Mechanisms of microbial movement in subsurface materials

The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of microbial movement in subsurface materials.

The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of magnetotactic bacteria in a rotating magnetic field

The dynamics of the motile magnetotactic bacterium Magnetospirillum gryphiswaldense in a rotating magnetic field is investigated experimentally and analyzed by a theoretical model. These elongated bacteria are propelled by single flagella at each bacterial end and contain a magnetic filament formed by a linear assembly of approximately 40 ferromagnetic nanoparticles. The movements of the bacteria in suspension are analyzed by consideration of the orientation of their magnetic dipoles in the field, the hydrodynamic resistance of the bacteria, and the propulsive force of the flagella. Several novel features found in experiments include a velocity reversal during motion in the rotating field and an interesting diffusive wandering of the trajectory curvature centers. A new method to measure the magnetic moment of an individual bacterium is proposed based on the theory developed.     

Light-Scattering Spectrum Due to Wiggling Motions of Bacteria

Simple models are used to calculate the inelastic light scattering spectrum of motile bacteria when wiggling motions are included in addition to translational displacement. Computations of spectra lead to the conclusion that nontranslational motions can be neglected when swimming speeds are deduced from light-scattering data for normal vigorously motile strains. On the other hand, for slowly translating bacteria, or for strains exhibiting noticeable wiggling motion when viewed in a microscope, additional spectral components may be significant. Such components are best distinguished when measurements are made at small and intermediate scattering angles; at large angles the spectra have approximately the same scaling properties (functionals of Qt, Q being the Bragg wave vector) as those associated with simple translational motility.     

Study of the motion of magnetotactic bacteria

Motion of flagellate bacteria is considered from the point of view of rigid body mechanics. As a general case we consider a flagellate coccus magnetotactic bacterium swimming in a fluid in the presence of an external magnetic field. The proposed model generalizes previous approaches to the problem and allows one to access parameters of the motion that can be measured experimentally. The results suggest that the strong helical pattern observed in typical trajectories of magnetotactic bacteria can be a biological advantage complementary to magnetic orientation. In the particular case of zero magnetic interaction the model describes the motion of a non-magnetotactic coccus bacterium swimming in a fluid. Theoretical calculations based on experimental results are compared with the experimental track obtained by dark field optical microscopy. Correspondence to: H. G. P. Lins de Barros     

Characterizing the adhesion of motile and nonmotile Escherichia coli to a glass surface using a parallel-plate flow chamber

A parallel-plate flow chamber was used to measure the attachment and detachment rates of Escherichia coli to a glass surface at various fluid velocities. The effect of flagella on adhesion was investigated by performing experiments with several E. coli strains: AW405 (motile); HCB136 (nonmotile mutant with paralyzed flagella); and HCB137 (nonmotile mutant without flagella). We compared the total attachment rates and the fraction of bacteria retained on the surface to determine how the presence and movement of the flagella influence transport to the surface and adhesion strength in this dynamic system. At the lower fluid velocities, there was no significant difference in the total attachment rates for the three bacterial strains; nonmotile strains settled at a rate that was of the same order of magnitude as the diffusion rate of the motile strain. At the highest fluid velocity, the effect of settling was minimized to better illustrate the importance of motility, and the attachment rates of both nonmotile strains were approximately five times slower than that of the motile bacteria. Thus, different processes controlled the attachment rate depending on the parameter regime in which the experiment was performed. The fractions of motile bacteria retained on the glass surface increased with increasing velocity, whereas the opposite trend was found for the nonmotile strains. This suggests that the rotation of the flagella enables cells to detach from the surface (at the lower fluid velocities) and strengthens adhesion (at higher fluid velocities), whereas nonmotile cells detach as a result of shear. There was no significant difference in the initial attachment rates of the two nonmotile species, which suggests that merely the presence of flagella was not important in this stage of biofilm development.     

Factors affecting the irreversible attachment of Pseudomonas aeruginosa to stainless steel

To better understand the interaction between bacteria and surfaces, we studied the irreversible attachment of Pseudomonas aeruginosa to a common surfacing material. When brought into contact with the steel, cells began to attach in less than 1 min and the number adhering increased with time. An important physiological variable in attachment was cell motility since adherence decreased at least 90% when flagella were removed by blending. This treatment was shown to be effective because it caused motility loss and not because it removed a structure necessary for adherence. Cell viability was less important since adherence decreased only 50% when the number of viable cells was reduced 4.7 logs by heating or formaldehyde treatment. Significant environmental variables included turbulence and ionic strength. Attachment of motile cells was reduced 90% by agitation, although agitation had little effect on adherence of nonmotile cells. Both motile and nonmotile cells adhered poorly in distilled water with attachment increasing as CaCl2 or NaCl concentration increased to 10 mM. At 100 mM, attachment decreased. Viable cells, both motile and nonmotile, adhered best at a pH of 7 to 8, whereas nonviable cells attached most rapidly at a low pH.     

SUSCEPTIBILITY AND PROTECTIVE MECHANISMS OF MOTILE AND NON MOTILE CELLS OF HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) TO PHOTOOXIDATIVE STRESS1

The life cycle of the unicellular green alga Haematococcus pluvialis consists of motile and nonmotile stages under typical growing conditions. In this study, we observed that motile cells were more susceptible than nonmotile cells to high light, resulting in a decrease in population density and photo‐bleaching. Using two Haematococcus strains, CCAP 34/12 (a motile cell dominated strain) and SAG 34/1b (a nonmotile cell dominated strain), as model systems we investigated the cause of cell death and the protective mechanisms of the cells that survived high light. The death of motile cells under high light was attributed to the generation of excess reactive oxygen species (ROS), which caused severe damage to the photosynthetic components and the membrane system. Motile cells were able to dissipate excess light energy by nonphotochemical quenching and to relax ROS production by a partially up‐regulated scavenging enzyme system. However, these strategies were not sufficient to protect the motile cells from high light stress. In contrast, nonmotile cells were able to cope with and survive under high light by (i) relaxing the over‐reduced photosynthetic electron transport chain (PETC), thereby effectively utilizing PETC‐generated NADPH to produce storage starch, neutral lipid, and astaxanthin, and thus preventing formation of excess ROS; (ii) down‐regulating the linear electron transport by decreasing the level of cytochrome f; and (iii) consuming excess electrons produced by PSII via a significantly enhanced plastid terminal oxidase pathway.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

A new type of microscope illumination and its use in microbiology

To overcome problems with taking photographs of motile microorganisms we used a new type of microscope illumination by means of a light-emitting diode (LED), which fully preserves the Kohler illumination principle. Oblique illumination or relief contrast after the Hostounsk, microscopy method were used for observing and photographing bacteria, flagellates and infusorians having different motion velocities with sufficient image contrast. The device, which permitted us to use short shutter time (1/30, 1/60 or 1/125 s), was successfully used in recording different motile microorganisms including their internal architecture.     

Chemotaxis of Yersinia pseudotuberculosis as a mechanism in its search for target tissues of the host organism

Y. pseudotuberculosis cells grown at biologically low temperature have been shown capable of chemotaxis with respect to carbohydrates and amino acids. During cultivation at 36-37 degrees C Y. pseudotuberculosis cells retain this property for 10-15 hours and then lose it. The mechanism of chemotaxis makes it possible for Y. pseudotuberculosis to "find" human and animal tissues and can facilitate the realization of the pathogenicity potential of these bacteria. When administered orally to mice motile bacteria, i. e. those grown at 6-8 degrees C, have been more virulent for the animals than nonmotile ones cultivated at 36-37 degrees C.     

Flagellar Motility Confers Epiphytic Fitness Advantages upon Pseudomonas syringae

The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain.     

A Study of Magnetic Properties of Magnetotactic Bacteria

The first direct measurements of magnetic properties of magnetotactic bacteria from natural samples are presented. Measurements were made at 4.2 K, using a Superconducting Quantum Interfering Device (SQUID) magnetometer. From the magnetization results an anisotropy is obtained that is typical of magnetized ferro- or ferri-magnetic materials. The average magnetic moment of the bacteria determined from the results is in good agreement with the estimated moment from electron microscopy.     

Life cycle,behavior and morphology of a new tide pool dinoflagellate,Gymnodinium pyrenoidosum sp. nov. (Gymnodiniales,Pyrrhophyta)

A new tide pool dinoflagellate,Gymnodinium pyrenoidosum Horiguchi et Chihara sp. nov. is described from central Japan. It was found to form dense blooms with a characteristic greenish color from April to November. The species exhibits a characteristic diurnal vertical migration and an alternation of a motile with a nonmotile phase, which are dependent on light intensity and tidal movement. Cells of the motile phase are unarmored and relatively small. They have a single, reticulate chloroplast, orange stigma situated near the sulcus and conspicuous pyrenoid in epicone. The alga reproduces itself by means of zoospores which are produced by the bipartition of protoplasm within the parent cell wall during the nonmotile stage which occurs at night. The occurrence of another type of motile cell, termed a macroswarmer, which differs from normal zoospore in size and shape has also been demonstrated.     

Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment

Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was nonmotile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis.     

Quantifying the magnetic advantage in magnetotaxis

Magnetotactic bacteria are characterized by the production of magnetosomes, nanoscale particles of lipid bilayer encapsulated magnetite, that act to orient the bacteria in magnetic fields. These magnetosomes allow magneto-aerotaxis, which is the motion of the bacteria along a magnetic field and toward preferred concentrations of oxygen. Magneto-aerotaxis has been shown to direct the motion of these bacteria downward toward sediments and microaerobic environments favorable for growth. Herein, we compare the magneto-aerotaxis of wild-type, magnetic Magnetospirillum magneticum AMB-1 with a nonmagnetic mutant we have engineered. Using an applied magnetic field and an advancing oxygen gradient, we have quantified the magnetic advantage in magneto-aerotaxis as a more rapid migration to preferred oxygen levels. Magnetic, wild-type cells swimming in an applied magnetic field more quickly migrate away from the advancing oxygen than either wild-type cells in a zero field or the nonmagnetic cells in any field. We find that the responses of the magnetic and mutant strains are well described by a relatively simple analytical model, an analysis of which indicates that the key benefit of magnetotaxis is an enhancement of a bacterium's ability to detect oxygen, not an increase in its average speed moving away from high oxygen concentrations.     

Energetics of an rf SQUID Coupled to Two Thermal Reservoirs

We study energetics of a Josephson tunnel junction connecting a superconducting loop pierced by an external magnetic flux (an rf SQUID) and coupled to two independent thermal reservoirs of different temperature. In the framework of the theory of quantum dissipative systems, we analyze energy currents in stationary states. The stationary energy flow can be periodically modulated by the external magnetic flux exemplifying the rf SQUID as a quantum heat interferometer. We also consider the transient regime and identify three distinct regimes: monotonic decay, damped oscillations and pulse-type behavior of energy currents. The first two regimes can be controlled by the external magnetic flux while the last regime is robust against its variation.     

Interactions between motile Escherichia coli and glass in media with various ionic strengths, as observed with a three-dimensional-tracking microscope.

Escherichia coli bacteria have been observed to swim along a glass surface for several minutes at a time. Settling velocities of nonmotile cells and a computer simulation of motile cells confirmed that an attractive force kept the bacteria near the surface. The goal of this study was to evaluate whether this attractive force could be explained by reversible adhesion of E. coli to the surface in the secondary energy minimum, according to the theory of Derjaguin, Landan, Verwey, and Overbeek (DLVO theory). This theory describes interactions between colloidal particles by combining attractive van der Waals forces with repulsive electrostatic forces. A three-dimensional-tracking microscope was used to follow both wild-type and smooth-swimming E. coli bacteria as they interacted with a glass coverslip in media of increasing ionic strengths, which corresponded to increasing depths of the secondary energy minimum. We found no quantifiable changes with ionic strength for either the tendencies of individual bacteria to approach the surface or the overall times bacteria spent near the surface. One change in bacterial behavior which was observed with the change in ionic strength was that the diameters of the circles which the smooth-swimming bacteria traced out on the glass increased in low-ionic-strength solution.     

Mound-cell movement and morphogenesis in Dictyostelium

To examine the mechanisms of cell locomotion within a three-dimensional (3-D) cell mass, we have undertaken a systematic 3-D analysis of individual cell movements in the Dictyostelium mound, the first 3-D structure to form during development of the fruiting body. We used time-lapse deconvolution microscopy to examine two strains whose motion represents endpoints on the spectrum of motile behaviors that we have observed in mounds. In AX-2 mounds, cell motion is slow and trajectories are a combination of random and radial, compared to KAX-3, in which motion is fivefold faster and most trajectories are rotational. Although radial or rotational motion was correlated with the optical-density wave patterns present in each strain, we also found small but significant subpopulations of cells that moved differently from the majority, demonstrating that optical-density waves are at best insufficient to explain all motile behavior in mounds. In examining morphogenesis in these strains, we noted that AX-2 mounds tended to culminate directly to a fruiting body, whereas KAX-3 mounds first formed a migratory slug. By altering buffering conditions we could interchange these behaviors and then found that mound-cell motions also changed accordingly. This demonstrates a correlation between mound-cell motion and subsequent development, but it is not obligatory. Chimeric mounds composed of only 10% KAX-3 cells and 90% AX-2 cells exhibited rotational motion, suggesting that a diffusible molecule induces rotation, but many of these mounds still culminated directly, demonstrating that rotational motion does not always lead to slug migration. Our observations provide a detailed analysis of cell motion for two distinct modes of mound and slug formation in Dictyostelium.     

Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.