Dynamics of cyanophage-like particles and algicidal bacteria causing Microcystis aeruginosa mortality

The dynamics of cyanophage-like particles and algicidal bacteria that infect the bloom-forming cyanobacterium Microcystis aeruginosa was followed in a hyper-eutrophic pond from September 1998 to August 1999. The densities of M. aeruginosa ranged between 4.0 × 10⁵ and 1.9 × 10⁷ cells ml⁻¹, whereas those of algicidal bacteria were between 4.0 and 5.1 × 10² plaque-forming units (PFU) ml⁻¹ and those of cyanophage-like particles were between <5.0 × 10² and 7.1 × 10³ PFU ml⁻¹. A significant relationship was found between the densities of algicidal bacteria and M. aeruginosa (r = 0.81, n = 69, P < 0.001), suggesting that the dynamics of the algicidal bacteria may regulate the abundance of M. aeruginosa. Occasional peaks of density of cyanophage-like particles were detected in October, June, and August, when sharp declines in M. aeruginosa cell densities were also observed. The densities of cyanophage-like particles became undetectable when the abundance of M. aeruginosa was low, suggesting the density-dependent infection of M. aeruginosa by cyanophage-like particles. Thus, we suggest that infections of both algicidal bacteria and cyanophage-like particles are important biological agents that decompose blooms of M. aeruginosa in freshwater environments. Received: August 31, 2000 / Accepted: December 6, 2000     

Rapid Bead-Based Antimicrobial Susceptibility Testing by Optical Diffusometry

This study combined optical diffusometry and bead-based immunoassays to develop a novel technique for quantifying the growth of specific microorganisms and achieving rapid AST. Diffusivity rises when live bacteria attach to particles, resulting in additional energy from motile microorganisms. However, when UV-sterilized (dead) bacteria attach to particles, diffusivity declines. The experimental data are consistent with the theoretical model predicted according to the equivalent volume diameter. Using this diffusometric platform, the susceptibility of Pseudomonas aeruginosa to the antibiotic gentamicin was tested. The result suggests that the proliferation of bacteria is effectively controlled by gentamicin. This study demonstrated a sensitive (one bacterium on single particles) and time-saving (within 2 h) platform with a small sample volume (~0.5 μL) and a low initial bacteria count (50 CFU per droplet ~ 10⁵ CFU/mL) for quantifying the growth of microorganisms depending on Brownian motion. The technique can be applied further to other bacterial strains and increase the success of treatments against infectious diseases in the near future.     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."     

Grazing protozoa and magnetosome dissolution in magnetotactic bacteria

Magnetotactic bacteria show an ability to navigate along magnetic field lines because of magnetic particles called magnetosomes. All magnetotactic bacteria are unicellular except for the multicellular prokaryote (recently named 'Candidatus Magnetoglobus multicellularis'), which is formed by an orderly assemblage of 17-40 prokaryotic cells that swim as a unit. A ciliate was used in grazing experiments with the M. multicellularis to study the fate of the magnetosomes after ingestion by the protozoa. Ciliates ingested M. multicellularis, which were located in acid vacuoles as demonstrated by confocal laser scanning microscopy. Transmission electron microscopy and X-ray microanalysis of thin-sectioned ciliates showed the presence of M. multicellularis and magnetosomes inside vacuoles in different degrees of degradation. The magnetosomes are dissolved within the acidic vacuoles of the ciliate. Depending on the rate of M. multicellularis consumption by the ciliates the iron from the magnetosomes may be recycled to the environment in a more soluble form.     

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. aviumintracellulare). Other possible applications of this method are discussed.     

Production of Metallogenium-like particles by heterotrophic manganese-oxidizing bacteria collected from a lake

As a model of chemically stratified structure of environment typical to the chemocline of lakes, a semisolid gradient medium was prepared to cultivate heterotrophic manganese-oxidizing bacteria originally collected from a lake. The bacteria growing under the conditions described produced extracellularly Metallogenium-like particles similar to those which are often detected in the chemocline of lakes. This suggested that the naturally occurring Metallogenium-like particles originated in activities of such heterotrophic manganese-oxidizing bacteria. The manganese oxidation activity usually appeared only at the stationary phase of bacterial growth. The oxidation of Mn²⁺ and the formation of Metallogenium-like particles by the bacteria were observed at neutral or slightly acidic pH. not under alkaline conditions, which is a conspicuous difference from the inorganic oxidation of Mn²⁺ by O₂. Bacterial manganese oxidation was stimulated by bicarbonate (50 or 500 M). An experiment of addition of H₂O₂ to the incubation tubes isolated from atmosphere failed to confirm the availability of externally added H₂O₂ as the electron acceptor, suggesting that the bacterial manganese oxidation required the presence of O₂.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles. Part II. Cleavage and Uptake of Carbohydrates

Abstract The bacterial colonization and development of the ectoenzymatic glucosidase activity and glucose uptake were followed together with bacterial growth (measured as thymidine incorporation) in laboratory experiments, using phytoplankton-derived particles incubated in rolling tanks. Bacterial colonization of the particles was rapid. In the particles, bacterial turnover rates (production/biomass) were low (0.02 to 0.14 d⁻¹). In the ambient water, turnover rates increased from 0.1 d⁻¹ to 23.3 d⁻¹, until the end of the experiment. In the control, lacking any particles, turnover of bacteria ranged from 0.3 to 7.6 d⁻¹. Similarly, glucose uptake rates, per bacterium, were 1 to 2 orders of magnitude lower for particle-attached bacteria than for their free-living counterparts. Generally, K_m values for glucosidase activity declined, over the incubation period, in particles and free-living bacteria until 168 h, and slightly increased, thereafter, to values of approximately 0.1 μM. Particle-attached bacteria exhibited significantly lower uptake rates of both thymidine and glucose, per bacterium, throughout the incubation. The per-cell ectoenzymatic activity was similar in particle-associated and free-living bacteria during the initial phase of the experiment, but was significantly higher after ≈200 h. Dissolved total (TCHO), as well as monomeric carbohydrates (MCHO), declined continuously in both particles and ambient water; they remained constant in the control; TCHO comprised about 50% of the dissolved organic carbon (DOC) in the particles. In ambient water TCHO contribution to DOC varied, with only one exception, between 25 and 45%; and in the control, between 20 and 50%. The shift detectable in the relation between ectoenzymatic activity and uptake of glucose between free-living and attached bacteria over the incubation period may reflect changes in the physiological status of the bacteria. Received: 3 February 1997; Accepted: 6 November 1997     

Trimethylamine and methylamine as growth substrates for rumen bacteria andMethanosarcina barkeri

Trimethylamine and methylamine were found to be used as methanogenic substrates byMethanosarcina barkeri or by bacteria found in low dilutions of rumen contents. When these substrates were used as the only added carbon and nitrogen source, up to 80% of the theoretical amount of methane production was obtained.Methanosarcina were enumerated from rumen contents at 10⁵–10⁶ bacteria/ml. Pure cultures of the various major rumen bacterial species, includingMethanobacterium ruminantium strain M1, were not able to utilize these substrates as energy and/or nitrogen sources. It is suggested that, in the rumen, trimethylamine and methylamine are primarily degraded byMethanosarcina, resulting in release of ammonia which then can be utilized by other rumen bacteria.     

Defective pyocin particles produced by some mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, defective in the production of active R-type pyocins, were isolated from pyocinogenic strains and their products were characterized. Polysheath-like structures were found in induced lysates of 29 out of 42 mutants. Two mutants (strain P15-16 and M189) were found to produce special defective particles, which were characterized in detail. The other 11 mutants did not produce significant amounts of any structure visible under an electron microscope. Serum blocking powers were found in lysates from P15-16 and M189 to significant amounts. Defective particle produced by strain P15-16 lacked the sheath component, whereas M189 had morphological defects at the junction between sheath and baseplate, and also in the architecture of baseplate. Both defective particles could adsorb to the surface of bacteria, that were sensitive to pyocin, at the tip of their fibers without killing cells. All M189 particles attached to the bacteria had the extended sheaths. Therefore, attachment to the bacteria by fibers is not sufficient to kill cells, and contraction of sheath must occur after the initial adsorption by fibers for pyocin to express its biological activity. Defective particles of strain P15-16, which was derived from strain P15 (a pyocin R1 producer), could be converted to active forms by an in vitro complementation reaction with extracts from certain mutants originated from strain PAO (a pyocin R2 producer). This result indicated the exchangeability of components between R-type pyocins belonging to the different groups.     

Microrheological aspects of adhesion of Escherichia coli on glass

The adhesion of both live and fixed bacteria (Escherichia coli) on glass has been studied under well-defined hydrodynamic conditions, created in an impinging jet apparatus. With this technique one can accurately measure the initial deposition rate jo on the surface, the average lifetime of a bacterium on the surface, tau esc, and the surface area blocked per deposited bacterium, normalized by its projected area, gamma. The experimental results are compared to theoretical results for equivalent spheres. It is found that near the stagnation point the deposition rate jo is mainly controlled by convective diffusive transport which, for rod-shaped Eschericia coli, with an axis ratio of about 2, is found to be equal to that for spheres. No differences in jo and tau esc were found between live and fixed bacteria at low flow rates. At high flow rates fixed bacteria adhered to the surface at a slower rate. In both systems jo was found to decrease suddenly at a distance of about 150 microns from the stagnation point, in contrast to systems of spherical particles for which jo is uniform over the surface. Most likely this is due to the rotation of the rod-shaped particles, which vary their distance to the surface periodically with time. The main difference between live and fixed bacteria, besides different deposition rates in strong flows, is that gamma is about 30% larger for fixed bacteria than for live ones, resulting in a much lower final coverage for fixed bacteria. These results imply a larger repulsion between fixed bacteria than between living ones. From detachment experiments we can conclude that not all bacteria stick to the surface with the same bond strength. The variation in the bond strength is due to the aging of the bonds between the bacteria and the surface. The average bond strength corresponds to an energy of about 13-15 kT.     

Bacterial transport through porous solids: interactions betweenMicrococcus luteus cells and sand particles

Summary As part of an evaluation of microbial systems for potential application in enhanced oil recovery (EOR) in situ, the behaviour of bacteria within the labyrinths of porous structures was investigated. Sandpacks were utilised as models of reservoir formations. UsingMicrococcus luteus (NCIB 8553) cells under non-growing conditions, the interaction between the bacteria and sand particles was investigated by a simple shake flask system. The attachment of bacterial cells to sand was time-dependent and reversible. With increasing density of cell suspensions, competivive effects on binding to sand were observed, indicating a possible interaction between two subpopulations of cells. Similar effects occurred when suspensions ofM. luteus cells were pumped through sandpacks. Shake flask experiments indicated that the maximum binding of bacteria to sand was about 2×10⁹ cells · g^–1, representing a total coverage of approximately 20% of the surface of sand particles by bacterial cells.     

内生细菌B10对稻瘟病菌抑制作用的初步研究

目的:研究内生细菌B10对水稻稻瘟病菌的抑制作用,为菌株的应用提供理论依据。方法:采用菌丝生长速率法、孢子萌发法和田间试验测定内生细菌发酵上清液和菌液对稻瘟病菌的抑制作用。结果:内生细菌B10发酵上清液对稻瘟病菌菌丝生长和孢子萌发有较强的抑制作用,100倍稀释液对菌丝生长的抑制率为79.37%,对孢子萌发的抑制率为63.42%,对稻瘟病的田间防治效果达70.2%以上。结论:内生细菌B10对稻瘟病有较强的抑菌作用。     

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.     

Use of superparamagnetic particles for isolation of cells

This report describes the preparation and characterization of synthetic ferritin-like particles produced by precipitation of magnetite from a mixture of ferrous and ferric ions in the presence of dextran. The 3-nm diameter particles, containing magnetite cores surrounded by chemisorbed dextran, had a magnetization of 46.7 emu/g of iron with M?ssbauer quadrupole splitting of 2 delta = 0.76 mm/s. The application of these particles as a laboratory reagent for isolation of Legionella from other water bacteria was successfully tested. A 400-fold enrichment for Legionella was obtained.     

Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

Vesicular stomatitis virus (VSV) particles formed at early times after infection contain only one-third the amount of viral glycoportein (G protein), relative to the major internal structural proteins M and N, as is found in particles released later. These "early" particles also have a lower density in equilibrium sucrose gradients than do those formed later; however, the sedimentation velocity and specific infectivity of these two classes of particles are the same. VSV-infected cells also release virus-like particles which sediment considerably faster than authentic virions and contain a higher-than-normal proportion of the VSV G protein relative to internal VSV proteins. These particles have a reduced specific infectivity but a normal density in sucrose gradients. All classes of VSV virions contain a constant proportion of M and N polypeptides. The ratio of G protein to M or N protein, in contrast, can vary over a sixfold range; this implies that an interaction between a precise number of surface G proteins with either of the underlying M and N proteins is not a prerequisite for budding of infectious viral particles from the cell surface.     

Agglutinins in the hemolymph of the hard clam, Mercenaria mercenaria.

Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substances with particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms.     

Use of Genetically Marked Minicells as a Probe in Measurement of Predation on Bacteria in Aquatic Environments

Minicells produced by Escherichia coli M2141 were used as probes to measure predation on pelagic bacteria in situ. The minicells, labeled with [³⁵S]methionine in one specific protein, were shown to disappear in the presence of a microflagellate (Ochromonas sp.), as seen by a decrease in the amount of labeled marker protein with time. Incubation in filtered (pore size, 0.2 μm) and autoclaved seawater did not affect the amount of labeled marker protein in the minicell. The generation time of flagellates feeding on minicells was determined to be similar to that found for flagellates grown on seawater bacteria or living E. coli NC3. Data indicate that minicells are seen as true food particles by the flagellates. The minicell probe was used in recapture experiments, in which predation in situ on pelagic bacteria was demonstrated. The rate of bacterial production showed a clear covariation with the rate of predation, both in different sea areas and in depth profiles. The obtained results (11 field experiments) showed that the rate of predation, on average, accounts for the consumption of 62% of the bacteria produced.     

Endophytic bacteria of <Emphasis Type="Italic">Mammillaria fraileana</Emphasis>, an endemic rock-colonizing cactus of the southern Sonoran Desert

The small cactus Mammillaria fraileana is a pioneer rock-colonizing plant harboring endophytic bacteria with the potential for nitrogen fixation and rock weathering (phosphate solubilization and rock degradation). In seeds, only a combination of culture-independent methods, such as fluorescence in situ hybridization, scanning electron microscopy, and fluorescence vital staining, detected significant amounts of non-culturable, but living, endophytic bacteria distributed underneath the membrane covering the embryo, in the undifferentiated tissue of the embryo, and in the vascular tissue. Large populations of culturable endophytic bacteria were detected in stems and roots of wild plants colonizing rocks in the southern Sonoran Desert, but not in seeds. Among 14 endophytic bacterial isolates found in roots, four isolates were identified by full sequencing of their 16S rRNA gene. In vitro tests indicated that Azotobacter vinelandii M2Per is a potent nitrogen fixer. Solubilization of inorganic phosphate was exhibited by Pseudomonas putida M5TSA, Enterobacter sakazakii M2PFe, and Bacillus megaterium M1PCa, while A. vinelandii M2Per, P. putida M5TSA, and B. megaterium M1PCa weathered rock by reducing the size of rock particles, probably by changing the pH of the liquid media. Cultivated seedlings of M. fraileana, derived from disinfected seeds and inoculated with endophytic bacteria, showed re-colonization 105 days after inoculation. Their densities decreased from the root toward the stem and apical zones. Functional traits in planta of culturable and non-culturable endophytic bacteria in seeds remain unknown.     

Circadian rhythm of air-borne bacteria and dust particles in a mouse breeding room

The circadian rhythm of air-borne bacteria and dust particles in a mice breeding room was studied at 1-hour intervals on the first, third and fifth day after accommodation of the animals. The numbers of air-borne bacteria in the room increased day by day after accommodation, and showed a circadian rhythm which went down at about noon and rose with three peaks at about 20:00, 1:00 and 8:00. The numbers of dust particles tended to decrease from day to day, and they showed almost the same circadian rhythm as the air-borne bacteria. Correlations between air-borne bacteria and dust particles were not significant for each particle level on the first day, but were significant for all the particle levels of 0.3, 1, 2, and 5 microns at the third day, and were also significant at particles levels of 0.5 micron or more on the fifth day.