Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium

Abstract

Fully deuterated components from autotrophic cell lysate are useful materials for labeling of heterotrophs with deuterium. To facilitate the faster production of deuterated algal lysate, we selected a mutant Chlorella strain that grows faster in heavy water than the wild type. The mutant DR-17 was found to have a higher level of Hsp60 and an elevated level of protein synthesis. We previously isolated a deuterium-resistant yeast cell line that was also found to express elevated level of Hsp70 (K. Unno, T. Kishido, M. Morioka, S. Okada, and N. Oku, Biol. Pharm. Bull. 26:799-802, 2003). This suggests that the overexpression of heat shock proteins is required to compensate for the deuterium isotope effect.Stable isotope labeling is an essential tool for studying the functional structure and assembly of proteins by nuclear magnetic resonance (NMR). While high deuteration levels of nonexchangeable sites in a protein are beneficial for main chain assignment, the complete exchange of D in exchangeable sites to H is required for proton detection. If amide protons involved in strong hydrogen bonds or buried inside the protein are not accessible to H in the solvent, the amides will remain in the deuterated form. Although this problem may be resolved by unfolding the protein using chemical denaturants followed by refolding in the presence of H 2 O, complete exchange and refolding is not always possible.The expression of proteins in fully deuterated algal lysate medium in 100% H 2 O has recently been described (6). Using this technique, deuterated samples were uniformly protonated at their amide sites, irrespective of the solvent exchange characteristics of the folded protein. We isolated a mutant clone to facilitate the production of deuterated growth media from algal Chlorella. 14 C-␣ aminoisobutyric acid (␣-AIB), which is an unavailable amino acid for protein synthesis, in 60% D 2 O medium at 25°C. The relative radioactivity of each precursor was 18.5 kBq/0.2 M/ml. The incubated cells were washed with icecold water. The incorporation of amino acid into cells was determined from the radioactivity of cells using a liquid scintillation counter (LSC). That of nucleic acids was determined from the radioactivity in the ethanol-insoluble fraction of cells. MATERIALS AND METHODS PreparationThe effect of D 2 O on protein synthesis was investigated using 35 S-L-methionine ( 35 S-Met; 185 kBq/0.2 M/ml) in 60 or 100% D 2 O medium. Labeled cells (2 ϫ 10 8 cells) were homogenized with glass beads. The homogenate was centrifuged at 200 ϫ g for 10 min and then at 9,000 ϫ g for 30 min, and the supernatant was applied to a Sephadex G-25 column (PD-10; Pharmacia Biotech). The radioactivity and protein concentration of these fractions were measured using LSC and a protein assay kit (Bio-Rad), respectively.Carbon fixation and amount of photosynthetic pigments. Fixation of 14 C was measured as described previously (13). Briefly, Chlorella cells (2 ϫ 10 7 cells/190 l) were preilluminated with 2.5 klx for 3 min at 25°C, and then 10 l of 10 mM Na...

To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii

“…The data presented show a decreased growth rate for cultures grown in 70% D2O or 90% D2O (Figure 2A-C). Similar findings were reported for Chlorella [16,35], Scenedesmus [36], Dunaliella [37], and Spirulina [38], as well as for Chlamydomonas itself [39]. Even though most reports show a concentrationdependent relationship between decreased growth rate of the culture and increased deuterium concentration, some studies report a stimulating effect of intermediate D2O concentrations (usually around 70% D2O) on observed growth rates [36].…”

Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription

“…After all, the cell has a wide range of tools available to minimize the effect, in particular DNA/RNA proofing and repair mechanisms, while the T7 polymerase used in the cell-free assay is unable to perform corrections. For a mutant Chlorella algae it was recently shown [ 39 ] that gene expression becomes less sensitive to the presence of high D 2 O concentrations by the overexpression of certain heat shock proteins (Hsp60 and Hsp70). This suggests that misfolding and prolonged maturation of proteins may become a more important factor after repair mechanisms at the transcription and translation level kicked in.…”

Directed microbial biosynthesis of deuterated biosurfactants and potential future application to other bioactive molecules

“…Methods were subsequently developed to adapt or 'train' organisms to increasing levels of deuterated substrates in an effort to produce labelled products (Paliy et al 2003;Newo et al 2004). An alternative approach was employed with Chlorella vulgaris using alkylating agents to produce mutant strains which were able to tolerate deuterium (Unno et al 2005) More recent work has focussed on trying to manipulate deuterium labelling in polyhydroxyoctanoate produced by Pseudomonas oleovorans for neutron scattering studies (Foster et al 2006;Russell et al 2007;Holden et al 2008). Overall, interest in producing deuterated metabolites in microorganisms has been limited since their only application appeared to be for basic research studies (Myles 2006), but this is an area of increasing interest.…”