Progress 10/01/02 to 09/30/03
Outputs
In the first of two publications (Yu et al., 2003) we reported the identification of a gene that regulates the amount of PS I in cyanobacterial cells. We uncovered this gene as a second-site suppressor to mutations involving the Cys ligands to the FA/FB clusters in PsaC. The C14SPsaC primary mutant strain was unable to grow photoautotrophically owing to suppressed levels of PS I. We isolated suppressor strains (described in an earlier progress report) that were capable of photoautotrophic growth at moderate light intensity (20 microE m-1 s-2). Two separate strains of C14SPsaC, termed C14SPsaC-R62 and C14SPsaC-R18, were found to have mutations in a previously uncharacterized open reading frame of the Synechocystis sp. strain PCC 6803 genome named sll0088. C14SPsaC-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482-->C change, and C14SPsaC-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys207 as the result of a
TGGTTATTT duplication at T690. These suppressor strains showed near-wild-type levels of Chl a and PS I. We proposed that the two mutations `broke' the function of the Sll0088 protein, thereby turning on genes that elevate the level of PS I in the mutant cells. In our second publication (Wang et al., 2004) we reported that the sll0088 gene functions as a transcriptional repressor of the adjacent suf operon. The suf operon was first described in E. coli in 2002, and based on sequence similarity, we predicted that that the adjacent sll0074, sll0075, sll0076 and sl0077 genes in Synechocystis sp. PCC 6803 were homologous to sufB, sufC, sufD and sufS of E. coli. The sll0088 gene in Synechocystis sp. PCC 6803 is located near the 5' end of the suf operon, but is divergently transcribed from it. The protein encoded by sll0088 has two significant features: a DNA-binding domain, and four highly conserved cysteine residues. The protein has high sequence similarity to transcription regulatory
proteins with a conserved DNA binding domain and can be classified in the DeoR family of helix-loop-helix proteins. The protein falls into a further subclass that contains a C-(X)12-C-(X)13-C-(X)14-C motif near the C-terminus, which binds an Fe/S cluster as shown by optical and EPR spectroscopy. Compared to the wild type, expression levels of the sufBCDS genes were elevated when cells were grown under conditions of oxidative and iron stress, and were even more elevated in a null mutant of Synechococcus sp. PCC 7002 in which the sll0088 homolog was insertionally inactivated. In agreement with the proposed role of the sufBCDS genes in iron metabolism, the sll0088 deletion mutant exhibited a significantly faster growth rate than the wild type under iron-limiting conditions. We proposed that the de-repression of the suf operon leads to a higher rate of Fe/S cluster biosynthesis, thereby explaining the higher PS I content of the C14SPsaC-R62 and C14SPsaC-R18 mutants. We have renamed
sll0088 gene sufR; it the first instance of a specific transcriptional regulator of the suf operon in any organism.
Impacts
We have identified the transcriptional regulator of the suf operon, which codes for proteins involved in Fe/S cluster assembly, including the FX, FB and FA clusters in Photosystem I. These genes are involked during periods of oxidative stress. A detailed understanding of the regulation of the biogenesis of reaction centers and their cofactors, as well as the impact of oxidative stress, is necessary for attempts to increase the efficiency of photosynthesis in plants.
Publications
- Yu J, Shen G, Wang T, Bryant DA, Golbeck JH, McIntosh L (2003) `Suppressor mutations in the study of Photosystem I biogenesis: sll0088 is a previously unidentified gene involved in reaction center accumulation in Synechocystis sp. strain PCC 6803'. J Bacteriol, 185, 3878-3887.
- Wang T, Shen G, Balasubramanian R, McIntosh L, Bryant, D. A., Golbeck JH (2004) `The sufR gene (sll0088) in Synechocystis sp. PCC 6803 functions as a repressor for the expression of the sufBCDS operon in Fe/S cluster biogenesis in cyanobacteria. J Bacteriol, 186, 956-967.
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Progress 10/01/00 to 09/30/01
Outputs
A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of the Synechocystis sp. PCC 6803 gene slr2034 and Arabidopsis thaliana HCF136, and the psbEFLJ operon, was cloned from the cyanobacterium Synechococcus sp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type Photosystem (PS) II levels. Inactivation of the rubA gene of Synechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was 80% of the wild-type level. RubA contains a domain of 50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane _-helix at its C-terminus. Overproduction of the water-soluble rubredoxin domain in Escherichia coli led to a product
with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type (40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis or stabilization of PS I. The properties of Photosystem I complexes were characterized spectroscopically after insertionally
inactivating the rubA gene in Synechococcus sp. PCC 7002. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, FX, FA and FB, are missing in whole cells, thylakoids, and Photosystem I (PS I) complexes of the rubA mutant. The flash-induced decay kinetics of both P700+ in the visible and A1- in the near-UV show that charge recombination occurs between P700+ and A1- in both thylakoids and PS I complexes. In agreement, the spin-polarized X-band EPR spectrum of P700+ A1- at low temperature shows that cyclic electron transfer between A1- and P700+ occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a fraction of the electrons promoted are irreversibly transferred to [FA/FB]. In contrast to the loss of FX, FB and FA, the Rieske iron-sulfur protein and the non-heme iron in Photosystem II are intact. We propose that rubredoxin is specifically required for the assembly of the FX iron-sulfur cluster but that FX is not
required for the biosynthesis of trimeric P700-A1 cores. Since the PsaC protein requires the presence of FX for binding, the absence of FA and FB may be an indirect result of the absence of FX.
Impacts
We have discovered a gene that codes for a rubredoxin protein that is involved in the assembly of the FX iron-sulfur cluster in photosystem I. Photosystem I is one of the two reaction centers that convert sunlight into chemical energy in plants. Rubredoxins are small, non-heme iron proteins that have been known and studied for decades. In spite of the fact that they are present in all organisms whose genomes have been sequenced, there is very little knowledge of their cellular functions. The importance of this discovery is that among the dozens of iron-sulfur proteins in the cell, the FX cluster, and only the FX cluster, fails to assemble when the gene that codes for rubredoxin is deleted. If the FX cluster is not present, Photosystem I cannot carry out photosynthesis. Hence, for the first time, a specific role is assigned to a plant rubredoxin. The long-term impact of this finding is that manipulation of the genes that carry out photosynthesis should allow us to
modify the photosynthetic reaction centers so that they an be more efficient at converting sunlight to chemical energy in plants. The discovery of all of the genes involved in assembling and degrading the photosynthetic reaction centers is the first step in being able to control their function. Since all of the foodstuffs and many of the natural fibers used by mankind are produced ultimately from plants, it is in our long-term interest to uncover those genes that allow high levels of photosynthetic reaction centers to be maintained in plants.
Publications
- Shen, G., Zhao, J., Reimer, S., Cai, Q., Golbeck, J. H. and Bryant, D. A. (2000) `Assembly of [4Fe-4S] Clusters in Photosystem I. I. Inactivation of the Gene Encoding a membrane-Associated Rubredoxin in the Cyanobacterium Synechococcus sp. PCC 7002' , J. Biol. Chem. (in review).
- Shen, G., Antonkine, M. L., van der Est, A. J., Vassiliev, I. R., Brettel, K., Zhao, J. Stehlik, D., Bryant, D. and Golbeck, J. H. (2000) `Assembly of [4Fe-4S] Clusters in Photosystem I. II. Rubredoxin Is Required for Assembly of FX as Shown by Optical and EPR Spectroscopy' J. Biol. Chem. (in review).
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