DnaK2 Mediates a Negative Feedback Regulation of the Heat Shock Responsive Hik2-Rre1 Two-Component System in the Cyanobacterium Synechococcus Elongatus PCC 7942.

The two-component system (TCS) is a conserved signal transduction module in bacteria. The Hik2-Rre1 system is responsible for transcriptional activation upon high-temperature shift as well as plastoquinone-related redox stress in the cyanobacterium Synechococcus elongatus PCC 7942. As heat-induced de novo protein synthesis was previously shown to be required to quench the heat-activated response, we investigated the underlying mechanism in this study. We found that the heat-inducible transcription activation was alleviated by the overexpression of dnaK2, which is an essential homolog of the highly conserved HSP70 chaperone and whose expression is induced under the control of the Hik2-Rre1 TCS. Phosphorylation of Rre1 correlated with transcription of the regulatory target hspA. The redox stress response was found to be similarly repressed by dnaK2 overexpression. Considered together with the previous information, we propose a negative feedback mechanism of the Hik2-Rre1-dependent stress response that maintains the cellular homeostasis mediated by DnaK2.

Conserved Two-component Hik2-Rre1 Signaling Is Activated Under Temperature Upshift and Plastoquinone-reducing Conditions in the Cyanobacterium Synechococcus elongatus PCC 7942.

The highly conserved Hik2-Rre1 two-component system is a multi-stress responsive signal-transducing module that controls the expression of hsp and other genes in cyanobacteria. Previously, we found in Synechococcus elongatus PCC 7942 that the heat-inducible phosphorylation of Rre1 was alleviated in a hik34 mutant, suggesting that Hik34 positively regulates signaling. In this study, we examined the growth of the hik34 deletion mutant in detail, and newly identified suppressor mutations located in rre1 or sasA gene negating the phenotype. Subsequent analyses indicated that heat-inducible Rre1 phosphorylation is dependent on Hik2 and that Hik34 modulates this Hik2-dependent response. In the following part of this study, we focused on the mechanism to control the Hik2 activity. Other recent studies reported that Hik2 activity is regulated by the redox status of plastoquinone (PQ) through the 3Fe-4S cluster attached to the cyclic GMP, adenylyl cyclase, FhlA (GAF) domain. Consistent with this, Rre1 phosphorylation occurred after the addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone but not after the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea to the culture medium, which corresponded to PQ-reducing or -oxidizing conditions, respectively, suggesting that the Hik2-to-Rre1 phosphotransfer was activated under PQ-reducing conditions. However, there was no correlation between the measured PQ redox status and Rre1 phosphorylation during the temperature upshift. Therefore, changes in the PQ redox status are not the direct reason for the heat-inducible Rre1 phosphorylation, while some redox regulation is likely involved as oxidation events dependent on 2,6-dichloro-1,4-benzoquinone prevented heat-inducible Rre1 phosphorylation. On the basis of these results, we propose a model for the control of Hik2-dependent Rre1 phosphorylation.