Regulatory mechanism of expression of chaperone protein genes(hsp genes) in cyanobacteria
Kan TANAKA
Institute of Innovative Research, Tokyo Institute of Technology
Research objective
Cyanobacteria are a large group of bacteria that have dramatically changed the earth's
environment through oxygenic photosynthesis. Furthermore, the photosynthetic function ofplants is also derived from the symbiosis of cyanobacteria. The photosynthetic light reaction
is an electron transport system driven by light and is inherently vulnerable to changes in theexternal environment, thus cyanobacteria have evolved strong stress response abilities andhave thrived in extremely diverse environments. However, many aspects of this control
mechanism are still unclear, and the purpose of this research is to elucidate the expression
regulation mechanism of the chaperone genes (hsp genes) that support stress tolerance incyanobacteria. Elucidation of the control system in cyanobacteria will lead to the elucidationof the mechanism that protects photosynthesis and is expected to provide basic informationfor various applications of photosynthetic function and maintenance of the global environment.
Regarding the regulation of the expression of chaperone proteins in bacteria,transcriptional activation by heat shock sigma factor (σH) in the heat shock response ofEscherichia coli has been analyzed in the most detail. On the other hand, σH does not exist in
Bacillus subtilis, and instead it has been shown that the heat shock response is due to therelease of a repressor that binds to the promoter. A heat shock response mechanism based on
Bacillus subtilis-type disinhibition was initially discovered in cyanobacteria, but this onlyaffected the response of a small number of genes, and the molecular mechanism of the original
heat shock response remains unclear. In this context, we found in the model cyanobacterium
Synechococcus elongatus strain PCC 7942 that the two-component regulatory system Hik2Rre1, which is universally conserved in cyanobacteria, is directly involved in the heat shock
response. That is, the response regulator Rre1 was phosphorylated by the sensory kinase Hik2
during heat shock and bound to the promoter regions of many chaperone protein genes toactivate transcription ref 1). Furthermore, it was revealed that temperature information is notdirectly recognized by Hik2 kinase, but that Hik2 is involved in the state of the photosynthetic
light reaction system that changes due to heat shock, especially the reduction state ofplastoquinone ref 2). In this study, we advanced our understanding of this Hik2-Rre1-dependent
heat shock response and analyzed a newly discovered Hik2-Rre1-independent transcriptional
activation mechanism of chaperone genes.

Methods
The promoter of the hspA gene, which encodes a small chaperone protein, was mainly used as
the regulatory target for analysis. The phosphorylation state of the response regulator Rre1
was detected by phos-tag gel electrophoresis, and the interaction between DNA-binding
proteins and promoters was analyzed by chromatin immunoprecipitation.
Results
The results obtained from this study are as follows.
1) The induction of expression of chaperone genes by heat shock response rapidly peaks andcontinues for about 30 minutes, and the expression declines to the basal level. At this time, if
a protein synthesis inhibitor such as chloramphenicol was added to the medium during the heatshock, the induction continued without decline after the heat shock induction. This means thatsome protein synthesized after heat shock negatively regulates the response. In this study, wehypothesized that the Hsp70-type chaperone DnaK2 is a negative regulatory factor, based on
previous findings in other organisms, and conducted verification experiments. When weconstruct a strain that can forcefully overexpress DnaK2 and apply heat shock while
overexpressing DnaK2, Rre1 phosphorylation, binding to the promoter region, anddownstream hspA gene expression are suppressed. In addition, since in silico analysis using
AlphaFold2 suggested an interaction between Hik2 and DnaK2, we constructed a regulatorymodel in which DnaK2 accumulated upon heat shock induction suppresses sensory kinase
Hik2 activity ref 3).
2) Over-oxidation of plastoquinone in the electron transport chain is induced by the inhibitorof photosystem II as DCMU. At this time, transcriptional activation of chaperone genes wasalso observed, but it was unclear whether Hik2-Rre1 was involved here as well. In this study,
we analyzed the phosphorylation status of Rre1 and its binding to the promoter and found thatno phosphorylation or DNA binding of Rre1 was observed. This result suggests that a signaltransduction system different from Hik2-Rre1 is involved in the response to plastoquinone
over-oxidation (manuscript in preparation).
3) In the process of analyzing the heat shock response system, we obtained an unknown
temperature-sensitive mutant strain. As a result of analyzing the whole genome sequence, apoint mutation in the gene encoding leu-tRNA was identified. Since a mutation that suppresses
temperature sensitivity was mapped to a specific RNA degrading enzyme gene, it wasconcluded that this tRNA undergoes degradation due to changes in its 3D structure at hightemperatures, making cell growth temperature sensitive ref 4).
Conclusion
Heat shock response is a temporary response accompanying a shift to high temperature in thegrowth environment. In previous studies, we have elucidated the induction mechanism of theheat shock response in cyanobacteria, and in this study, we have revealed the termination

mechanism of the heat shock response. Furthermore, two additional pathways were suggestedto induce the expression of chaperone genes independent of the two-component regulatory
system Hik2-Rre1. In summary, in cyanobacteria, various stresses are mainly recognized aschanges in the photosynthetic light reaction system, and an overall picture of inducing
chaperone genes through multiple signal transduction systems was revealed.
References
1) Kobayashi, I., Watanabe, S., Kanesaki, Y., Shimada, T., Yoshikawa, H., and *Tanaka, K.
(2017) Conserved two-component Hik34-Rre1 module directly activates heat-stressinducible transcription of major chaperone and other genes in Synechococcus elongatus
PCC 7942. Mol. Microbiol. 104: 260-277.
2) Bairagi, N., *Tanaka, K. et al. (2022) Conserved two-component Hik2-Rre1 signaling isactivated under high temperature upshift and plastoquinone-reducing conditions in thecyanobacterium Synechococcus elongatus PCC 7942. Plant Cell Physiol. 63:176-188.
3) Hasegawa, H., Kobayashi, I., Bairagi, N., Watanabe, S., and *Tanaka, K. (2023) DnaK2Mediates a Negative Feedback Regulation of the Heat Shock Responsive Hik2-Rre1 Twocomponent System in the Cyanobacterium Synechococcus elongatus PCC 7942. Plant Cell
Physiol. 65:120-127. (doi: 10.1093/pcp/pcad129).
4) Hasegawa, H., Kanesaki, Y., Watanabe, S., and *Tanaka, K. (2023) A high-temperaturesensitivity of Synechococcus elongatus PCC 7942 due to a tRNA-Leu mutation. J. Gen.
Appl. Microbiol. 69:167-174. (doi: 10.2323/jgam.2023.03.001).
5) Luo, Y., Imamitsu, H., Tsurumaki, T., and *Tanaka, K. (2024) Structure of the SigF1dependent pilA1 gene promoter and the light activation mechanism in the cyanobacteriumSynechococcus elongatus PCC 7942. (Submitted for publication).