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Targeted deletion and mutational analysis of the essential (2Fe–2S) plant‐like ferredoxin in Synechocystis PCC6803 by plasmid shuffling
Author(s) -
Poncelet Mireille,
CassierChauvat Corinne,
Leschelle Xavier,
Bottin Hervé,
Chauvat Franck
Publication year - 1998
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1998.00844.x
Subject(s) - biology , plasmid , ferredoxin , synechocystis , gene , mutant , genetics , replicon , microbiology and biotechnology , biochemistry , enzyme
The genes encoding (2Fe–2S) plant‐like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedI gene (ssl0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light‐induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150, negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150, sll1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange showed that the C18–C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the FedI carboxy terminus is important for effective PSI/FedI interactions. The plasmid‐shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.