TARGETED DELETION AND MUTATIONAL ANALYSIS OF THE ESSENTIAL (2FE-2S) PLANT-LIKE FERREDOXIN IN SYNECHOCYSTIS PCC6803 BY PLASMID SHUFFLING

The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedI gene (ssl0 020) coding for the most abundant ferredoxin product was found to be e xpressed strongly as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828) or moderately exp ressed as polycistronic transcripts regulated by either light fluence (slr0150, negative control) or glucose availability (sll1382), fedl wa s found to be critical to Synechocystis PCC6803 viability in spite of slr0150, sll1382 or flavodoxin induction, even after the addition of g lucose that compensates for the loss of photosynthesis. Nevertheless, fedl could be deleted from all chromosome copies in cells propagating a fedl gene (even of heterologous origin) on a replicating plasmid, Th is strain was used as the host for the subsequent introduction of fedl mutant alleles propagated on a second vector. Analysis of the fedl mu tant strains generated after plasmid exchange showed that the C18-C85 disulphide bridge is not central either to the tight compaction of fer redoxin I or to its reduction by photosystem I and demonstrated that t he length of the Fedl carboxy terminus is important for effective PSI/ Fedl interactions. The plasmid-shuffling strategy presently described has general applicability for mutational analysis of essential genes i n many organisms, as it is based on promiscuous plasmids.