Regulatory roles for IscA and SufA in iron homeostasis and redox stress responses in the cyanobacterium Synechococcus sp. strain PCC 7002

Abstract

SufA, IscA, and Nfu have been proposed to function as scaffolds in the assembly of Fe/S clusters in bacteria. To investigate the roles of these proteins further, single and double null-mutant strains of Synechococcus sp. strain PCC 7002 were constructed by insertional inactivation of genes homologous to sufA, iscA, and nfu. Demonstrating the nonessential nature of their products, the sufA, iscA, and sufA iscA mutants grew photoautotrophically with doubling times that were similar to the wild type under standard growth conditions. In contrast, attempts to inactivate the nfu gene only resulted in stable merodiploids. These results imply that Nfu, but not SufA or IscA, is the essential Fe/S scaffold protein in cyanobacteria. When cells were grown under iron-limiting conditions, the iscA and sufA mutant strains exhibited less chlorosis than the wild type. Under iron-sufficient growth conditions, isiA transcript levels, a marker for iron limitation in cyanobacteria, as well as transcript levels of genes in both the suf and isc regulons were significantly higher in the iscA mutant than in the wild type. Under photosynthesis-induced redox stress conditions, the transcript levels of the suf genes are notably higher in the sufA and the sufA iscA mutants than in the wild type. The growth phenotypes and mRNA abundance patterns of the mutant strains contradict the proposed scaffold function for the SufA and IscA proteins in generalized Fe/S cluster assembly and instead suggest that they play regulatory roles in iron homeostasis and the sensing of redox stress in cyanobacteria.

FIG. 1.

Constructions for attempted inactivation of the sufA, iscA, and nfu genes and PCR analysis of transformants. (A) Physical map showing insertional inactivation of the sufA gene by a kanamycin resistance cartridge (aphII) and verification of transformants by PCR analysis. Arrows indicate gene orientations and primers used to check for segregation of alleles. Lane 1, wild type; lane 2, sufA mutant; lane 3, iscA mutant; and lane 4, sufA iscA mutant. (B) Physical map showing insertional inactivation of the iscA gene with a gentamicin resistance cartridge (aacC1) and verification of transformants by PCR analysis. Lanes are the same as for panel A. (C) Physical map showing the construction for the deletion of nfu and PCR analysis of transformants. PCR primers P3 and P4 were used to amplify the nfu loci in DNA isolated from the wild type (lane 1), a nfu transformant in the wild-type background (lane 2), and an nfu transformant in a PS I-less (ΔpsaAB) background (lane 3).

FIG. 2.

Sequence alignment of SufA and IscA proteins from cyanobacterium Synechococcus sp. strain PCC 7002, Synechocystis sp. strain PCC 6803, Arabidopsis thaliana, Escherichia coli, Erwinia chrysanthemi (SufA), and Azotobacter vinelandii (IscA). The conserved cysteine residues are highlighted and numbered C1 through C5 (2).

FIG. 3.

Expression profile of the genes in the suf and isc regulons. RT-PCR was performed on RNA isolated from the wild type (WT) (lane 1), the sufA mutant (lane 2), the iscA mutant (lane 3), and the sufA iscA mutant (lane 4), cells grown under normal photoautotrophic growth condition. The rimM gene serves as the loading control.

FIG. 4.

Comparison of expression profiles of the genes in the suf and isc regulons in the wild-type and mutant strains when cells were grown under (lanes 1) normal photoautotrophic growth conditions and (lanes 2) iron-limiting photoautotrophic growth conditions. The rimM gene serves as the loading control.

FIG. 5.

Comparison of expression profiles of the genes in the suf and isc regulons in the wild-type and mutant strains when cells were grown under (lanes 1) normal photoautotrophic growth conditions and (lanes 2) redox stress conditions induced by addition of methyl viologen. The rimM gene serves as the loading control.

FIG. 6.

A model for the regulation and assembly of Fe/S clusters in cyanobacteria. Solid lines indicate known processes, while dashed lines indicate proposed processes for the model. The SUF system plays an important role in the Fe/S cluster assembly. SufS and SufE mediate the release of sulfur from cysteine. SufB, SufC, and SufD may play a role in the iron mobilization and Fe/S cluster formation. The Nfu protein may function as a scaffold protein and plays an essential role in the transient assembly and delivery of Fe/S clusters to target proteins. We propose that Fe/S cluster homeostasis in the cell is maintained by a mechanism that involves the assembly of Fe/S clusters on SufA and IscA for the purpose of sensing and transmitting information about the cluster status of the cell to regulatory proteins like SufR. apo, protein with no Fe/S clusters; SH, reduced form of cysteine.