Response of neutrophilic Shewanella violacea to acid stress: growth rate, organic acid production, and gene expression.

Neutrophilic Shewanella violacea is isolated from deep-sea sediments and its response to high pressure and high salinity has been investigated. Here, the pure effects of acidic pH on S. violacea physiology were examined, aiming at further understanding of its stress response mechanism. S. violacea could grow at initial pH of 5.0-7.0 without pH adjustment during the test at atmospheric pressure, and the lowest growth rate was obtained at pH 5.0. The pH of the same growth culture with an initial pH of 5.0 rose toward a neutral pH of ~ 7.0 at the exponential growth phase, indicating that S. violacea has a mechanism for acid neutralization. When S. violacea cells were grown at the fixed pH of 5.0, about five times higher concentrations of butyric and isovaleric acids were produced than at pH 7.0. The expression level of the genes encoding three enzymes for isovaleric acid synthesis from L-leucine was also found to be upregulated in S. violacea cells grown at the fixed pH of 5.0 compared with at pH 7.0 through RNA-seq analysis. Therefore, S. violacea at least produces isovaleric acid in its response to acid stress, which further deepens our understanding of the stress response mechanism inherent in this bacterium.

Stability of cytochromes c' from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure.

The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.

Differences in biochemical properties of two 5'-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions.

Deep-sea Shewanella violacea 5'-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions. Abbreviations: NTase: 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase; SVNTase: Shewanella violacea 5'-nucleotidase; CD: circular dichroism.

Transcriptome analysis of Chironomus sulfurosus larvae living in acidic environments: Insights into molecular mechanisms for acid tolerance.

Larvae of chironomid Chironomus sulfurosus mainly live in acidic rivers near hot springs, suggesting that they naturally select acidic environments as preferred habitats. Here we showed that C. sulfurosus larvae moved toward acidic areas and stayed alive on agar gels with a pH gradient of H2SO4, and the body fluid pH of the homogenized larvae was near neutral even acclimated under the acidic conditions, indicating mechanisms for acid tolerance. In order to gain insights into this mechanism at the molecular level, de novo RNA-seq analysis was performed on C. sulfurosus larvae. As a result, 1,208 genes were found to be significantly up-regulated in larvae acclimated at pH 2.0 compared to controls at pH 7.0. Among the up-regulated genes, ones encoding cuticle proteins, peritrophic matrix proteins, mucus-forming proteins, F-type ATPase subunits, glutathione S transferases, ß-1,3-D-glucan synthetase, hemoglobin, and cytochrome P450 were identified. This transcriptome analysis in conjunction with behavioral and biochemical assays expands our knowledge of gene expression in C. sulfurosus larvae living in acidic environments, which will provide a basis for further studies to elucidate the molecular mechanisms for acid tolerance employed by organisms in nature.

Designing easy DNA extraction: Teaching creativity through laboratory practice.

Subject material concerning Deoxyribose Nucleic Acid (DNA) structure in the format of creativity-driven laboratory practice offers meaningful learning experience to the students. Therefore, a laboratory practice in which utilizes simple procedures and easy-safe-affordable household materials should be promoted to students to develop their creativity. This study aimed to examine whether designing and conducting DNA extraction with household materials could foster students' creative thinking. We also described how this laboratory practice affected students' knowledge and views. A total of 47 students participated in this study. These students were grouped and asked to utilize available household materials and modify procedures using hands-on worksheet. Result showed that this approach encouraged creative thinking as well as improved subject-related knowledge. Students also demonstrated positive views about content knowledge, social skills, and creative thinking skills. This study implies that extracting DNA with household materials is able to develop content knowledge, social skills, and creative thinking of the students. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):216-225, 2017.