Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production.

Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low risk of contamination. However, genetic tools to modify the metabolism of Halomonas for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for Halomonas, named pUCpHAw and pHA1AT_32, consisting of ori regions from two plasmids isolated from Halomonas sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the Halomonas sp. KM-1 (KM-1). A protein that was highly and constitutively accumulated was identified as a hemolysin coregulated protein (Hcp) based on proteome analysis of KM-1. Using the hcp promoter, various genes, such as phaA and EGFP, were highly expressed. To establish a gene disruption system, the Streptococcus pyogenes cas9 gene and guide RNA for the pyrF gene, a yeast URA3 homologue, were expressed in pUCpHAw and pHA1AT_32, respectively. As a result, gene disruption mutants were isolated based on phenotypes, 5-fluoroorotic acid resistance, and uracil auxotrophy. A combination of KM-1 and these vectors could be a suitable platform for industrial chemical and protein production.

Identification of New Halomonas Strains from Food-related Environments.

Halomonas species, which are aerobic, alkaliphilic, and moderately halophilic bacteria, produce diverse biochemicals. To identify food-related Halomonas strains for bioremediation and the industrial production of biochemicals, 20 strains were isolated from edible seashells, shrimp, and umeboshi (pickled Japanese plum) factory effluents. All isolates were phylogenetically classified into a large clade of Halomonas species. Most isolates, which grew in wide pH (6-13) and salt concentration (0-14%) ranges, exhibited the intracellular accumulation of poly(3-hydroxybutyrate) granules. The characteristics of these isolates varied. A020 isolated from umeboshi factory effluents exhibited enhanced stress tolerance and proliferation and comprised two plasmids. IMZ03 and A020 grew to more than 200 OD600, while IMZ03 produced 3.5% 3-hydroxybutyrate in inorganic medium supplemented with 10% sucrose. The mucus of TK1-1 cultured on agar medium comprised approximately 64| |mM of ectoine. Whole-genome sequencing of A020 was performed to elucidate its origin and genomic characteristics. The genome ana-lysis revealed a region exhibiting synteny with a large virus genome isolated from the ocean, but did not identify any predictable pathogenic genes. Therefore, saline foods and related materials may be suitable resources for isolating Halomonas strains exhibiting unique, useful, and innocuous features.

Experimental test of the viscous anisotropy hypothesis for partially molten rocks.

Chemical differentiation of rocky planets occurs by melt segregation away from the region of melting. The mechanics of this process, however, are complex and incompletely understood. In partially molten rocks undergoing shear deformation, melt pockets between grains align coherently in the stress field; it has been hypothesized that this anisotropy in microstructure creates an anisotropy in the viscosity of the aggregate. With the inclusion of anisotropic viscosity, continuum, two-phase-flow models reproduce the emergence and angle of melt-enriched bands that form in laboratory experiments. In the same theoretical context, these models also predict sample-scale melt migration due to a gradient in shear stress. Under torsional deformation, melt is expected to segregate radially inward. Here we present torsional deformation experiments on partially molten rocks that test this prediction. Microstructural analyses of the distribution of melt and solid reveal a radial gradient in melt fraction, with more melt toward the center of the cylinder. The extent of this radial melt segregation grows with progressive strain, consistent with theory. The agreement between theoretical prediction and experimental observation provides a validation of this theory.

Seismic evidence for sharp lithosphere-asthenosphere boundaries of oceanic plates.

The mobility of the lithosphere over a weaker asthenosphere constitutes the essential element of plate tectonics, and thus the understanding of the processes at the lithosphere-asthenosphere boundary (LAB) is fundamental to understand how our planet works. It is especially so for oceanic plates because their relatively simple creation and evolution should enable easy elucidation of the LAB. Data from borehole broadband ocean bottom seismometers show that the LAB beneath the Pacific and Philippine Sea plates is sharp and age-dependent. The observed large shear wave velocity reduction at the LAB requires a partially molten asthenosphere consisting of horizontal melt-rich layers embedded in meltless mantle, which accounts for the large viscosity contrast at the LAB that facilitates horizontal plate motions.