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

Abstract

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.

Involvement of the NADH oxidase-encoding noxA gene in oxidative stress responses in Corynebacterium glutamicum

“…Because Park et al noted that SpiA played both positive and negative roles and that some whcA-regulated genes are not under the control of the spiA gene, we can speculate that the noxA gene is not likely to be part of the spiA-regulon. This hypothesis is further supported by the fact that, while the growth of spiA mutants is affected by both menadione and diamide (Park et al 2011), the growth of noxA mutants is only affected by menadione. Moreover, unlike in the case of spiA mutants, the expression of the trxB gene encoding thioredoxin reductase was not affected in the noxA mutants-a finding that further supports our hypothesis.…”

“…It is interesting to note that SpiA is not required for the DNA-binding of WhcA. Park et al (2011Park et al ( , 2012 postulated that the WhcA protein forms a complex with the SpiA protein and that the SpiA-WhcA protein complex then binds to its target promoters to repress gene expression in The enzymes were induced by growing C. glutamicum AS019E12 cells to late-exponential phase in MCGC minimal medium. Cells were harvested, disrupted, and assayed for activity as described in the "Materials and methods".…”

“…The whcE gene plays a positive role in the survival of cells exposed to oxidative and heat stresses (Kim et al 2005), whereas the whcA gene plays a negative role in the expression of genes associated with the oxidative stress response (Choi et al 2009). The WhcA protein interacts with SpiA, an oxidoreductase-like protein (Park et al 2011, and the interaction is labile to oxidants. The whcB gene plays a regulatory role in the stationary phase of bacterial growth, particularly in electron transfer reactions .…”

Role of Corynebacterium glutamicum sprA Encoding a Serine Protease in glxR-Mediated Global Gene Regulation

“…Glucose and acetate were added as carbon sources to the MCGC minimal medium at 1% and 2% (w/v), respectively. Selective and nonselective broths (BacterioMatch II Two-Hybrid System, Agilent Technologies) for E. coli XL1-Blue MRF′ kan were prepared as described [29]. Antibiotics were added at the following concentrations (μg mL −1 ): 50 ampicillin, 5 tetracycline, 20 chloramphenicol, and 25 kanamycin.…”

“…Polymerase chain reaction (PCR) was performed as previously described [6]. cDNA conversion and real-time quantitative PCR (RT-qPCR) were also performed as previously described [29]. CFX96 Real-Time PCR Detection System (Bio-Rad) was used for gene expression analysis.…”