Single-cell Raman Spectroscopy Reveals the Inorganic Carbon Source Utilization Preference of Synechocystis sp. PCC7942

doi:10.13560/j.cnki.biotech.bull.1985.2025-1469

Abstract

Abstract:

Objective The supply form of inorganic carbon (Ci) is a critical environmental factor regulating photosynthetic efficiency in cyanobacteria. However, the inorganic carbon utilization preference of cyanobacteria at the single-cell level and the corresponding molecular regulatory mechanism remain unclear under the coexistence of CO₂ and $H C O 3 -$ . This study used the model strain Synechococcus sp. PCC7942 to reveal its utilization preference for different inorganic carbon sources at the single-cell level. Method This study integrated ¹³C stable isotope probing, single-cell Raman spectroscopy (Raman-SIP), and transcriptome sequencing. Six carbon source treatments were set, including ambient ¹²C-NaHCO₃, ¹³C-NaHCO₃, air, ¹²C-CO₂, ¹²C-CO₂ + ¹³C-NaHCO₃, and ¹³C-CO₂ + ¹²C-NaHCO₃. Cells were cultured in BG11 medium under controlled light and temperature. The ¹³C assimilation and carbon source utilization characteristics were quantitatively characterized by detecting the shift of the carotenoid v1 peak using single-cell Raman spectroscopy. Transcriptome sequencing was performed on samples grown on single carbon sources (¹²C-NaHCO₃ and ¹²C-CO₂). The regulatory mechanism of carbon metabolism pathways was dissected through data quality control, genome alignment, screening of differentially expressed genes, and GO/KEGG enrichment analysis. Result In the CO₂/ $H C O 3 -$ coexistence system, the strain predominantly utilizes CO₂—an observation quantitatively confirmed by dynamic redshifts in the Raman carotenoid v1 peak. RNA-seq analysis revealed 2 750 differentially expressed genes (DEGs) with annotations in the CO₂-treated group, among which 1 083 were significantly differentially expressed. Most genes encoding enzymes of the Calvin-Benson cycle showed downregulated transcription, whereas rbcLS expression was upregulated. Conclusion This study integrates single-cell Raman spectroscopy and transcriptomics to demonstrate that Synechococcus sp. PCC7942 preferentially utilizes CO₂ when CO₂ and $H C O 3 -$ coexist, and such preference can be quantitatively characterized by Raman peak redshift. The related gene expression profiles are consistent with the metabolic phenotypes, uncovering the inorganic carbon utilization mechanism. This work establishes a systematic linkage among carbon source preference, Raman spectral characterization, and transcriptional regulation at the single-cell level.

Key words: Synechococcus elongatus PCC7942, single-cell Raman spectroscopy, inorganic carbon utilization preference, isotope probing, transcriptome, Calvin-Benson cycle, carotenoid, Raman shift

LIU Jia, MENG Yu, JING Xiao-yan. Single-cell Raman Spectroscopy Reveals the Inorganic Carbon Source Utilization Preference of Synechocystis sp. PCC7942[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1469.

Figures/Tables 6

Table 1 Composition of BG11 Medium

序号 Number	BG11培养基 BG11 medium			A₅（微量金属溶液）A₅ trace metal solution
序号 Number	成分 Component	含量 Content （mL/L）	母液质量浓度 Concentration （g/L）	成分 Component	母液质量浓度 Concentration （g/L）
1	NaNO₃	10	150.0	H₃BO₃	2.86
2	K₂HPO₄	10	4.0	MnCl₂·4H₂O	1.86
3	MgSO₄·7H₂O	10	7.5	ZnSO₄·7H₂O	0.22
4	CaCl₂·2H₂O	10	3.6	Na₂MoO₄·2H₂O	0.39
5	柠檬酸	10	0.6	CuSO₄·5H₂O	0.08
6	柠檬酸铁铵	10	0.6	Co（NO₃）₂·6H₂O	0.05
7	EDTANa₂	10	0.1
8	Na₂CO₃	10	2.0
9	A₅	1

Fig. 1 Growth curve of Synechococcus elongatus PCC7942 in liquid BG11 media supplied with different carbon sourcesA: Incubation device without CO2 as carbon source. B: Incubation device with CO2 as carbon source. C: Growth curve of Synechococcus elongatus PCC7942 in liquid BG11 media supplied with different carbon sources

Fig. 2 Dynamic red shift of S. elongatus PCC7942 (mean Raman spectra) supplied with different carbon sourcesA-E: Dynamic red shift of S. elongatus PCC7942 (mean Raman spectra) supplied with 13C-NaHCO3 (A)、13C-CO2 plus 5 mmol/L 12C-NaHCO3 (B)、12C-CO2 (C)、12C-NaHCO3 (D)、12C-CO2 plus 5 mmol/L 13C-NaHCO3 (E) as the carbon source, respectively. F-G: Dynamic red shift of S. elongatus PCC7942 (v1) supplied with 13C-NaHCO3 (F) and 13C-CO2 plus 5 mmol/L 12C-NaHCO3 (G) as the carbon source, respectively

Fig. 3 Generalized model for the cyanobacterial CO2-concentrating mechanism (CCM) and protein dynamic changes in its photosynthesis pathway

Fig. 4 KEGG functional enrichment analysis

Fig. 5 Protein dynamics in the Calvin cycle pathway, glycolysis pathway and citrate cycle pathwayA: Protein dynamics in the Calvin cycle pathway. B: Protein dynamics in the glycolysis pathway and the citrate cycle pathway

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