Hisao Morisaki

Isolation and Characterization of Endophytic Bacteria from Wild and Traditionally Cultivated Rice Varieties

Abstract Endophytic bacteria were isolated from surface-sterilized stems, seeds, and leaf sheaths of wild and traditionally cultivated rice varieties. Phylogenetic analyses based on 16S rDNA revealed a wide divergence among the isolates. However, the most frequently isolated groups were Methylobacterium sp. in the α-subdivision of Proteobacteria, and Curtobacterium sp. in the high G+ C Gram-positive group. Various phenotypic traits that are expected to be involved in the persistence and functions of the bacteria were analyzed: Most of the isolates from rice excreted pectinase, were motile, and showed an osmotic resistance to 0.6 M sucrose. These traits may be involved in endophytic characteristics in rice. About 50% of the isolates showed a cellulase activity. A few isolates fixed nitrogen, produced indole-3-acetic acid, and formed capsules. These activities were partially correlated with the phylogenetic group.

The effect of motility and cell-surface polymers on bacterial attachment

Recently it was shown that motility of Vibrio alginolyticus facilitated cell attachment to glass surfaces. In the present study the same relationship between motility and cell attachment was confirmed for Alcaligenes and Alteromonas spp. These findings clearly answer a long-standing question: does motility facilitate attachment? However, they are contradictory to a general view on cell attachment that the energy barrier due to electrostatic repulsion between negatively charged bacterial cells and a glass surface is much greater than both the thermal kinetic energy of the bacterial cell and the bacterial swimming energy. It is shown that the energy barrier becomes far less than that usually estimated when bacterial cells are rich in polymers at their surfaces. This finding reasonably explains the dependence of bacterial attachment rate on cell motility and demands reconsideration of the mechanism of bacterial attachment.

SURFACE CHARACTERISTICS OF THE MICROBIAL CELL OF PSEUDOMONAS SYRINGAE AND ITS RELEVANCE TO CELL ATTACHMENT

Abstract The cell surface of four strains of Pseudomonas syringae pv. atropurpurea NIAES 1309 was characterized by analyzing the dependence of the electrophoretic mobility of the microbial cells on pH and ionic strength. Among the strains, the wild type (WT) had the least electric charge, while carrying the softest polymer layer, at the surface. The cell surface of the WT strain was determined, by chemical analysis, to be rich in sugar, and thus seems to be bulky because of hydration. The number of cells attached to a glass plate was least for the WT strain. These findings led to the assumption that a soft and hydrated polymer layer at the cell surface may hinder the cell attachment.

Cell-surface hydrophobicity and scum formation of Rhodococcus rhodochrous strains with different colonial morphologies.

Rhodococcus rhodochrous has been reported to be one of the micro‐organisms responsible for the formation of scum which is thick and viscous biological foam in activated sludge plants. The hydrophobicity of mycolic acids present on the cell surface and the long‐branched shape of the hyphae have been thought to contribute to the scum formation. Cell surface hydrophobicity and scum formation of four R. rhodochrous strains with different colony morphologies were determined, and the results showed that the two rough strains had strong cell surface hydrophobicity and produced scum, whereas the weakly hydrophobic smooth strain and the hydrophilic mucoidal strain did not. All four strains displayed long, branched hyphae, and their electrophoretic mobilities were similar, between pH 4 and 9. These data suggest that changes in the cell surface hydrophobicity of the R. rhodochrous result in changes in the culture characteristics and the formation of scum.

Relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus

To elucidate the function of extracellular polysaccharide (EPS) produced by bacteria with hydrophobic surfaces we investigated relationships among colony morphotypes, cell-surface properties and bacterial adhesion to substrata in Rhodococcus. Rough strains adhered well to various materials and formed cell aggregates, while their mucoidal derivatives did so poorly. Cell surfaces of the rough strains were more hydrophobic than those of the corresponding mucoidal strains. Production of the EPS by the rough strains was far less than that by the corresponding mucoidal strains. Adhesion of the rough strains to quartz, glass or hydrocarbon was inhibited by the addition of EPS. Thus, rhodococcal EPS plays an important role in determination of the cell-surface hydrophobicity by acting as a hydrophilin, and its addition to the rough strains resulted in inhibition of their adhesion to the substrata.

Measurement of the force necessary for removal of bacterial cells from a quartz plate

Summary: The force necessary to remove the cells of a bacterium, an isolate (Bacillus sp.) from grassland soil, from a quartz plate was investigated in phosphate buffer solution by using a well-defined liquid flow caused by electroosmosis. The cells were not removed at a specific strength of removal force but removed over a wide range of force, about 10-14-10-12 N per cell. An exponential relation was observed between the removal force and the number of removed cells in media of pH 7·0,80 and 9·0; more cells were removed by a smaller force. Experiments in media of different pH values in the range 3·0 to 9·0 showed that greater force was necessary to remove the cells in media of lower pH. The smaller electrostatic repulsion between the bacterial cells and the quartz plate at lower pH values was considered to result in the greater force needed for removal. The relation between the strength of the removal force and the strength of the adhesion force of the bacterial cells was considered. The findings showed that the cell adhesion was reversible and that the adhesion site was located near the end of the cell.

Virtual metagenome reconstruction from 16S rRNA gene sequences

Microbial ecologists have investigated roles of species richness and diversity in a wide variety of ecosystems. Recently, metagenomics have been developed to measure functions in ecosystems, but this approach is cost-intensive. Here we describe a novel method for the rapid and efficient reconstruction of a virtual metagenome in environmental microbial communities without using large-scale genomic sequencing. We demonstrate this approach using 16S rRNA gene sequences obtained from denaturing gradient gel electrophoresis analysis, mapped to fully sequenced genomes, to reconstruct virtual metagenome-like organizations. Furthermore, we validate a virtual metagenome using a published metagenome for cocoa bean fermentation samples, and show that metagenomes reconstructed from biofilm formation samples allow for the study of the gene pool dynamics that are necessary for biofilm growth.

Electrophoretic Mobility of Bacillus subtilis Knockout Mutants with and without Flagella

Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3. The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength. The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene. The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment. It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy. These results indicate that the flagella are a key factor influencing cell surface properties.

Direct Observation and Analysis of Bacterial Growth on an Antimicrobial Surface

ABSTRACT Cells of Escherichia coli NBRC 3972 and Staphylococcus aureus NBRC 12732 were inoculated onto an agar (1.5%) medium varying in nutrient concentration from full strength of the nutrient broth (NB) to 1/10 NB. Immediately thereafter, the inoculated agar was placed on antimicrobial and nonantimicrobial surfaces in such a way that the microbial cells came into contact with these surfaces. Cell growth was directly observed under a microscope, and the growth rate constant of the cells was measured based on the increase in the area of the colonies formed by the growing cells. On the antimicrobial surface, the growth rate constant decreased at lower nutrient concentrations for both E. coli and S. aureus cells, whereas it showed little change on the nonantimicrobial surface. It was supposed that either the nutrient uptake or the nutrient utilization efficiency was retarded by the antimicrobial surface. At the lowest nutrient concentration examined in the present study, 1/10 NB, the cells could hardly grow on the antimicrobial surface, indicating that the surface would be sufficiently active in preventing bacterial growth under normal usage conditions, such as the wet areas of a kitchen. It was also revealed that the antimicrobial surface could prevent the division of cells either during the growth stage or before the onset of growth.

Bacterial attachment over a wide range of ionic strengths.

The number of bacterial cells adhered on a glass surface was counted over a wide range of ionic strengths. The counted number increased linearly with the square root of time. The rate of attachment increased with the increase in ionic strength and then plateaued. The rate of attachment was analyzed on the basis of the potential barrier between the surface of the bacterial cell and that of the substratum. An equation for formulating the dependence of the attachment rate on the ionic strength was proposed, which seems to be useful for the systematic understanding of bacterial attachment in various environments, from terrestrial to marine.