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.
In previous work, some members of our group isolated mutant strains of Synechocystis sp. strain PCC 6803 in which point mutations had been inserted into the psaC gene to alter the cysteine residues to the F(A) and F(B) iron-sulfur clusters in the PsaC subunit of photosystem I (J. P. Yu, I. R. Vassiliev, Y. S. Jung, J. H. Golbeck, and L. McIntosh, J. Biol. Chem. 272:8032-8039, 1997). These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll a and photosystem I. In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 micromol m(-2) s(-1)). Two separate suppressor strains of C14S(PsaC), termed C14S(PsaC)-R62 and C14S(PsaC)-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the Synechocystis sp. strain PCC 6803 genome named sll0088. C14S(PsaC)-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482-->C change in sll0088, and C14S(PsaC)-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in sll0088. These suppressor strains showed near-wild-type levels of chlorophyll a and photosystem I, yet the serine oxygen ligand to F(B) was retained as shown by the retention of the S > or = 3/2 spin state of the [4Fe-4S] cluster. The inactivation of sll0088 by insertion of a kanamycin resistance cartridge in the primary C14S(PsaC) mutant produced an engineered suppressor strain capable of photoautotrophic growth. There was no difference in psaC gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an sll0088 deletion mutant. The sll0088 gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms, including Arabidopsis thaliana. The protein contains a typical helix-turn-helix DNA-binding motif and can be classified as a negative regulator by phylogenetic analysis. This suggests that the product of sll0088 has a role in regulating the biogenesis of photosystem I.
Yu
Jianping
J
MSU-DOE Plant Research Laboratory and Biochemistry and Molecular Biology Department, Michigan State University, East Lansing, Michigan 48824, USA.
Shen
Gaozhong
G
Wang
Tao
T
Bryant
Donald A
DA
Golbeck
John H
JH
McIntosh
Lee
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013489:Suppression, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins