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The Corynebacterium glutamicum WhcA protein, which inhibits the expression of oxidative stress response genes, is known to interact with the SpiA protein. In this study, we constructed and analyzed spiA mutant cells with the goal of better understanding the function of the spiA gene. A C. glutamicum strain overexpressing the spiA gene showed retarded cell growth, which was caused by an increased sensitivity to oxidants. Expression of the spiA and whcA genes was repressed by oxidant diamide, indicating coordinate regulation and dispensability of the genes in cells under oxidative stress. In the spiA-overexpressing cells, the trx gene, which encodes thioredoxin reductase, was severely repressed. Deletion of whcA in spiA-overexpressing cells (or vice versa) produced phenotypes similar to the wild-type strain. Collectively, these data demonstrate a negative regulatory role of the spiA gene in whcA-mediated oxidative stress response and provide additional clues on the mechanism by which the whcA gene is regulated.

The function of whcB, one of the four whiB homologues of Corynebacterium glutamicum, was assessed. Cells carrying the P(180)-whcB clone, and thus overexpressing the whcB gene, showed retarded growth, probably due to increased sensitivity to oxidants, whereas cells lacking whcB (ΔwhcB) did not. However, growth retardation was not observed in cells with additionally whcE deleted. Furthermore, the ΔwhcE phenotype, characterized by slow growth and sensitivity to oxidants, was reversed in cells carrying P(180)-whcB. Like the whcE gene, which is also known as a whiB homologue, the whcB gene was preferentially expressed in stationary phase. Determination of the genes under regulation of whcB using two-dimensional polyacrylamide gel electrophoresis identified several genes involved in electron transfer reactions that were regulated in cells carrying P(180)-whcB. Collectively, these findings indicate that whcB function requires whcE. Furthermore, whcB and whcE are paralogues but perform distinct regulatory roles during growth under oxidative stress.

The Corynebacterium glutamicum whcA gene is known to play a negative role in the expression of genes responding to oxidative stress. The encoded protein contains conserved cysteines, which likely coordinate the redox-sensitive Fe–S cluster. To identify proteins which may interact with WhcA, we employed a two-hybrid system utilizing WhcA as ‘bait’. Upon screening, several partner proteins were isolated from the C. glutamicum genomic library. Sequencing analysis of the isolated clones revealed out-of-frame peptide sequences, one of which showed high sequence homology with a dioxygenase encoded by NCgl0899. In vivo analysis of protein interaction using real-time quantitative PCR, which monitors his3 reporter gene expression, demonstrated that the interaction between NCgl0899-encoded protein and WhcA was specific. The interaction was labile to oxidants, such as diamide and menadione. Based on these data, NCgl0899 was named spiA (stress protein interacting with WhcA). Physical association and dissociation of the purified His6–WhcA and GST–SpiA fusion proteins, as assayed by in vitro pull-down experiments, were consistent with in vivo results. These data indicated that the interaction between WhcA and SpiA is not only specific but also modulated by the redox status of the cell and the functionality of the WhcA protein is probably modulated by the SpiA protein.

The Corynebacterium glutamicum whcA gene is known to play a negative role in the expression of genes responding to oxidative stress. The encoded protein contains conserved cysteines, which likely coordinate the redox-sensitive Fe-S cluster. To identify proteins which may interact with WhcA, we employed a two-hybrid system utilizing WhcA as 'bait'. Upon screening, several partner proteins were isolated from the C. glutamicum genomic library. Sequencing analysis of the isolated clones revealed out-of-frame peptide sequences, one of which showed high sequence homology with a dioxygenase encoded by NCgl0899. In vivo analysis of protein interaction using real-time quantitative PCR, which monitors his3 reporter gene expression, demonstrated that the interaction between NCgl0899-encoded protein and WhcA was specific. The interaction was labile to oxidants, such as diamide and menadione. Based on these data, NCgl0899 was named spiA (stress protein interacting with WhcA). Physical association and dissociation of the purified His(6)-WhcA and GST-SpiA fusion proteins, as assayed by in vitro pull-down experiments, were consistent with in vivo results. These data indicated that the interaction between WhcA and SpiA is not only specific but also modulated by the redox status of the cell and the functionality of the WhcA protein is probably modulated by the SpiA protein.