Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σHin Corynebacterium glutamicum

The extracytoplasmic function sigma factor σ(H) is responsible for the heat and oxidative stress response in Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, previous transcriptome analyses have not been able to discriminate between direct and indirect targets of σ(H). Here, we determined the direct genome-wide targets of σ(H) using chromatin immunoprecipitation with microarray technology (ChIP-chip) for analysis of a deletion mutant of rshA, encoding an anti-σ factor of σ(H). Seventy-five σ(H)-dependent promoters, including 39 new ones, were identified. σ(H)-dependent, heat-inducible transcripts for several of the new targets, including ilvD encoding a labile Fe-S cluster enzyme, dihydroxy-acid dehydratase, were detected, and their 5' ends were mapped to the σ(H)-dependent promoters identified. Interestingly, functional internal σ(H)-dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin and affected expression of Zn-responsive genes, possibly through intracellular Zn overload, indicating new physiological roles of σ(H). Furthermore, sigA encoding the primary σ factor was identified as a new target of σ(H). Reporter assays demonstrated that the σ(H)-dependent promoter upstream of sigA was highly heat inducible but much weaker than the known σ(A)-dependent one. Our ChIP-chip analysis also detected the σ(H)-dependent promoters upstream of rshA within the sigH-rshA operon and of sigB encoding a group 2 σ factor, supporting the previous findings of their σ(H)-dependent expression. Taken together, these results reveal an additional layer of the sigma factor regulatory network in C. glutamicum.