Effect of compatible solutes on marine halophilic Vibrio sp. under hyper salt stress

1. Effect of compatible solutes on marine halophilic Vibrio sp. under hyper salt stress Graduate school of maritime sciences, Kobe university Yin Yue

3. Introduction of halophilic and halotolerant bacteria

4. Marine bacteria • Prokaryotic single celled organisms that live in the ocean without chlorophyll and phycocyanin. • The most widely distributed and largest species of organisms in marine environment. • Usually the diameter is less than 1 micron. • Spherical, rod, spiral, and branchedfilamentous shapes. • Halophilic, Psychrophilic, Thermophilic, Barophilic,etc.

5. classification of bacteria according to the response to nacl Vibrio sp. September 1 Larsen, H. (1986) Halophilic and halotolerant microorganisms−an overview and historical perspective. FEMSMicrobiol. Rev., 39, 3−7. (Slightly modified by Professor Mimura)

6. Introduction of Vibrio sp.

7. Colored scanning electron micrographs of Vibrio sp. Vibrio vulnificus b Vibrio cholerae a a: https://fineartamerica.com/featured/1-vibrio-cholerae-dennis-kunkel-microscopyscience-photo-library.html b; (Vibriovulnificus - Wikipedia) https://en.wikipedia.org/wiki/Vibrio_vulnificus

8. Morphological Characteristics rod

9. Growth conditions

10. (Hood, M.A., J.B. Guckert, D.C. White and F. Deck. 1986. Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae. Appl. Environ. Microbiol. 52: 788–793.) https://tpwd.texas.gov/huntwild/hunt/wma/find_a_wma/maps/?action=getMap&region=4

11. Dickson, A.G., and Goyet, C.. Handbook of methods for the analysis of the various parameters of the carbon dioxide system in sea water. Version 2. United States: N. p., 1994. Web.

12. Roles of K+ played in bacteria cells (Wolfgang, 2003)

13. K+ transport https://www2.estrellamountain.edu/faculty/farabee/biobk/BioBooktransp.html

14. Heat stress Salt stress Environmental stresses

15. SALT STRESS(HYPER): is the exposure of microbesto a higher concentration of salinity than intracellular. • Salt stress is primarily osmotic stress. • OSMOTIC STRESS: is physiologic dysfunction caused by a sudden change in the solute concentration around a cell , which causes a rapid change in the movement ofwater across its cell membrane.

16. Compatible solutes Osmoprotectants (compatible solutes): are small organic molecules with neutral charge and low toxicity at high concentrations that act as osmolytes and help organisms survive osmotic stress.

17. Osmoprotectant compounds (Amino acids) L-Citrulline L-Proline D-Proline

18. Glycine betaine from marine phytoplankton Glycine betaine (Astrid, 2012) https://www.uts.edu.au/research-and-teaching/our-research/climate-change-cluster/events/c3-colloquium-functional-genetics

19. Experiments and results

20. (Mimura, et al., 2005)

21. Growth experiment Growth medium (培地) Nutrients Bacto peptone: 5 g yeast extract: 1 g Salts NaCl and/or KCl Dissolved in 50 mM HEPES-TMAH buffer, pH 7.8. Incubation (培養条件) Preincubation: Nutrients and 0.5 M NaCl at 30ºC. Incubation: Cell suspension was added to each medium to give one-thousandth dilution.

22. Vibrio sp. September 1株の増殖実験結果 (前培養は0.5 M NaCl添加の栄養培地at 30ºC) KCl stress NaCl stress ○：0.5 M NaCl at 30ºC ●：0.5 M NaCl at 37ºC ▲：1.2 M NaCl at 37ºC ■：1.5M NaCl at 37ºC ○: 0.5 M KCl at 37ºC ●: 0.5 M KCl + 50 mMNaCl at 37ºC △: 0.8 M KCl at 37ºC ▲: 0.8 M KCl + 50 mMNaCl at 37ºC

23. Intracellular free amino acids analysis

24. Analysis of free amino acids pooled in the cells

26. Surviving Experiments

27. PREPARE • Cells were grown in the nutrient medium containing 0.5 M NaCl at 30ºC. • Harvested at early stationary phase of growth and washed twice with HEPES-TMAH buffer, pH 7.5, containing 0.5 M NaCl. • Suspended in the same mixture as used for washing. • Cellssuspension were pipetted to each mixture containing various concentrations of NaCl, or KCl, supplemented with osmoprotectant(50 mM) or not, at 30 or 37ºC. • At given time, an aliquot (100 μL) was taken and spread onto nutrients agar plates after serial dilution with the same mixture used in the last step. • After 2 days incubation at 30ºC, colonies formed on the plates were counted. CULTURE COUNT

28. Changes in the survivability of resting Vibrio sp. cellsunderhyper NaCl stress ●: 0.5 M NaCl at 30ºC ▲:1.2 M NaCl at 30ºC ■: 1.5 M NaCl at 30º ◆: 1.8 M NaCl at 30ºC

29. Changes in survivability of resting Vibrio sp. cells under hyper KCl stress ○: 1.2 M KCl alone at 30ºC, △: 1.2 M KCl + 50 mMNaCl at 30℃ (n=3), ●: 1.2 M KCl + 50 mMcitrulline at 30ºC, ▲: 1.2 M KCl + 50 mMNaCl + 50 mMcitrulline at 30℃ (n=3).

30. Enhanced Repressed Slightly enhanced

31. Conclusion • Vibrio sp. September 1 could not grow in high salinity of KClwithout NaCl externally added. • Citrulline was accumulated in the cells grown in the presence of 0.8 M KCl and 50 mMNaCl,but not used as compatible solute for osmoadaptation to hyper K+ stress. • Proline and betaine are able to improve the survivability of cells to cope with both Na+ and K+ salt stress.

32. summary • Vibrio sp. September 1 can grow in concentration up to 0.8 M of KCl. • This strain require Na+ for growth under hyper KCl stress. • High salinity of KCl might interfere the uptake of compatible solutes from external environment. • Proline and betaine are major osmoprotectants for Vibrio sp. September 1. coping with salt stress.

33. Future study • Brevibacterium sp., a genus of halotolerant bacteria. • Growth and surviving experiments under hyper KCl stress • Effect of ectoine on

34. References [1] Robert A. Macleod and E. Onofrey. 1956. Nutrition and metabolism of marine bacteria. J. Bacteriol., 71(6), 661-667. [2] Wolfgang Epstein. 2003. The Roles and regulation of potassium in bacteria. Progress in Nucleic Acid Research and Molecular Biology, 75, 293-320. [3] Haruo Mimura, Ryo Katakura, Hiroshi Ishida. 2005. Changes of microbial populations in a ship’s ballast water and sediments on a voyage from Japan to Qatar. Marine Pollut. Bul., 50, 751-757. [4] Zobell, C. E. and Upham, H. C. 1944. A list of marine bacteria including descriptions of sixty new species. Bull. Scripps Inst. Oceanog., Univ. Calif., 5, 239-292. [5] MacLeod, R. A. and E. Onofrey. 1957. Nutrition and metabolism of marine bacteria. III. The relation of sodium and potassium to growth. J. Cell. Comp. Physiol., 50, 389- 401. [6] Xiuping Fu, Weili Liang, Pengcheng Du, Meiying Yan and Biao Kan. 2014. Transcript changes in Vibrio cholerae in response to salt stress. Gut Pathogens, 6, 1-6. [7] Erina FujiwaraNagata, MitsuruEguchi. 2004. Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition. FEMS Microbiology Letters, 234: 163–167. [8] Astrid Spielmeyer, Georg Pohnert. 2012. Influence of temperature and elevated carbon dioxide on the production of dimethylsulfoniopropionate and glycine betaine by marine phytoplankton. Marine Environmental Research, 73, 62-69. [9] M.Ghoul, J.Minet, T.Bernard, E. Dupray, M. Cormie. 1995. Marine Macroalgae as a Source for Osmoprotectionfor Escherichia coli. Microb. Ecol., 30, 171-181.

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