Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function |
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| Authors: | Wim F. J. Vermaas Gaozhong Shen Itzhak Ohad |
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| Affiliation: | (1) Department of Botany, Arizona State University, 85287-1601 Tempe, AZ, USA;(2) Center for the Study of Early Events in Photosynthesis, Arizona State University, 85287-1601 Tempe, AZ, USA;(3) Department of Biological Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel;(4) Present address: Department of Molecular and Cell Biology, Pennsylvania State University, 16802 University Park, PA, USA |
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| Abstract: | Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of QB–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Fv variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N -(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - QA primary electron-accepting plastoquinone in Photosystem II - QB secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate |
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| Keywords: | gene replacement Photosystem II photosynthesis thylakoid membranes transformation |
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