SpiE interacts with Corynebacterium glutamicum WhcE and is involved in heat and oxidative stress responses

Abstract

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.

The actinobacterial WhiB‐like (Wbl) family of transcription factors

“…Several Wbl paralogues have been shown to interact with the primary sigma factor, SigA. Other protein partners for Wbls have also been identified; SpiE interacts with WhcE in Corynebacterium (Park et al , ) and SpiA interacts with WhcA/WblA in Corynebacterium and Streptomyces (Park et al , ; Kim et al , ). In Streptomyces , WhiB has been shown to co‐control the expression of genes required for sporulation with an unrelated protein, WhiA.…”

“…In C. glutamicum , deletion of the orthologous gene, whcE leads to an increased sensitivity to heat and oxidative stress (Kim et al , ). Oxidised WhcE Cg interacts with the efflux pump SpiE (Stress Protein Interacting with WhcE) and expression of the genes encoding both proteins increases under heat and oxidative stress, suggesting that WhcE Cg and SpiE collectively contribute to mediating the cellular response under these conditions (Park et al , ).…”

Trehalose biosynthetic gene otsB of Corynebacterium glutamicum is regulated by whcE in response to oxidative stress

“… C. glutamicum HL1485 and HL1491 carry a Δ spiE mutation and pSL551, which overexpresses the spiE gene, respectively [16]. Escherichia coli DH10B (Invitrogen) was used for the construction and propagation of the plasmids, and E.…”

“…C. glutamicum HL810 and HL1108 carry a ΔwhcE mutation and pSL395, which overexpresses the whcE gene, respectively [18]. C. glutamicum HL1485 and HL1491 carry a ΔspiE mutation and pSL551, which overexpresses the spiE gene, respectively [16]. Escherichia coli DH10B (Invitrogen) was used for the construction and propagation of the plasmids, and E. coli BL21 DE3 (Merck) was used for the expression of whcE (His 6 -WhcE).…”

“…The whcE gene plays a positive role in the survival of cells exposed to oxidative and heat stress [15]. Under oxidative stress conditions, the WhcE protein interacts directly with SpiE, which plays a positive role in whcE-mediated oxidative stress defence networks [16]. The whcA gene plays a negative role in the expression of genes involved in the oxidative stress response [17][18][19].…”