Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

Abstract

In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex. An insertion mutation introduced in the psaC locus of the filamentous cyanobacteriumAnabaena variabils ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide. In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark. However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased. In intact cells of T398-1, bicarbonatedependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates. The low-temperature EPR signals attributed to the [4Fe-4S] centers FA and FB were absent in the mutant cells. Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells. Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI. Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB. These studies demonstrate thatAnabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.Photosystem I (PSI) is a membrane-bound pigment-protein complex that mediates electron transfer from reduced plastocyanin and cytochrome c553 to ferredoxin. Biochemical and biophysical analyses of thylakoid membranes and isolated PSI particles from cyanobacteria and higher plants have helped in formulating the functional organization of redox intermediates involved in the transfer of electrons from P700, the reaction-center chlorophyll(s) (Chl) of PSI, to ferredoxin.Five different electron-transfer intermediates-namely, A0, Al, Fx, FA, and FE-Nare known to be involved in electron transfer from P700 to ferredoxin (reviewed in ref.

Arabidopsis thaliana Plants Lacking the PSI-D Subunit of Photosystem I Suffer Severe Photoinhibition, Have Unstable Photosystem I Complexes, and Altered Redox Homeostasis in the Chloroplast Stroma

“…In contrast, a PSI-D knock-out mutant in Synechocystis accumulated wild-type levels of PSI-A/B (9). A similar discrepancy between prokaryotes and eukaryotes has been observed in mutants lacking PSI-C. Knock-out of the psaC gene resulted in the accumulation of a non-functional PSI complex (45), whereas a corresponding Chlamydomonas mutant showed a rapid turn-over of the PSI-A/B core subunits and no accumulation of PSI (46) Interestingly, in Synechocystis, the PSI complex without PSI-D is completely inactive with ferredoxin as acceptor, but is able to reduce flavodoxin at low rates (47). Plants do not contain flavodoxin and therefore the lethality of a complete down-regulation is not unexpected.…”

Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function.

“…In cyanobacteria and higher plants, the psaA and psaB genes are adjacent and are cotranscribed. In cyanobacteria, targeted deletion of PSI was found to correlate with an extreme light sensitivity of the resulting mutants; this is observed both in Anabaena variabilis ATCC 29413 (Mannan et al, 1991;Toelge et al, 1991) and Synechocystis 6803 (Smart et al, 1991;Smart and Mclntosh, 1993). Thus, mutants lacking PSI were grown in darkness (as can be done for Anabaena 29413); strains such as Synechocystis 6803, which cannot be propagated in complete darkness, were propagated by light-activated heterotrophic growth (LAHG).…”

Photosystem I

“…The absence of PsaC has different effects on the assembly of the core complex in different organisms. The PSI core is unstable in the PsaC-less mutant of C. reinhardtii (Takahashi et al, 1991) but is assembled and functional in charge separation in the cyanobacterial mutants (Mannan et al, 1991;Yu et al, 199513). The mutant strains of A. vaviabilis ATCC 29413, in which cysteinyl ligands for the iron-sulfur clusters were mutated to aspartate, can grow under photoautotrophic conditions (Mannan et al, 1996).…”