Identification of SpiA that interacts with Corynebacterium glutamicum WhcA using a two-hybrid system

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.