Poster: Oxidative Stress

Abs # 150: SufR functions in photosystem I biogenesis as the repressor of the suf operon in cyanobacteria

Presenter:	Wang, Tao , txw199@psu.edu
Authors	Wang, Tao  (A)   Shen, Gaozhong  (A)   Balasubramanian, Rama  (A)   McIntosh, Lee  (B)   Bryant, Donald A (A)   Golbeck, John H (A)
Affiliations:	(A): Penn State University (B): Michigan State University

The ORF sll0088, which is involved in Photosystem I assembly in Synechocystis sp. PCC 6803, was inactivated in Synechococcus sp. PCC 7002 by inserting the aphII gene at the unique StuI site in the sll0088 homolog. Growth experiments with 2,2'-dipyridyl, an Fe chelator, and streptonigrin, an Fe-activated antibiotic, indicate that sll0088 functions in Fe metabolism. Moreover, the amino acid sequence suggests that the sll0088 gene product be a transcriptional repressor. There are four suf genes immediately downstream of sll0088 in Escherichia coli and Erwinia chrysanthemi; these suf gene homologues constitute an operon along with the two other identified suf genes. It has been established in several organisms that Suf proteins function in Fe/S assembly under oxidative stress conditions. RT-PCR data show that the four suf genes in Synechococcus sp. PCC 7002 co-transcribe and therefore also form an operon. Northern blot and RT-PCR data confirm the prediction that the sll0088 gene product serves as the transcriptional repressor of suf operon. sll0088 is therefore renamed sufR. Under oxidative stress conditions induced by adding DCMU, DBMIB, or H₂O₂ to the growth media, the transcription of the suf genes increases, while that of sufR remains unchanged, suggesting that oxidative signals are not sensed by sufR at the level of transcription. The SufR protein was overexpressed in E coli and purified. Fe/S cluster reconstitution was achieved by incubating the SufR apoprotein with ferrous chloride and sodium sulfide. EPR spectroscopy clearly shows a [4Fe-4S] cluster, indicating that SufR itself is an Fe/S protein. Physiologically, this [4Fe-4S] cluster may serve as the sensor of oxidative stress. Funded by USDA Award 2001-35318-10125 to JHG and LM.