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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.

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 gene whcE in Corynebacterium glutamicum positively responds to oxidative and heat stress. To search for proteins that interact with WhcE, we employed a two-hybrid system with WhcE as the bait. Sequencing analysis of the isolated clones revealed peptide sequences, one of which showed high sequence identity to a hydrophobe/amphiphile efflux-1 family transporter encoded by NCgl1497. The interaction of the NCgl1497-encoded protein with WhcE in vivo was verified using reporter gene expression by real-time quantitative PCR (RT-qPCR). The WhcE protein strongly interacted with the NCgl1497-encoded protein in the presence of oxidative and heat stress. Furthermore, purified WhcE and NCgl1497-encoded proteins interacted in vitro, especially in the presence of the oxidant diamide, and the protein-protein interaction was disrupted in the presence of the reductant dithiothreitol. In addition, the transcription of NCgl1497 was activated approximately twofold in diamide- or heat-treated cells. To elucidate the function of the NCgl497 gene, an NCgl1497-deleted mutant strain was constructed. The mutant showed decreased viability in the presence of diamide and heat stress. The mutant strain also exhibited reduced transcription of the thioredoxin reductase gene, which is known to be regulated by whcE. Based on the results, NCgl1497 was named spiE (stress protein interacting with WhcE). Collectively, our data suggest that spiE is involved in the whcE-mediated oxidative stress response pathway of C. glutamicum.