Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling.

Abstract

The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedl gene (ssI0020) 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 (sIr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (sIr0150, negative control) or glucose availability (sII1382). fedl was found to be critical to Synechocystis PCC6803 viability in spite of sIr0150, sII1382 or flavodoxin induction, even after the addition of glucose 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. This strain was used as the host for the subsequent introduction of fedl mutant alleles propagated on a second vector. Analysis of the fedl 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 Fedl 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.