Nonlinearity in the Growth of Bacterial Colonies: Conditions and Causes

The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size. The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density. In this work, Pirt's model was tested on two bacteria—Alcaligenes sp. and Pseudomonas fluorescens—having markedly distinct cultural properties and grown on an agarized medium with pyruvate. The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (10⁵–10⁶ cells), and for different depths of the agar layer, determining the amount of available substrate. A linear growth mode was observed only with P. fluorescens and only in the case of growth from a microdroplet. When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of K _r (the rate of colony radius increase) occurring after 2–3 days of growth. The growth of P. fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp. turned out to be directly related to the medium layer thickness. The departure from linearity is consistently explained by a new kinetic scheme stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center. The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by the age of cells. The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.     

Cell Surface Hydrophobicity as a Pellicle Formation Factor in Film Strain of Saccharomyces

The film strain of Saccharomyces grows through non-film and film stages. The differences in cell surface hydrophobicity were examined at both stages. The degree of hydrophobic was quantitatively determined by comparing distribution ratios of cells between buffered aqueous and organic solvent phases. The cell surface in the film stage was more hydrophobic than that in the non-film stage, whereas the inherent non-film strain of Saccharomyces always showed low hydrophobicity. These results indicate that the change from non-film to film stage was due to a change in cells from hydrophilic to hydrophobic. The effects of growth conditions on hydrophobicity were further examined with the film strain Saccharomyces bayanus. Ethanol as sole carbon source more efficiently increased hydrophobicity than glucose. The increase in hydrophobicity seemed to depend upon respiration accompanying assimilation of ethanol. It was also found that the addition of a limited amount of biotin, as well as higher pH in medium lowered hydrophobicity. Variation in degree of pellicle formation was positively related to that of cell surface hydrophobicity.     

Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Variability in Pseudomonas aeruginosa lipopolysaccharide expression during crude oil degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Role of Cell Hydrophobicity on Colony Formation in Microcystis (Cyanobacteria)

Colony formation is highly import ant for the competitive advantage of the cyanobacterium Microcystis over other phytoplankton species. The laboratory‐grown colonial Microcystis strains isolated from Lake Taihu (China) maintained colonial forms under the low light condition (10 μE m^–2 s^–1). The cell surface hydrophobicities of the Microcystis colonies were measured by cyanobacterial adherence to xylene in comparison with unicellular Microcystis strains. The cells of the tested colonial strains were all hydrophobic, while the cells of the tested unicellular strains were all hydrophilic. Incubation under the higher light condition (75 μE m^–2 s^–1) leaded to the significant decrease in the cell hydrophobicities of the colonial Microcystis and the transition from colonial forms to unicellular forms. These findings indicated that the cell hydrophobicity of Microcystis may play a role in cell‐cell adherence and colony formation. Phosphate‐limitation, nitrate‐limitation and pH did not affect cell hydrophobicities of colonial Microcystis. Treatment with proteolytic enzymes had no effect on the cell hydrophobicity, indicating that cell surface proteins did not contribute to high cell hydrophobicity. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

CYANOPHYTE CEPHALODIA IN THE LICHEN GENUS NEPHROMA

The lichens, Nephroma expallidum (Nyl.) Nyl. and N. arcticum (L.) Torss., consistently have at least two symbionts in a single thallus: a green alga in the algal layer and a blue-green alga in the internal cephalodia. The cephalodia originate from algal cells in contact with the lower surface of the lichen, in the zone of rhizine formation. The rhizines surround the epiphytic algal colony and form a second cortical layer; following dissociation of the original lower cortex, further growth of the two organisms results in the cyanophyte colony being enveloped by a compact layer of fungal tissue and positioned in the lichen medulla. The colony may eventually assume a superior or inferior position in relation to the lichen thallus, depending in part on the lichen species. Nephroma anticum may have two distinct morphological forms of blue-green algae in the same thallus and occasionally in the same cephalodium. It appears that the relationship that exists between the cephalodial algae and the lichen thallus is antagonistic and results, in some cases, in the exclusion of the green algal layer and death to the cephalodial cyanophytes.     

Nonlinearity in the growth of bacterial colonies: conditions and causes

The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size. The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density. In this work, Pirt's model was tested on two bacteria--Alcaligenes sp. and Pseudomonas fluorescens--having markedly distinct cultural properties and grown on agarized medium with pyruvate. The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (10(5)-10(6) cells), and for different depths of the agar layer, determining the amount of available substrate. A linear growth mode was observed only with P. fluorescens and only in the case of growth from a microdroplet. When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of Kr (the rate of colony radius increase) occurring after 2-3 days of growth. The growth of P. fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp. turned out to be directly related to the medium layer thickness. The departure from linearity is consistently explained by a new kinetic chart stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center. The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by age of cells. The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.     

Bacterial degradation of polycyclic aromatic hydrocarbons on agar plates: the role of biosurfactants

Summary Colonies of a polycyclic aromatic hydrocarbon (PAH) degrading and biosurfactant producing strain of Pseudomonas marginalis PD-14B, pre-incubated on nutrient agar plates, formed zones of clearing when the agar surface was coated with phenanthrene. Application of a drop of the cell-free biosurfactant solution to the agar surface, followed by coating with phenanthrene film also produced a clear zone against the opaque background of the PAH coating. The results indicate that bacterial colonies generate transparent haloes not only as a result of PAH degradation, as is generally concluded from such tests, but also by solubilization of these hydrophobic compounds, mediated by biosurfactants released by the cells into the agar zone surrounding the colony.     

APPLICATION OF THE 22.5 MEV DEUTERON MICROBEAM TO THE STUDY OF MORPHOGENETIC PROBLEMS WITHIN THE SHOOT APEX OF OSMUNDA CLAYTONIANA

Excised shoot apices of Osmunda claytoniana were grown under controlled sterile conditions. Histological examination of the normal shoot apex shows that it is comprised of: (1) a promeristem, which possesses 1 or more apical initiating cells at its center; (2) a prestelar tissue consisting of an incipient vascular tissue which flanks the pith-mother-cell zone; the pith-mother-cell zone gives rise to the pith rib meristem and subsequently to the fundamental parenchyma of the pith; (3) the fundamental parenchyma of the cortex and the fundamental parenchyma of the dermal system both arising from flank cells of the promeristem. Apical initial cells of meristems irradiated with a 127,000 rad acute exposure of a deuteron beam having a diameter of 25μ, histologically examined at 7-day intervals for a 12-week period, as early as 3 weeks’ postirradiation, showed the apical initiating cell(s) together with certain of the cells of the pith-mother-cell zone to be destroyed. A wound response develops peripherally to the destroyed initials. In addition, an isolated, organized growth center is observed to develop from normal promeristem cells. Incipient vascular tissue and a new pith-mother-cell zone are also observed to develop in association with the new center of growth. Implications of the role of the interrelationships between apical initiating cell(s) and other cells of the meristem and the role they may play in maintenance of meristematic integrity within the shoot meristem are discussed.     

Role of hydrophobicity in adhesion of wild yeast isolated from the ultrafiltration membranes of an apple juice processing plant

The role of cell surface hydrophobicity in the adhesion to stainless steel (SS) of 11 wild yeast strains isolated from the ultrafiltration membranes of an apple juice processing plant was investigated. The isolated yeasts belonged to four species: Candida krusei (5 isolates), Candida tropicalis (2 isolates), Kluyveromyces marxianus (3 isolates) and Rhodotorula mucilaginosa (1 isolate). Surface hydrophobicity was measured by the microbial adhesion to solvents method. Yeast cells and surfaces were incubated in apple juice and temporal measurements of the numbers of adherent cells were made. Ten isolates showed moderate to high hydrophobicity and 1 strain was hydrophilic. The hydrophobicity expressed by the yeast surfaces correlated positively with the rate of adhesion of each strain. These results indicated that cell surface hydrophobicity governs the initial attachment of the studied yeast strains to SS surfaces common to apple juice processing plants.     

High surface hydrophobicity of hemagglutinatingVibrio cholerae and other vibrios

Surface hydrophobicity of hemagglutinatingVibrio cholerae, Vibrio parahaemolyticus, and NAG vibrios has been investigated. Most strains caused mannose-sensitive hemagglutination of monkey, guinea pig, chicken, and mannose-resistant hemagglutination of human erythrocytes with different degrees of hemagglutinating activity. Hemagglutinating strains adsorbed to a hydrophobic gel (Octyl Sepharose), whereas nonhemagglutinating strains failed to adsorb.Vibrio cholerae and other vibrios investigated seem to have pronounced surface hydrophobicity as estimated by Octyl Sepharose and they correspondingly autoaggregated into visible cell clumps in ammonium sulfate solution at low molarity (0.2–0.4 M). Nonhemagglutinating strains did not aggregate even at high (2 M) ammonium sulfate concentration. The presence of surface hemagglutinins of vibrios is growth-media-dependent. Strains, grown in four different liquid media, produced hemagglutinins and expressed pronounced surface hydrophobicity. Studies with electron microscopy revealed the presence of fimbriae on the vibrio cells. The number of fimbriae on the cells varied from strain to strain. Some strains possessed more than 300 fimbriae/cell whereas others had less than 10 fimbriae/cell. Vibrio hemagglutinins are easily detached from the cell surface by heating or sonication, and their cell surface hydrophobicity decreased simultaneously.     

The role of regulation of cell speed in the behavior of Physarum polycephalum amoebae

Studies on the behavior of wild-type and mutant Physarum polycephalum amoebae have revealed that regulation of cell speed results in different patterns of cell dispersion in different environments and have shown that P. polycephalum can be used for genetic studies of the mechanisms responsible for this element of cell behavior. Colonies generated by clonal populations of amoebae growing on E. coli display alternate colony morphologies depending on the pH of the culture medium and the presence of live E. coli as a nutrient. In the larger ‘spreading colonies’ cells at the outside of a colony are dispersed over a wide band of bacteria while in the smaller ‘aggregate ring colonies’ most cells moving on bacteria are aggregated in a regularly shaped ring on a narrow band of bacteria at the border of the bacterial lawn created when amoebae completely consume the bacteria available in the colony center. Measurements of cell growth, the rate of colony expansion, and the rate of single cell movement show that cells in contact with bacteria move more slowly in aggregate ring than in spreading colonies. Moreover, since in aggregate ring colonies the rate of movement of cells in contact with bacteria is also reduced relative to that of cells moving on adjacent regions of the agar surface, inhibition of cell speed appears to be at least partially responsible for generating the aggregate ring morphology. Characterization of the behavior of a single locus mutant which generates spreading colonies under conditions where aggregate ring colonies are normally formed has provided additional evidence that a specific mechanism is involved in controlling the distribution of amoebae through regulation of cell speed. Furthermore, the studies of this mutant have shown that aberrant colony morphology can be used as an easily recognized phenotype for identifying and studying mutants with defects which affect the regulation of cell speed.     

The position of cleavage furrow in cultured <Emphasis Type="Italic">L-929</Emphasis> and <Emphasis Type="Italic">CHO</Emphasis> cells

The position of the cleavage furrow (random or otherwise) was studied on cultured L-929 (NCTC, clone 929) and CHO cells. CHO cells were seeded uniformly on the surface of Petri dishes; L-929 cells were grown as colonies so that migrating cells could be watched. Cell behavior was registered by time-lapse imaging. Two parameters were analyzed on captured images: the angle between the cell polarization axis and cleavage furrow and the angle between the cell polarization axis or cleavage furrow and the horizontal axis of the image field. It was shown that the position of CHO cells in the dish plane and the value of the angle between the cell polarization axis and the cleavage furrow were random. The L-929 cells migrating from the colony were orientated such that their polarization axis was directed to the colony center and the cleavage furrow was perpendicular to this axis. The nonrandom position of cultivated cells during mitosis and their cleavage furrow during the telophase are discussed.     

Effect of milk on surface properties of Staphylococcus aureus from bovine mastitis

Abstract The surface hydrophobicity of cells of Staphylococcus aureus strains isolated from bovine mastitis grown on conventional agar and broth media was drastically reduced after incubation with bovine milk. Strains grown in high carbohydrate-high salt media yielded cells with reduced surface hydrophobicity compared to cells grown in conventional media, and adding bovine milk to minimal medium also yielded cells with reduced surface hydrophobicity, as determined by hydrophobic interaction chromatography and the salt aggregation test. Incubation of strains in milk and growth in a medium supplemented with bovine milk also significantly changed bacterial surface charge as determined by free-zone electrophoresis. Strains with high or with decreased adsorptive and aggregating properties did not produce surface capsule or slime. Heat treatment (60° C or 80° C) of the bacterial suspensions did not significantly change their adsorptive and aggregating properties.     

Two continuum models for the spreading of myxobacteria swarms

We analyze the phenomenon of spreading of a Myxococcus xanthus bacterial colony on plates coated with nutrient. The bacteria spread by gliding on the surface. In the first few hours, cell growth is irrelevant to colony spread. In this case, bacteria spread through peninsular protrusions from the edge of the initial colony. We analyze the diffusion through the narrowing reticulum of cells on the surface mathematically and derive formulae for the spreading rates. On the time scale of tens of hours, effective diffusion of the bacteria, combined with cell division and growth, causes a constant linear increase in the colony's radius. Mathematical analysis and numerical solution of reaction-diffusion equations describing the bacterial and nutrient dynamics demonstrate that, in this regime, the spreading rate is proportional to the square root of both the effective diffusion coefficient and the nutrient concentration. The model predictions agree with the data on spreading rate dependence on the type of gliding motility.     

Potentiation of bone marrow colony growth in vitro by the addition of lymphoid or bone marrow cells

Colony formation and growth in vitro by C57B1 mouse bone marrow cells were analysed following stimulation by a standard dose of serum colony stimulating factor. Under restricted conditions, colony crowding was observed to potentiate colony growth rates. The addition of thymic or lymph node lymphoid cells or nonviable bone marrow cells also potentiated colony growth. Extensive reutilisation of nuclear material by bone marrow colony cells was observed when labeled lymphoid and bone marrow cells were added to the culture system. The results provide evidence that lymphocytes can exert trephocytic effects on proliferating hematopoietic cells.     

Enzyme release from heat-stressed cell membranes as a function of hydrophobicity evaluated by using aqueous two-phase systems

The release behavior of a periplasmic enzyme, acid phosphatase, from heat-stressed Escherichia coli cells was characterized by using kinetic analyses when the cells were treated by Triton X-100–EDTA. The hydrophobicity of the cell surface and the release-rate of the enzyme were not influenced by heat treatment at temperatures between 30 and 50°C. However, these values varied above 55°C. The release-rate constants were found to correspond to the net and local hydrophobicity of the outer membrane surface, evaluated by aqueous two-phase partitioning.     

Role of hydrophobicity in bacterial adherence to carbon nanostructures and biofilm formation

The role of cell and surface hydrophobicity in the adherence of the waterborne bacterium Mycobacterium smegmatis to nanostructures and biofilm formation was investigated. Carbon nanostructures (CNs) were synthesized using a flame reactor and deposited on stainless steel grids and foils, and on silicon wafers that had different initial surface hydrophobicities. Surface hydrophobicity was measured as the contact angle of water droplets. The surfaces were incubated in suspensions of isogenic hydrophobic and hydrophilic strains of M. smegmatis and temporal measurements of the numbers of adherent cells were made. The hydrophobic, rough mutant of M. smegmatis adhered more readily and formed denser biofilms on all surfaces compared to its hydrophilic, smooth parent. Biofilm formation led to alterations in the hydrophobicity of the substratum surfaces, demonstrating that bacterial cells attached to CNs are capable of modifying the surface characteristics.