Functional Proteomic Research Of The Histidine Kinase Hik33 In Synechocystis Sp. PCC 6803

Cyanobacteria are widely distributed in almost every terrestrial and aquatic habitat,and thus have developed an efficient system to cope with versatile and ever-changing adverse environmental conditions during the long evolutional process. It has been well accepted that the two-component systems (TCS), the dominant signal transduction systems in prokaryotes,are critically important for cyanobacteria to perceive the environment changes and translate them to intracellular signals,which in turn regulate differential expression of proteins that are important for stress acclimation and trophic growth. The typical TCS are usually composed of a histidine kinase (Hik) acting as the sensor and a cognate response regulator (Rre).The most frequently occurring environment stresses cyanobacteria have to cope with in their natural habitats include HL irradiation, high or low temperature, high salt,acid, and drought. The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses. Hik33 is highly conserved in cyanobacteria but not in other prokaryotes,suggesting that the functions of Hik33 are specialized to coordinate photosynthesis and stress response.Unfortunately, despite the tremendous amount of attempts in elucidating the working mechanism of Hik33, how Hik33 perceives the divergent environmental signals and regulates the corresponding cellular response in cyanobacteria remain largely unknown.Characterization of Hik3 3-dependent differential gene expression can provide important information for the understanding how Hik33 operates in stress response.Nevertheless, such information is only available at transcription level and limited to only a single stress condition in each study. Because transcription levels are usually not well correlated with the protein levels, and because Hik33 operates in multiple types of stress responses, such transcription information without enough generality is still not sufficient for elucidating the working mechanism of Hik33 in regulating stress responses.Using a hik33-deficient strain of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we carried out our research from different aspects, and have made some progresses as follows:1. Hik33 regulates the response of synechosystis cells through the common stress-responsive (CSR) proteins.By using quantitative proteomics and bioinformatics analyses, we found that depletion of Hik33 significantly downregulated 208 and upregulated 369 proteins.Among these, 60 proteins belong to the common stress-responsive (CSR) proteins which we have defined in our research.Among the downregulated CSR-proteins the most prominently enriched function is photosynthesis,particularly PS I and PBS,which are important for propagation of Synechocystis by providing material and energy necessary for cell growth.Downregulation of such a function indicates that Ahik33 and stressed WT cells need to cope with the deleterious intracellular conditions resulting from Hik33 depletion and stresses, respectively, through sacrificing propagation.In contrast, a number of heat shock proteins,chaperons,proteases,and proteins involved in the regulation of intracellular redox states were upregulated in response to Hik33 depletion or abiotic stresses. Protein denaturation occurs in almost all abiotic stresses,resulting in accumulation of denatured proteins in the cytosol. This in turn evokes the stress response. Upregulation of heat shock proteins, chaperons, and proteases such as HspA, HtpG, GroEL1, DnaJ, and C1pC could be important to repair such a damage and is important for cell survival. Moreover, abiotic stresses, and deletion of hik33 as well, could induce intracellular redox changes and production of ROS. Consequently, proteins involved in redox regulation and ROS scavenging, which include MrgA (Slrl894), the peroxiredoxin S110755 and Slr0242, Gpxl (Slr1711) and Gpx2 (Slr1922), SodB, KatF (S111987), and Tpx (S110755), were also upregulated.Finally, damage to PS II is more likely to occur in stresses or in Ahik33, which requires more proteins such as FtsH and OCP for protection and repair. Together, the functions of upregulated proteins, though divergent, are critical for protecting the cyanobacterium from damage caused by abiotic stresses or Hik3 3-depletion.The coordinated upregulation and downregulation of the functions critical for propagation and survival, respectively, could be an evolved strategy of the cyanobacterium to survive in stress conditions,and Hik33 is a general but not the sole regulator for the implementation of such a strategy. The systematically characterized Hik33-regulated cyanobacterial proteome,which is largely involved in stress responses,builds the molecular basis for Hik33 as a general regulator of stress responses.2. ?hik33 has higher efficiency in utilizing glucose through upregulation of the OPP pathwayPhenotypic experiments showed that supplying the Hik33 mutant with glucose dramatically improved its growth. To uncover the proteomic basis underlying the fast photomixotrophic growth of Ahik33 with a rate nearly comparable to that of the WT as we and others observed,we compared the differential expression patterns of carbon assimilation-related proteins between ?hik3 3 and the WT in the normal and the HG conditions.The proteomic data showed that all proteins involved in the NADPH-producing part of the OPP pathway, which include Zwf, OpcA, Gnd, and Pgl, are reversed from downregulation or slight downregulation to upregulation or slight upregulation in the mutant by glucose, indicating that the mutant could acquire extra ability in utilizing glucose. Indeed, the mutant grows much faster than the WT in the light-activated heterotrophic growth (LAHG) condition,where glucose,the only carbon and energy source, is catabolized mainly through the OPP pathway. This observation confirms that?hik33 has higher efficiency in utilizing glucose through upregulation of the OPP pathway. Noteworthy, the Ch1 content of the mutant is still lower than that of the WT in the LAHG condition, suggesting that hik33-deletion-induced stress response is not eliminated in the LAHG condition.Together, these findings strongly support that the mutant can better utilize glucose,most likely through upregulation of the OPP pathway but not through elimination of the hik33-deletion-induced stress response.Based on the proteomic results and phenotypic observations, we proposed a model explaining how glucose is catabolized in ?Hik33 in the photomixotrophic growth condition. In the WT,operation of the OPP pathway is nearly negligible as previously determined by the metabolic flux analysis using 13C-labeled glucose, whereas in the mutant, the OPP pathway become functional, at least partially, to provide NADPH and RuBP for CO2 assimilation.3. Upregulation of PetE and downregulation of PetJ represent a strategy for Ahik33 to grow fast in high concentration CO2 condition.Supplying the Hik33 mutant with high concentration CO2 (HC) also dramatically improved the growth of the mutant. We phenotypically examined Ahik33 supplemented with high concentration CO2 (HC), and quantitatively compared its proteome with that of the wild type (WT) strain cultured in the same conditions.To answer why HC drastically promotes photoautotrophic growth of the mutant as observed, we compared the differential expression pattern of proteins involved in photoautotrophic growth including proteins in photosynthesis and CO2 assimilation with that in the normal condition.Compared with the WT, the differential expression of the majority of proteins in this category observed in the normal condition was not significantly altered by HC with a few exceptions including CupB, NdhD4, PetE, and PetJ. The downward shift of NdhD4 and CupB could be an indicator that CO2 supply is no longer limiting the growth of ?hik33 in the HC condition. Nevertheless, the changes in CO2 assimilation system as represented by CupB and NdhD4 are not able to promote CO2 assimilation and photoautotrophic growth.NADPH is a final product of photosynthetic electron transport (PET) occurring on the thylakoid membrane, where the respiratory electron transport (RET) also resides.The two electron transport chains are interwoven with each other and share several components such as the plastoquinone pool, the Cyt b6f complex, and soluble electron carriers. Hence the non-NADPH-producing RET could compete with PET for electrons that are otherwise used for NADPH production and subsequent CO2 fixation.PetE and PetJ are the two only soluble carriers transporting electrons from the cytochrome (Cyt) b6/f complex to PS I and/or the terminal oxidase on the respiratory electron transport chain. Both proteins are not differentially expressed between the two strains in the normal condition. However, in HC, PetE is significantly upregulated while PetJ is significantly downregulated in the mutant.Upregulation of PetE and downregulation of PetJ may represent a strategy for the mutant to grow fast in HC, The fast growth of the mutant in HC condition requires large amount of photosynthetically-produced NADPH for fixation of CO2, and the availability of CO2 is no longer a limiting factor for the growth of Ahik33 in the HC condition. However, downregulation of the major photosynthetic machineries such as PsbO, PBS, and PS I in the mutant was not remarkably altered by HC, which is still the bottleneck of PET-dependent NADPH production. A potential way to overcome this bottleneck is to maximally utilize photosynthetically-generated electrons for NADPH production by minimizing the amount of electrons flowing to the RET chain. Both PET and RET localize on the thylakoid membrane and share the soluble electron carriers such as PetE and PetJ. Upregulation of PetE and downregulation of PetJ could be critically important to direct the majority of electrons to PS I instead of the terminal oxidase to produce sufficient amount of NADPH necessary for CO2 assimilation in the HC condition.