Nitrogen fixation, along with photosynthesis is the basis of all life on earth. Current understanding suggests that no plant fixes its own nitrogen. Some plants （mainly legumes） fix nitrogen via symbiotic anaerobic ...Nitrogen fixation, along with photosynthesis is the basis of all life on earth. Current understanding suggests that no plant fixes its own nitrogen. Some plants （mainly legumes） fix nitrogen via symbiotic anaerobic microorganisms （mainly rhizobia）. The nature of biological nitrogen fixation is that the dinitrogenase catalyzes the reaction-splitting triple-bond inert atmospheric nitrogen （N2） into organic ammonia molecule （NH3）. All known nitrogenases are found to be prokaryotic, multi-complex and normally oxygen liable. Not surprisingly, the engineering of autonomous nitrogen-fixing plants would be a long-term effort because it requires the assembly of a complex enzyme and provision of anaerobic conditions. However, in the light of evolving protein catalysts, the anaerobic enzyme has almost certainly been replaced in many reactions by the more efficient and irreversible aerobic version that uses O2. On the other hand, nature has shown numerous examples of evolutionary convergence where an enzyme catalyzing a highly specific, O2-requiring reaction has an oxygen-independent counterpart, able to carry out the same reaction under anoxic conditions. In this review, I attempt to take the reader on a simplified journey from conventional nitrogenase complex to a possible simplified version of a yet to be discovered light-utilizing nitrogenase.展开更多
The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein pro- file changes ...The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein pro- file changes to facilitate the cells differentially propagate in different modes has not been comprehensively investigated. Using isobaric labeling-based quantitative proteomics, we simultaneously identified and quantified 45% Synechocystis proteome across four different trophic modes, i.e., autotrophic, heterotro- phic, photoheterotrophic, and mixotrophic modes. Among the 155 proteins that are differentially expressed across four trophic modes, proteins involved in nitrogen assimilation and light-independent chlorophyll synthesis are dramatically upregulated in the mixotrophic mode, concomitant with a dramatic increase of PII phosphorylation that senses carbon and nitrogen assimilation status. Moreover, functional study us- ing a mutant defective in light-independent chlorophyll synthesis revealed that this pathway is important for chlorophyll accumulation under a cycled light/dark illumination regime, a condition mimicking day/night cycles in certain natural habitats. Collectively, these results provide the most comprehensive information on trophic mode-dependent protein expression in cyanobacterium, and reveal the functional significance of light-independent chlorophyll synthesis in trophic growth.展开更多
PCR amplified ORF 469 fragment from Synechocystis sp . PCC 6803 was cloned into pUC118 and a construct was made in which part of ORF 469 was deleted and replaced by erythromycin resistance cassette. Tran...PCR amplified ORF 469 fragment from Synechocystis sp . PCC 6803 was cloned into pUC118 and a construct was made in which part of ORF 469 was deleted and replaced by erythromycin resistance cassette. Transformation of wild type strain of Synechocystis sp . PCC 6803 with this construct yielded a mutant in which ORF 469 was deleted. In the resulting mutant, the light independent pathway of chlorophyll biosynthesis was inactivated and availability of chlorophyll was fully dependent on light. When propagated the mutant in dark, the chlorophyll was non detectable and protochlorophyllide with 645?nm fluorescence emission peak was accumulated. Meanwhile, the fluorescence emission peaks (excited at 435?nm) of thylakoids at 685?nm, 695?nm and 725?nm, which represented relative chlorophyll\|binding proteins, disappeared. Upon return of dark\|grown ORF 469 mutant to the light, greening occurred and chlorophyll was synthesized to assembly fluorescence emission components in photosystems. Newly synthesized chlorophyll combined the fluorescence component of 685?nm at first, then 725?nm and 695?nm at last, which indicates a pecking order for biogenesis of chlorophyll binding proteins when availability of chlorophyll is limited. The mutant lacking ORF 469 in Synechocystis sp . PCC 6803 was suggested as an excellent cyanobacterial system for studies on the interactions between chlorophyll and chlorophyll binding proteins in photosystems. 展开更多
文摘Nitrogen fixation, along with photosynthesis is the basis of all life on earth. Current understanding suggests that no plant fixes its own nitrogen. Some plants （mainly legumes） fix nitrogen via symbiotic anaerobic microorganisms （mainly rhizobia）. The nature of biological nitrogen fixation is that the dinitrogenase catalyzes the reaction-splitting triple-bond inert atmospheric nitrogen （N2） into organic ammonia molecule （NH3）. All known nitrogenases are found to be prokaryotic, multi-complex and normally oxygen liable. Not surprisingly, the engineering of autonomous nitrogen-fixing plants would be a long-term effort because it requires the assembly of a complex enzyme and provision of anaerobic conditions. However, in the light of evolving protein catalysts, the anaerobic enzyme has almost certainly been replaced in many reactions by the more efficient and irreversible aerobic version that uses O2. On the other hand, nature has shown numerous examples of evolutionary convergence where an enzyme catalyzing a highly specific, O2-requiring reaction has an oxygen-independent counterpart, able to carry out the same reaction under anoxic conditions. In this review, I attempt to take the reader on a simplified journey from conventional nitrogenase complex to a possible simplified version of a yet to be discovered light-utilizing nitrogenase.
文摘The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein pro- file changes to facilitate the cells differentially propagate in different modes has not been comprehensively investigated. Using isobaric labeling-based quantitative proteomics, we simultaneously identified and quantified 45% Synechocystis proteome across four different trophic modes, i.e., autotrophic, heterotro- phic, photoheterotrophic, and mixotrophic modes. Among the 155 proteins that are differentially expressed across four trophic modes, proteins involved in nitrogen assimilation and light-independent chlorophyll synthesis are dramatically upregulated in the mixotrophic mode, concomitant with a dramatic increase of PII phosphorylation that senses carbon and nitrogen assimilation status. Moreover, functional study us- ing a mutant defective in light-independent chlorophyll synthesis revealed that this pathway is important for chlorophyll accumulation under a cycled light/dark illumination regime, a condition mimicking day/night cycles in certain natural habitats. Collectively, these results provide the most comprehensive information on trophic mode-dependent protein expression in cyanobacterium, and reveal the functional significance of light-independent chlorophyll synthesis in trophic growth.
基金the National Nature Science Foundation of China !(No. 39870 0 6 4and 495 2 5 2 0 5 )partly by Na tional Nature Science
文摘PCR amplified ORF 469 fragment from Synechocystis sp . PCC 6803 was cloned into pUC118 and a construct was made in which part of ORF 469 was deleted and replaced by erythromycin resistance cassette. Transformation of wild type strain of Synechocystis sp . PCC 6803 with this construct yielded a mutant in which ORF 469 was deleted. In the resulting mutant, the light independent pathway of chlorophyll biosynthesis was inactivated and availability of chlorophyll was fully dependent on light. When propagated the mutant in dark, the chlorophyll was non detectable and protochlorophyllide with 645?nm fluorescence emission peak was accumulated. Meanwhile, the fluorescence emission peaks (excited at 435?nm) of thylakoids at 685?nm, 695?nm and 725?nm, which represented relative chlorophyll\|binding proteins, disappeared. Upon return of dark\|grown ORF 469 mutant to the light, greening occurred and chlorophyll was synthesized to assembly fluorescence emission components in photosystems. Newly synthesized chlorophyll combined the fluorescence component of 685?nm at first, then 725?nm and 695?nm at last, which indicates a pecking order for biogenesis of chlorophyll binding proteins when availability of chlorophyll is limited. The mutant lacking ORF 469 in Synechocystis sp . PCC 6803 was suggested as an excellent cyanobacterial system for studies on the interactions between chlorophyll and chlorophyll binding proteins in photosystems.
