5:15 PM - 6:30 PM
[BCG03-P06] Physiological Response to the Extraordinary Iron Stress in the Unicellular Red Alga Galdieria sulphuraria
Keywords:Cyanidiophyceae, Galdieria sulphuraria, Iron stress, mRNA-seq
Introduction
Cyanidiophyceae is a family of extremophiles adapted to high temperature and acidic conditions (pH 2, 42°C), such as hot springs. Galdieria sulphuraria is the most stress tolerant member in Cyanidophyceae and the dominant species occupied over 90% of the biomass in their habitats (Schönknecht et al. 2013, Science 339:1207).
Cyanidiophyceae have been reported to live in volcanic areas and rivers around mining areas where contains iron over several hundred mM (Baker et al. 2004, AEM 70:6264) because high temperature and acidity in such areas promote the leaching of metals from the environment. In the laboratory, it has been reported to grow in the presence of 200 mM Al (Yoshimura et al. 1999, Soil Sci Plant Nutr 45:721). Thus, Cyanidiophyceae exhibits a highly metal tolerance that is several hundred to a thousand times higher than that of many organisms living in neutral regions.
Regarding the metal tolerance mechanism, the metal chelation of polyphosphate has been reported in Cyanidophyceae as known as in other organisms (Nagasaka et al. 2003, BioMetals 16:465). However, the behavior of metals under acidic environment is completely different from that in neutral pH in their chemical form and their adsorption kinetics, suggesting the existence of unknown metal tolerance mechanisms in addition to the known mechanisms. Whole-genome sequencing suggests that horizontal gene transfer from archaea and bacteria facilitates the adaptation to extreme environments (Schönknecht et al. 2013), but the functions of the genes acquired by horizontal gene transfer are mostly unknown. The mechanism which enable G. sulphuraria to survive under the extraordinary metal stress has not been elucidated. In this study, we focused on iron which is essential but toxic at high concentration and aimed to clarify the tolerance mechanism to excess iron in G. sulphuraria.
Results and Discussions
At first, the growth of G. sulphuraria were examined in the normal culture media (2x Allen’s medium) containing 0.03, 0.3, 3, 30, 100, and 300 mM FeSO4. The results showed that the growth rate and final OD of G. sulphuraria were similar to those of the normal culture conditions up to 100 mM, whereas G. sulphuraria did not grow at 300 mM. Since the addition of 300 mM H2SO4 does not affect the growth of G. sulphuraria, the growth inhibition observed under 300 mM FeSO4 would be due to the excess amounts of Fe.
The Fe concentration of the culture supernatant dropped sharply to 0.3 ± 0.1 mM between 1 and 2 days after the addition of 3 mM FeSO4, while after the addition of 100 mM FeSO4, the Fe concentration in the culture supernatant after 2 days was kept as 98 ± 3 mM and decreased to 77 ± 3 mM on the 6th day as increasing the number of the cells. These results suggest the two different mechanisms underlying the growth in the presence of 3 mM and 100 mM FeSO4. After the addition of 3 mM FeSO4, cells absorbed Fe in the culture medium within two days, lowering the extracellular Fe concentration and maintaining the growth similar to the normal growth conditions. In the presence of 100 mM FeSO4, cells seemed to absorb Fe for 6 days, but the extracellular Fe concentration remained high even if they absorbed Fe in the medium. Therefore, cells will need a mechanism that allows them to maintain the growth in the addition of Fe absorbing mechanism in the presence of 100 mM Fe.
Microscopic observation showed the extracellular structure after the addition of more than 3 mM FeSO4. This structure was stained with several lectins-FITC, including ConA, suggesting that they contain polysaccharides. The extracellular structures started to form within 6 hours after the addition of 100 mM FeSO4. In order to understand the tolerance mechanism to excess amounts of Fe, including the formation of extracellular structures, we are currently conducting mRNA-seq analysis using RNA sampling at 0, 1, 3, 6, 24, and 48 hours after the addition of 100 mM FeSO4.
Cyanidiophyceae is a family of extremophiles adapted to high temperature and acidic conditions (pH 2, 42°C), such as hot springs. Galdieria sulphuraria is the most stress tolerant member in Cyanidophyceae and the dominant species occupied over 90% of the biomass in their habitats (Schönknecht et al. 2013, Science 339:1207).
Cyanidiophyceae have been reported to live in volcanic areas and rivers around mining areas where contains iron over several hundred mM (Baker et al. 2004, AEM 70:6264) because high temperature and acidity in such areas promote the leaching of metals from the environment. In the laboratory, it has been reported to grow in the presence of 200 mM Al (Yoshimura et al. 1999, Soil Sci Plant Nutr 45:721). Thus, Cyanidiophyceae exhibits a highly metal tolerance that is several hundred to a thousand times higher than that of many organisms living in neutral regions.
Regarding the metal tolerance mechanism, the metal chelation of polyphosphate has been reported in Cyanidophyceae as known as in other organisms (Nagasaka et al. 2003, BioMetals 16:465). However, the behavior of metals under acidic environment is completely different from that in neutral pH in their chemical form and their adsorption kinetics, suggesting the existence of unknown metal tolerance mechanisms in addition to the known mechanisms. Whole-genome sequencing suggests that horizontal gene transfer from archaea and bacteria facilitates the adaptation to extreme environments (Schönknecht et al. 2013), but the functions of the genes acquired by horizontal gene transfer are mostly unknown. The mechanism which enable G. sulphuraria to survive under the extraordinary metal stress has not been elucidated. In this study, we focused on iron which is essential but toxic at high concentration and aimed to clarify the tolerance mechanism to excess iron in G. sulphuraria.
Results and Discussions
At first, the growth of G. sulphuraria were examined in the normal culture media (2x Allen’s medium) containing 0.03, 0.3, 3, 30, 100, and 300 mM FeSO4. The results showed that the growth rate and final OD of G. sulphuraria were similar to those of the normal culture conditions up to 100 mM, whereas G. sulphuraria did not grow at 300 mM. Since the addition of 300 mM H2SO4 does not affect the growth of G. sulphuraria, the growth inhibition observed under 300 mM FeSO4 would be due to the excess amounts of Fe.
The Fe concentration of the culture supernatant dropped sharply to 0.3 ± 0.1 mM between 1 and 2 days after the addition of 3 mM FeSO4, while after the addition of 100 mM FeSO4, the Fe concentration in the culture supernatant after 2 days was kept as 98 ± 3 mM and decreased to 77 ± 3 mM on the 6th day as increasing the number of the cells. These results suggest the two different mechanisms underlying the growth in the presence of 3 mM and 100 mM FeSO4. After the addition of 3 mM FeSO4, cells absorbed Fe in the culture medium within two days, lowering the extracellular Fe concentration and maintaining the growth similar to the normal growth conditions. In the presence of 100 mM FeSO4, cells seemed to absorb Fe for 6 days, but the extracellular Fe concentration remained high even if they absorbed Fe in the medium. Therefore, cells will need a mechanism that allows them to maintain the growth in the addition of Fe absorbing mechanism in the presence of 100 mM Fe.
Microscopic observation showed the extracellular structure after the addition of more than 3 mM FeSO4. This structure was stained with several lectins-FITC, including ConA, suggesting that they contain polysaccharides. The extracellular structures started to form within 6 hours after the addition of 100 mM FeSO4. In order to understand the tolerance mechanism to excess amounts of Fe, including the formation of extracellular structures, we are currently conducting mRNA-seq analysis using RNA sampling at 0, 1, 3, 6, 24, and 48 hours after the addition of 100 mM FeSO4.