The sufR Gene (sll0088 in Synechocystis sp. Strain PCC 6803) Functions as a Repressor of the sufBCDS Operon in Iron-Sulfur Cluster Biogenesis in Cyanobacteria

The biogenesis and assembly of fully functional photosystem I (PS I) require the assembly of FX, FA, and FB, which are [4Fe-4S] clusters, and soluble ferredoxin, which contains a [2Fe-2S] cluster. Iron-sulfur clusters can be assembled and inserted into proteins in vitro by incubating the apoprotein with iron, sulfide, and a thiol-containing reducing agent, such as 2-mercaptoethanol (22). This approach has been used in conjunction with bacterial expression systems to elucidate the structure and function of a variety of photosynthetic iron-sulfur proteins, including PsaC (reviewed in reference 11). In contrast, the in vivo biosynthesis of iron-sulfur clusters in photosynthetic complexes involves biochemical assembly processes that are poorly characterized. One known factor is a membrane-bound rubredoxin, which appears to be associated exclusively with the assembly of the FX iron-sulfur cluster (33, 34). Another known factor is the open reading frame sll0088 in Synechocystis sp. strain PCC 6803, which appears to have a role in regulating the biogenesis of PS I (43) and is the topic of this study. In nonphotosynthetic organisms, iron-sulfur cluster assembly is known to be a multistep process that involves cluster biosynthesis, insertion, and stabilization (9). The isc operon is implicated in generalized iron-sulfur cluster assembly in many organisms, including Azotobacter vinelandii (45), Escherichia coli (25), and Saccharomyces cerevisiae (31). Homologs of several genes in the isc operon, including iscS and iscA, have been identified in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 (16, 17). In fact, three distinct iscS-like homologs have been identified in genomes of Synechocystis sp. strain PCC 6803 (16, 18), Anabaena sp. strain PCC 7120 (15, 26), and Synechococcus sp. strain PCC 7002 (J. Zhao, T. Li, J. Marquardt, and D. A. Bryant, unpublished data). The presence of multiple iscS homologs hints at different functions or regulatory mechanisms in the biosynthesis of iron-sulfur clusters. Two additional iron-sulfur cluster assembly systems that are more specialized in function, nif and suf, exist in bacteria. The nif operon in A. vinelandii is involved in the biosynthesis of the nitrogenase iron-molybdenum cofactor (8). The suf operon has been shown to function in the assembly of iron-sulfur clusters under conditions of oxidative stress (24, 41). It is known that at least two of these systems, isc and suf, exist in mitochondria (31, 39) and in the chloroplasts of higher plants (20, 28, 40), respectively. However, virtually nothing is known concerning their regulation. As reported previously (43), a methodology of selecting suppressors to primary mutations in iron-sulfur proteins was used to isolate spontaneous suppressors from psaC site-directed mutants of Synechocystis sp. strain PCC 6803. The phenotypes of the C51DPsaC (FB) and C14SPsaC (FA) mutants of PsaC were such that the strains failed to grow photoautotrophically, yet electron throughput from cytochrome c6 to flavodoxin in isolated PS I complexes was similar to that of the wild type (14, 44). The mutants were sensitive to high light intensities and could not grow photomixotrophically under white-light intensities in the range from 20 to 60 μE m−2 s−1. The mutants had two additional phenotypes: the amount of PS I, but not PS II, was lower in the mutants than in the wild type on a per cell basis, and the mutants were able to grow photomixotrophically at a light intensity of 20 to 60 μE m−2 s−1 only in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of PS II. Thus, the failure to grow photoautotrophically was apparently due to the lowered ratio of PS I to PS II in the mutant cells rather than an inefficiency in forward electron transfer in any individual PS I complex. Screening the C51DPsaC and C14SPsaC mutants under high-light intensity resulted in the appearance of suppressor mutants. Two of the intergenic suppressor strains, C14SPsaC-R18 and C14SPsaC-R62, which were capable of photoautotrophic growth at normal light intensities, were selected for further study. These two suppressor strains retained the primary mutation, which was verified through amplification of the psaC gene fragment by PCR and sequencing. The suppressor mutations were mapped to a specific gene, sll0088, by phenotypic complementation and identified by DNA sequencing (43). To elucidate the function of the sll0088 gene homologs in cyanobacteria, we sought to characterize the physical properties of the protein encoded by this gene, to investigate the regulation of the sufBCDS operon, and to study the physiology of a sll0088 null mutant. We show that the protein encoded by sll0088 is an iron-sulfur protein that functions as a transcriptional repressor, regulating the expression of the sufBCDS operon. Thus, it is similar in function to IscR, which is an iron-sulfur protein that functions as a transcriptional repressor of the isc operon (32).