Doctoral Thesis

Response of deep-sea Shewanella violacea to acid stress(Summary)

LISA LISDIANA
Graduate School of Biosphere ScienceHiroshima University
September 2019

Chapter 1: Introduction  Physical and chemical changes in the environment might cause stress on a
microorganism and lead to metabolic disorders (Beales. Comprehensive Reviews in Food

Science and Food Safety. 3:1–20. 2004). Neutrophilic Shewanella species, which can beisolated from a wide range of aquatic environments, exhibit great potential for application
to bioremediation of pollutants and as microbial fuel cells (Hau and Gralnick. Annu Rev

Microbiol. 61:237–258. 2007; Sekiguchi et al. JAMSTEC Rep Res Dev. 11:33–41. 2010).
Amongst Shewanella species, Shewanella violacea thrives in extreme environments, andthus it has been extensively investigated in order to understand its biological stressresponse mechanism.
  In this study, S. violacea was specifically examined in the aspects of growth rate,organic acid production, and gene expression in order to gain a fundamental insight into its
physiological stress response to acidic pH. Atmospheric pressure (0.1 MPa) was adopted
throughout this study to examine the pure pH effects on S. violacea physiology.

Chapter 2: Growth of S. violacea cells under various pH conditions  The experiments aim to elucidate the ability of S. violacea to grow at various pH
conditions and observe the extracellular pH change during cell growth. S. violacea could
grow at an initial of pH 5.0–7.0 and the growth rate at initial pH 5.0 was slower than thoseat pH 6.0 and 7.0. In addition, the pH of the culture initially adjusted at pH 5.0 increasedtoward neutral during the cell cultivation, which indicates an acid neutralization
mechanism. However, genes encoding enzymes for ammonia production, which is highlyrelated to acid neutralization mechanism are not annotated in S. violacea genome. Thus,
acid neutralization mechanism in this bacterium remains unknown.

Chapter 3: Metabolite production of S. violacea cells grown at acidic and neutral pHs
  The experiments aim to elucidate the physiological response in S. violacea to acidstress through organic acid production. S. violacea cells grown at pH 5.0 produced higherconcentrations of butyric and isovaleric acids than those at pH 7.0. The ability of S.

violacea cells to grow at pH 5.0 (Chapter 2) may be related to the production of theseorganic acids.

Chapter 4: The expression level of genes related to organic acid production in Shewanella

violacea cells grown at acidic and neutral pHs
  The experiments aim to elucidate the expression level of genes related to organic acidproduction in S. violacea cells grown at pH 5.0 and 7.0. The expression level of genes
related to the synthesis of butyric acid as the response to acid stress was not entirely
detected in this experiment because three genes encoding β-hydroxybutyryl-CoA
dehydrogenase, phosphate butyryltransferase, and butyrate kinase are not annotated in thegenome of S. violacea . As for isovaleric acid, the expression level of three genes
encoding key enzymes related to the synthesis, namely branched amino acidaminotransferase, α-keto acid dehydrogenase, and acyl-CoA thioesterase was upregulated
at pH 5.0. Accordingly, the production of isovaleric acid is the response of S. violacea cells
to acid stress.

Chapter 5: Transcriptome analysis of S. violacea genes at pH 5.0 and 7.0
  The experiment aims to compare the expression level of each gene of S. violacea at
pH 5.0 and 7.0.  The expression level of 81 genes of S. violacea is upregulated  2-fold at
pH 5.0 than those at pH 7.0. Amongst the upregulated genes are related to universal stressresponse, i.e. genes encoding serine endonuclease, the disulfide bond formation family
proteins, and heat shock proteins, also genes encoding NADH-dehydrogenase andtwo-component systems. Meanwhile, other 110 genes are downregulated  2-fold at pH 5.0
than those at pH 7.0.  The most downregulated gene is encoding TonB-depending receptor
and other notable genes are encoding porin. These notable upregulated anddownregulated genes probably have a specific role in the response to acid stress in S.

violacea.

Chapter 6: Conclusion and future plan  This study revealed S. violacea can survive at the acidic pH of 5.0. Collectively, thepresent results at least showed that acid stress leads to isovaleric acid production in thisbacterium. This information deepens our understanding of the stress response mechanism
inherent in S. violacea cells. Response mechanism to acid stress in this bacterium should
be further studied, especially in the role of each gene found to be upregulated anddownregulated in the transcriptome analysis.