Yuki Tsuchiya

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.

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.

Analysis of the ion adsorption-desorption characteristics of biofilm matrices.

The characteristics of biofilm polymers formed on stone surfaces in Lake Biwa and ion adsorption and desorption to and from these biofilms were investigated. The results indicated that both positively and negatively charged sites exist in the biofilm polymer. A physicochemical interaction between these sites and ions in the surrounding water seems to promote the adsorption of ions to the biofilm through an attractive electrostatic interaction and an ion-exchange mechanism. The results also indicated that, in comparison with ion-exchange resins, ions were more loosely bound to and desorbed more easily from the biofilm polymer. This suggests that microbes in the biofilm can readily use these ions as nutrient ions. Our present findings indicate that the biofilm may play an important role in supplying nutrient ions to microbes in the biofilm and in the development of a nutrient-rich environment within the biofilm through both ion adsorption and desorption. This study shows for the first time that the inside of a biofilm can be a sustainable environment for microbes.

Cloth colorization caused by microbial biofilm

In this study, cloth disfeaturement was investigated biologically. To clarify whether or not microbes can cause cloth disfeaturement, and to identify the microbes causing the disfeaturement, worn cloth samples were incubated on sweat-ingredient agar medium. Non-sterilized cloth samples became yellow-colored during incubation, and bacterial strains belonging to the genera Bacillus, Brevibacterium, Kocuria, Micrococcus and Staphylococcus were isolated from the yellow-colored parts. Two major isolates close to the genera Bacillus and Micrococcus were inoculated separately or together on cloth samples to examine whether or not these isolates can cause colorization. When the isolate close to Micrococcus was inoculated on its own or mixed with the isolate close to Bacillus, the samples turned yellow to a greater extent and a biofilm-like structure was observed by SEM on the colored areas. In contrast, the isolate close to Bacillus alone barely caused any colorization, and no biofilm-like structure was observed. From the yellow-colored samples, bacterial strains with the same 16S rRNA gene sequences as those of the inoculated strains were re-isolated. These results strongly suggest that the bacterial strain belonging to genus Micrococcus causes cloth colorization by forming a biofilm structure.

Characterization of the internal ion environment of biofilms based on charge density and shape of ion

Biofilm polymers contain both electrically positively and negatively charged sites. These charged sites enable the biofilm to trap and retain ions leading to an important role of biofilm such as nutrient recycling and pollutant purification. Much work has focused on the ion-exchange capacity of biofilms, and they are known to adsorb ions through an exchange mechanism between the ions in solution and the ions adsorbed to the charged sites on the biofilm polymer. However, recent studies suggest that the adsorption/desorption behavior of ions in a biofilm cannot be explained solely by this ion exchange mechanism. To examine the possibility that a substantial amount of ions are held in the interstitial region of the biofilm polymer by an electrostatic interaction, intact biofilms formed in a natural environment were immersed in distilled water and ion desorption was investigated. All of the detected ion species were released from the biofilms over a short period of time, and very few ions were subsequently released over more time, indicating that the interstitial region of biofilm polymers is another ion reserve. The extent of ion retention in the interstitial region of biofilms for each ion can be determined largely by charge density, |Z|/r, where |Z| is the ion valence as absolute value and r is the ion radius. The higher |Z|/r value an ion has, the stronger it is retained in the interstitial region of biofilms. Ion shape is also a key determinant of ion retention. Spherical and non-spherical ions have different correlations between the condensation ratio and |Z|/r. The generality of these findings were assured by various biofilm samples. Thus, the internal regions of biofilms exchange ions dynamically with the outside environment.