Establishment of a reborn MMV-microarray technology: realization of microbiome analysis and other hitherto inaccessible technologies.

BACKGROUND: With the accelerating development of bioscience, the problem of research cost has become important. We previously devised and developed a novel concept microarray with manageable volumes (MMV) using a soft gel. It demonstrated the great potential of the MMV technology with the examples of 1024-parallel-cell culture and PCR experiments. However, its full potential failed to be expressed, owing to the nature of the material used for the MMV chip. RESULTS: In the present study, by developing plastic-based MMVs and associated technologies, we introduced novel technologies such as C2D2P (in which the cells in each well are converted from DNA to protein in 1024-parallel), NGS-non-dependent microbiome analysis, and other powerful applications. CONCLUSIONS: The reborn MMV-microarray technology has proven to be highly efficient and cost-effective (with approximately 100-fold cost reduction) and enables us to realize hitherto unattainable technologies.

Basidiomycetous Yeast, Glaciozyma antarctica, Forming Frost-Columnar Colonies on Frozen Medium.

The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at -1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water; pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.

Cold adaptation of fungi obtained from soil and lake sediment in the Skarvsnes ice-free area, Antarctica.

A total of 71 isolates were collected from lake sediment and soil surrounding lakes in the Skarvsnes area, Antarctica. Based on ITS region sequence similarity, these isolates were classified to 10 genera. Twenty-three isolates were categorized as ascomycetous fungi from five genera (Embellisia, Phoma, Geomyces, Tetracladium or Thelebolus) and 48 isolates were categorized as basidiomycetous fungi in five genera (Mrakia, Cryptococcus, Dioszegia, Rhodotorula or Leucosporidium). Thirty-five percent of culturable fungi were of the genus Mrakia. Eighteen isolates from eight genera were selected and tested for both antifreeze activity and capacity for growth under temperatures ranging from -1 to 25 °C. Rhodotorula sp. NHT-2 possessed a high degree of sequence homology with R. gracialis, while Leucosporidium sp. BSS-1 possessed a high degree of sequence homology with Leu. antarcticum (Glaciozyma antarctica), and these two isolates demonstrated antifreeze activity. All isolates examined were capable of growth at -1 °C. Mrakia spp., while capable of growth at -1 °C, did not demonstrate any antifreeze activity and exhibited only limited secretion of extracellular polysaccharides. Species of the genus Mrakia possessed high amounts of unsaturated fatty acids, suggesting that members of this genus have adapted to cold environments by increasing their membrane fluidity.

Paenibacillus macquariensis subsp. defensor subsp. nov., isolated from boreal soil.

Two Gram-variable, aerobic, motile, rod-shaped, endospore-forming bacterial strains, M4-2T and M4-1, were isolated from soil samples collected from Oblast Magadan, Russian Far East, as micro-organisms antagonistic to the psychrophilic phytopathogenic fungus Typhula ishikariensis. Strains M4-2T and M4-1 were identified as members of the genus Paenibacillus by phenotypic and phylogenetic analyses based on 16S rRNA gene sequences. The strains contained anteiso-C15:0 as the major fatty acid (63.0-64.7%) and MK-7 as the major isoprenoid quinone. The DNA G+C contents were 42.8 and 41.7 mol%, respectively. 16S rRNA gene sequence analysis showed that strains M4-2T and M4-1 exhibited high similarities with Paenibacillus macquariensis DSM 2T (99.5 and 99.7%, respectively) and Paenibacillus antarcticus LMG 22078T (99.4 and 99.5%, respectively). There were no clear differences in the phenotypic characteristics and chemotaxonomic and phylogenetic data between the novel isolates and P. macquariensis DSM 2T. DNA-DNA hybridization experiments between strain M4-2T and P. macquariensis DSM 2T and P. antarcticus LMG 22078T revealed reassociation values of 56 and 49%, respectively. Multilocus sequence analysis confirmed the differences between the new isolates and reference strains that were observed with the DNA-DNA hybridization studies. On the basis of the results described above, it is proposed that the isolates represent a novel subspecies of P. macquariensis, Paenibacillus macquariensis subsp. defensor subsp. nov. The type strain is M4-2T (=JCM 14954T=NCIMB 14397T).