A rapid procedure for selective enrichment of photosynthetic electron transport mutants.

Abstract

Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) is shown to be effective for the selective enrichment of mutants of Chlamydomonas reinhardtii that possess impaired photosynthetic electron transport. More than 99.91% of -type cells are killed when incubated in the presence of 6-10 mM metronidazole for 24 hr under illumination of 7500 lux. Survival of wild-type cells in darkness and of mutants that are blocked at different steps in photosynthetic electron transport is nearly 100% when incubated in the presence of the drug under identical conditions.The toxicity of metronidazole is demonstrated to depend upon its reduction by photosynthetic electron transport. Light-dependent oxygen utake mediated by metronidazole is shown to require active photosystem I in vitro and in vivo. Ferredoxin is necessary for metronidazole reduction by thylakoid membrane fractions enriched in photosystem I activity. We propose that the toxicity of metronidazole is due to the formation of lethal derivatives of the drug or to the accumulation of hydrogen peroxide, which could occur upon autooxidation of metronidazole reduced by one electron. Preparation of Thylakoid Membrane Fractions. Thylakoid membranes were purified as previously described (S). For assays of photosynthetic electron transport activities, the thylakoids were suspended in 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes)-KOH (pH 7.5).Preparation of PS I Particles. Preparations of thylakoid membrane particles enriched in PS I activities were obtained by digitonin treatment of crude membranes (10). Wild-type cells (8 X 108) were harvested by centrifugation at 2500 X g and washed with 10 mM potassium phosphate (pH 7.0)/20 mM KCI/2.5 mM MgCl2. Washed cells were resuspended in 40 ml of the same buffer and were disrupted at 00 by sonication for 1 min with a Heat-System Ultrasonics sonifier. Pellets obtained by centrifugation at 48,000 X gma. for 15 min were resuspended in 15 ml of the buffer and homogenized with a motor-driven Teflon pestle. The thylakoids (300 ,g of chlorophyll per ml) were treated with 0.5% (wt/vol) RESULTSMetronidazole (2-methyl-5-nitroimidazole-1-ethanol) is widely used clinically for the treatment of infections of anaerobic bacteria and protozoa (11). It is a nitroheterocyclic compound possessing a low redox potential (-325 mV at pH 6.9) (12). The toxicity of metronidazole is thought to depend upon reduction 610

Acclimation to Singlet Oxygen Stress in Chlamydomonas reinhardtii

“…Interestingly, pretreatment with RB increased methyl viologen sensitivity but had no impact on sensitivity to metronidazole. This could be related to differences in chemical structure or sites of action between these two compounds (70). The experiment was also reversed, this time pretreating with neutral red, hydrogen peroxide, methyl viologen, metronidazole, and tert-butyl hydroperoxide, and challenging with RB.…”

“…1A). We tested hydrogen peroxide, tert-butyl hydroperoxide (an organic peroxide capable of triggering lipid peroxidation chain reactions), metronidazole (a compound which generates superoxide only in the chloroplast) (70), and RB (a xanthene dye that produces 1 O 2 ‫ء‬ upon excitation by light) for the ability to induce an acclimation response. The response to RB was the most dramatic, with a significant proportion of pretreated cells surviving concentrations that killed all unpretreated cells (Fig.…”

“…The arrays used in our analysis cover approximately 20% of the genome. Extrapolating from these results to the full genome yields an estimated 70 1 O 2 ‫-ء‬regulated genes in the C. reinhardtii genome.…”

Chloroplast RNA Stability in Chlamydomonas: Rapid Degradation of psbB and psbC Transcripts in Two Nuclear Mutants.

“…High fluorescent colonies of C. reinhardtii were obtained following UV treatment and enrichment for photosynthetic electron transport mutants with metronidazole (Schmidt et al, 1977). To identify strains with defects in photosystem II biogenesis, the isolates were characterized by pulse labeling cells with 35SO$-in the presence of cycloheximide.…”

The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

“…This drug is specifically reduced by low-potential electron donors, such as ferredoxins and flavodoxins, and the reduced derivatives are toxic to cells (44). Previous reports have shown that metronidazole enrichment for electron transport mutants can be successful (21,46).…”