia interpunctella. Only methyl bromide had an adverse effect: the granulosis virus was inactivated. Spores of B. thuringiensis washed from treated wheat after fumigation with methyl bromide did not produce colonies on nutrient agar plates, but the activity of the B. thuringiensis against Indian meal moths was not affected.     

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.     

Growth of Variants of Myxococcus virescens

Some observations on variant strains of Myxococcus virescens B2 with special emphasis on characteristics associated with the ability to grow in dispersion are reported. The isolated strains were divided into two major classes according to their mode of growth in shaken and static liquid cultures based on casitone and casamino acids media. Strains growing in dispersion were designated D⁺-strains and those growing in aggregates or as films, D^?-strains. Colony morphology, cell morphology, growth in liquid and on solid medium and morphogenesis were compared. The ability to grow in dispersion shown by D⁺-strains seemed to be associated with a smooth colony on casitone agar, inability to form typical fruiting bodies and a low linear growth rate of colonies on solid medium as compared with the D^?-strains. In contrast D^?-strains produced rough colonies on casitone agar, were able to fruit and evidently formed an adhesive slime in the form of fibrils extending from the cell surface. It is suggested that the observed differences depend on different envelopes of the cells in the two classes.     

1α,25-Dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃]at 10⁻⁸-10⁻⁷ M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)₂D₃ increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)₂D₃ and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)₂D₃ is an active metabolite which may be involved in supporting cartilage growth.     

A Modified Consumer Inkjet for Spatiotemporal Control of Gene Expression

This paper presents a low-cost inkjet dosing system capable of continuous, two-dimensional spatiotemporal regulation of gene expression via delivery of diffusible regulators to a custom-mounted gel culture of E. coli. A consumer-grade, inkjet printer was adapted for chemical printing; E. coli cultures were grown on 750 µm thick agar embedded in micro-wells machined into commercial compact discs. Spatio-temporal regulation of the lac operon was demonstrated via the printing of patterns of lactose and glucose directly into the cultures; X-Gal blue patterns were used for visual feedback. We demonstrate how the bistable nature of the lac operon''s feedback, when perturbed by patterning lactose (inducer) and glucose (inhibitor), can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this include sharp boundaries and the generation of traveling waves of mRNA expression. To our knowledge, this is the first demonstration of reaction-diffusion effects in the well-studied lac operon. A finite element reaction-diffusion model of the lac operon is also presented which predicts pattern formation with good fidelity.     

Different Mechanisms of Resistance to Bacillus thuringiensis Toxins in the Indianmeal Moth

Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of ¹²⁵I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688^s) and two resistant (198^r and Dpl^r) to Bacillus thuringiensis. Toxicological studies showed that the 198^r colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl^r colony was 4-fold more resistant to protoxin versus toxin. Binding results with ¹²⁵I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl^r colony showed a drastic reduction in binding affinity (60-fold higher K_d), although they had similar concentrations of binding sites. Insects from the 198^r colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K_d and ca. three-fold-lower R_t compared with the 688^s colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198^r colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473–23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.     

The Alternative Role of Enterobactin as an Oxidative Stress Protector Allows Escherichia coli Colony Development

Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress.     

A novel method for exploring elemental composition of microbial communities: Laser ablation-inductively coupled plasma-mass spectrometry of intact bacterial colonies

Bacterial colonies are spatially complex structures whose physiology is profoundly dependent on interactions between cells and with the underlying semi-solid substratum. Here, we use bacterial colonies as a model of a microbial community to evaluate the potential of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to delineate elemental distributions within colonies with minimal pre-treatment. To reduce water content of the colony and limit undesirable absorption of laser energy, we compared methods of preparing 24 h-old colonies of Escherichia coli TG1 on agar for laser ablation. Colonies on excised agar segments dried on chromatography paper were superior to colonies dried in a dessicator or by prolonged incubation, with respect to signal magnitude, signal:noise ratio and background signal. Having optimised laser scan speed (10 μm s^− 1) and laser beam diameter (100 μm), further improvements were achieved by growing colonies on nylon membranes over agar, which were then transferred to the ablation chamber without further treatment. Repeated line rasters across individual membrane-supported colonies yielded three-dimensional elemental maps of colonies, revealing a convex morphology consistent with visual inspection. By normalising isotope counts for P, Mn, Zn, Fe and Ca against Mg, the most abundant cellular divalent cation, we sought elemental heterogeneity within the colony. The normalised concentration of Mn in the perimeter was higher than in the colony interior, whereas the converse was true for Ca. LA-ICP-MS is a novel and powerful method for probing elemental composition and organisation within microbial communities and should find numerous applications in, for example, biofilm studies.     

Colony growth of Streptomyces coelicolor A3(2) under conditions of continuous nutrient supply

Colony growth of Streptomyces coelicolor A3(2) was studied on a cellophane membrane beneath which was passed a continuous supply of liquid medium. Colony development and differentiation occurred normally but hyphal extension rates and colony radial growth rates were reduced and branch formation was increased in comparison with colonies grown on the same medium solidified with agar. These changes are thought to result from continuous removal of staling compounds which would otherwise suppress branching at the colony margin. Glucose concentrations in the range of 0–1 g · l⁻¹ had little effect on radial growth and branching except at a concentration of 1 g glucose · l⁻¹, at which branching at the colony margin was suppressed. This concentration of glucose did not permit continued growth on solid medium.     

Entropy-driven motility of Sinorhizobium meliloti on a semi-solid surface

Sinorhizobium meliloti growing on soft agar can exhibit an unusual surface spreading behaviour that differs from other bacterial surface motilities. Bacteria in the colony secrete an exopolysaccharide-rich mucoid fluid that expands outward on the surface, carrying within it a suspension of actively dividing cells. The moving slime disperses the cells in complex and dynamic patterns indicative of simultaneous bacterial growth, swimming and aggregation. We find that while flagellar swimming is required to maintain the cells in suspension, the spreading and the associated pattern formation are primarily driven by the secreted exopolysaccharide EPS II, which creates two entropy-increasing effects: an osmotic flow of water from the agar to the mucoid fluid and a crowding or depletion attraction between the cells. Activation of these physical/chemical phenomena may be a useful function for the high molecular weight EPS II, a galactoglucan whose biosynthesis is tightly regulated by the ExpR/SinI/SinR quorum-sensing system: unlike bacterial colonies that spread via bacterium-generated, physical propulsive forces, S. meliloti under quorum conditions may use EPS II to activate purely entropic forces within its environment, so that it can disperse by passively ‘surfing’ on those forces.     

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Lack of Cross-Resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) Resistant to Cry Toxins from Bacillus thuringiensis subsp. israelensis

Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp. jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with B. thuringiensis subsp. jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp. jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.