Hik28-dependent and Hik28-independent ABC transporters were revealed by proteome-wide analysis of ΔHik28 under combined stress

In the present study, the effect of combined temperature and nitrogen stress on the cell growth of WT and MT strains was studied by measuring cell density, Chl a content and O2 evolution. When the ΔO2 evolution of the cells grown in the presence and absence of nitrogen was calculated (Table 1 and Suppl. Fig. 3), the higher value of ΔO2 evolution found in the MT cells indicated poorer adaptation to N stress. This result implies that Hik28 most likely plays a role in cell growth via the photosynthetic mechanism in response to N stress.

Moreover, the clustering results, shown in Fig. 2B, of the differentially expressed proteins obtained from comparative proteomic analysis of the WT and MT strains supported the critical role of Hik28 in response to the temperature downshift due to the absence of Hik28 led to similar protein expression pattern of MT under 16oC and that of the WT and MT strains under the optimal temperature. Sensor histidine kinases in two-component signal transduction systems, including Hik28, enable cyanobacteria to sense, respond, and adapt to environmental changes, stressors, and growth conditions. It is well known that in the response mechanism, the phosphoryl group is transferred from the autophosphorylated sensor histidine kinase to a response regulator (RR), which subsequently affects cellular physiology by regulating gene expression [14]. After signal retrieval, the activated RR binds to its target promoter regions and subsequently regulates the transcriptional machinery [15]. Moreover, in the stress response mechanism, the two-component system and ABC transporters are functionally related in the transportation of substrates, including peptides, amino acids, sugars and antibiotics [16,17,18,19].

Effects of Hik28 deletionTwo-component signal transduction system and ABC transporter:

Based on the proteomic data, a unique set of signaling and response regulator proteins were detected in the WT and mutant strains under temperature and combined stress (Fig. 3A-D and Suppl. Fig. 5A-D). The data clearly indicated the effect of Hik28 deletion at the level of abiotic stress sensing and the specific set of response regulators involved. Furthermore, a two-component system is known to tightly regulate ABC transporters, which are an important class of proteins that transport various extracellular substrates, including peptides, amino acids, sugars and antibiotics [20]. After the two-component system senses and transfers the signal from the environmental stress, proteins in the ABC transporter group are one of the immediate responses of the cells to the stress that is regulated by the TCS. The TCS induces a quick and specific response to stimuli, and both the TCS and the ABC transporter system have demonstrated their ability to sense biotic and abiotic stress and substrates, including peptides, amino acids, sugars and antibiotics; however, the exact mechanism is not fully established.

In Synechocystis sp. PCC6803, WT strain, there are a total of 73 ABC transporter proteins, and 15 of them were detected in the proteomic data. It has been reported that the genes encoding TCS proteins and the ABC transporters that they regulate are located close together in the genome [21, 22]. Therefore, the loci of the encoded TCS genes and the ABC transporters found in the two strains under each experimental condition are shown in Fig. 4. An illustration of the loci using CGview and genome feature information showed that the iron transporter, Slr0513: FutA2 or AfuA, was in a position upstream of Hik28 and that the protein was downregulated in the Hik28-deletion mutant in response to elevated temperature, whereas it was upregulated in the WT under the combined stress of high temperature and nitrogen depletion. Synechocystis sp. PCC6803 was reported earlier to have a 10-fold higher demand for iron than Escherichia coli to sustain photosynthesis. Thus, (i) the evidence supported the necessity of the iron-binding protein Afu/FutA2 for growth (Badaruh et al. 2008) in the WT, and the data showed that the regulation of this transporter was Hik28-dependent; (ii) the evidence showed that the genes coding for the TCS proteins, e.g., Hik28, might regulate ABC transporters, e.g., Afu/FutA2, whose genes are nearby in the genome.

Fig. 4figure 4

An illustration of the loci on Synechocystis sp. PCC6803 genome using CGview and genome feature information of the encoded TCS genes and the ABC transporters found in the two strains, WT and MT, under each experimental condition A zoom-in of the region surrounding Hik28-encoded gene and B Synechocystis sp. PCC6803 genome with Hik28-and ABC transporter- genes labeled

In addition to the iron transporter, the group of ABC transporters, whose regulation can be considered Hik28 dependent, were urea and α-glucoside transporters. These two transporters were downregulated in the Hik28-deletion mutant and had protein–protein interactions with proteins involved in the metabolic process to concentrate carbon dioxide. The two transporters were differentially expressed in response to low-temperature stress, supporting the finding that Hik28 plays a critical role as a signaling molecule in the low-temperature response mechanism [11].

Furthermore, it is noteworthy that the hypothetical and unknown proteins located upstream (Slr0516) and downstream (Sll0493) of Hik28 were found to have protein–protein interactions with Hik28 according to STRING [23]. In addition to Hik28, Slr0516 also interacts with biopolymer transporters, whereas Sll0473 interacts with nitrate and bicarbonate transporters (Suppl. Fig. 6A). Moreover, Slr0517, located downstream of Hik28, was functionally related to Hik28 and proteins in glutamine metabolic processes and purine biosynthesis in the PPI network (Suppl. Fig. 6A). In accordance with previous reports, the TCS and the ABC transporter located adjacent to it in the genome were related, thus supporting the finding that the TCS regulates nearby ABC transporter genes [21, 22].

The ABC transporters of the two strains, WT and MT, were compared under combined stress; in the mutant, which lacked Hik28, the ABC transporters responsible for molybdate/sulfate, xenobiotic, and phosphate transporter were revealed to be downregulated proteins (Fig. 3C-D and Suppl. Table 7). However, the deletion of Hik28 combined with temperature stress has negative effects on the iron, osmolyte and sugar transfer systems, whereas the urea transporter Sll0374: UrtE was downregulated after high-temperature exposure and vice versa under low-temperature stress. Interestingly, Slr0559, an ABC transporter for general L-amino acids, was upregulated in the mutant strain under high-temperature stress regardless of the nitrogen supply. However, in the WT under the three experimental conditions, low temperature, combined low-temperature and nitrogen stress, and combined high-temperature and nitrogen stress, and in the MT under the condition of combined low-temperature and nitrogen stress specifically, the proteomic data showed upregulation of the phosphate transporter Slr1247: PstS, suggesting the necessity of phosphate for cyanobacterial growth. Indeed, phosphate is a key growth limiting nutrient, particularly in freshwater cyanobacteria [24].

Other proteins in the class of ABC transporters involved in the control of the C/N ratio inside the cells are Slr0040: bicarbonate transporter and Sll1450: nitrate/nitrite/cyanate transporter. The nitrate/nitrite/cyanate transporter was differentially expressed only in the absence of Hik28 in response to high-temperature stress and in combination with nitrogen depletion. Moreover, the PPI network of these transporters showed interactions with proteins involved in nitrogen metabolism and iron and bicarbonate transport systems (Suppl. Fig. 6F). The bicarbonate transporter was downregulated under the combined stress of nitrogen depletion and low temperature in the Hik28-deletion mutant, whereas the protein expression level in the WT was decreased only under low-temperature stress, showing its Hik28-independent regulation. (Fig. 3A-D). The results suggested that (i) Hik28 possibly played a role in nitrogen assimilation and (ii) the bicarbonate requirement of the cells was reduced in response to low-temperature conditions. The evidence obtained from the proteome analysis supported the importance of the regulation of the C/N ratio in the survival and growth of cyanobacteria [11], especially under stress conditions. Furthermore, the proteins in the bacterial secretion system, HlyD and TolC, were differentially expressed under high temperature in the Hik28-deletion strain (Fig. 3B). The results indicated that the absence of Hik28 and exposure to the combined stress had direct effects on the group of ABC transporters that transfer nutrients across the periplasmic membrane.

Response of metabolic pathways to combined stress

The metabolic pathways affected by the combined stress of immediate temperature shift and nitrogen depletion were comparatively analyzed by strain and by growth condition, as shown in Fig. 3A-D and Suppl. Fig. 5A-D. Taken together, the proteomic data on differentially expressed proteins and the protein–protein interaction network demonstrated changes in the expression levels of N metabolism proteins, GlnA, GlnB and GlnN under temperature stress and combined stress in the absence of Hik28 (Suppl. Fig. 6B-I), supporting the report by Kurdrid et al. that Hik28 is critically involved in N metabolism. Moreover, the results at the proteome level showed the effect on fatty acid biosynthesis in mutant cells in response to the combined stress of high temperature and nitrogen depletion, which was in accord with the fatty acid biosynthesis data showing the drastic accumulation of C16:1Δ9 reported by Kurdrid et al. (2020). It is also noteworthy that the proteins involved in oxidative phosphorylation were significantly upregulated in the MT strain in response to temperature downshift and its combination with nitrogen stress. The upregulation of F-type H+-transporting ATPase and subunit a of ATP synthase suggested that the mutant cells had increased energy requirements under stress; therefore, the absence of Hik28 led to the requirement of ATP under low-temperature and combined stress (Suppl. Fig. 6B and D), which strongly supported the evidence that Hik28 played a crucial role in the low-temperature stress response mechanism [11].

According to a report by Kurdrid et al., oxygen evolution and chlorophyll a content decreased in mutant cells after a temperature shift to 16 °C in the presence of nitrogen, showing the negative effect of Hik28 deletion on the photosynthetic apparatus [11]. In the present study, the upregulation of proteins in PSI, PSII, the cytochrome b6f complex and photosynthetic electron transport was observed in the WT strain (Suppl. Fig. 6F). However, in the MT, under the combined stress of low temperature and N depletion, PetE, a protein in the photosynthetic electron transport system, was upregulated (Suppl. Fig. 6D), whereas it was downregulated after a temperature downshift (Suppl. Fig. 6B), supporting the evidence found by Kurdrid et al. [11]. In response to a temperature upshift or its combination with N stress, the proteins in PSII and the cytochrome b6f complex were upregulated. The comparative proteome data indicated that Hik28 may have an effect on PetE under low-temperature stress and in combination with N stress; however, the expression of other proteins in the photosynthetic system was independent of Hik28. Moreover, the expression level of ribose-5-phosphate isomerase, RpiA, during carbon fixation by this photosynthetic organism increased only in the MT strain after a temperature shift from 35 °C to 16 °C, suggesting that the mutant cells under low-temperature stress acquired higher levels of carbon than WT cells (Fig. 3A, Suppl. Fig. 6B and F). This evidence supported the finding by Hutchings et al. that RpiA, which plays a key role in the pentose phosphate pathway, is directly connected to fatty acid biosynthesis, which is regulated under temperature stress, by NADP/NADPH metabolism [25].

Another pathway involved in photosynthesis is porphyrin metabolism, in which the photosynthetic pigment chlorophyll a is synthesized. The enzymes involved in porphyrin metabolism, such as porphobilinogen synthase (HemE), were upregulated at elevated growth temperatures in the absence of Hik28 protein (Fig. 3B), whereas the expression level of protochlorophyllide oxidoreductase (Por) decreased after a temperature upshift in the WT strain (Suppl. Fig. 5B). The results showed that the proteins involved in porphyrin metabolism were regulated in response to elevated growth temperature and that Hik28 deletion caused differences in the regulation of porphyrin metabolism. Moreover, regardless of Hik28 deletion and stress conditions, the ribosomal proteins and the proteins in the oxidative phosphorylation pathway were upregulated, suggesting a need for protein biosynthesis and energy for the stress response mechanism and other metabolic processes.

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