Development of a novel single-cell omics platform for the investigation of microbial population heterogeneity during stress adaptation
Zusammenfassung der Projektergebnisse
The Sino-German project aimed towards a better understanding of molecular features and mechanisms of microbial population heterogeneity. For this purpose, single cell Raman and fluorescence profiling were envisioned, followed by detailed molecular analysis with proteomics. The research models chosen were "Corynebacterium glutamicum" and "Nannochloropsis oceanica", with high potential for industrial biotechnology utilization. Consequently, the proof of principle studies should exemplify obtaining superior strains in respect to biofuel production and stress adaptation besides new insight into basic biology. Proteomics sample preparation and analysis protocols were optimized for sorted cells, culminating in a procedure requiring ~ 1 m microbial cells for successful shotgun proteomics. Unfortunately, available Raman sorters were still too slow to deliver such cell numbers in a reasonable amount of time, though population heterogeneity in metabolic activity could be certainly detected for 13C-glucose consumption. Hence, FACS was employed for analysis of population heterogeneity in tandem with proteomics. For C. glutamicum the method was benchmarked with a mixture of two strains, the wild type and delta aceE mutant harbouring GFP reporter system for branched-chain amino acids. By means of cytometry and PCA of proteomes, efficient FACS separation of both strains could be demonstrated. Furthermore, proteome changes that contribute to the high production of branched chain amino acids (BCAA) in the delta aceE strain were disclosed. For Nannochloropsis, phenotypic heterogeneity of lipid storage under nitrogen deplete and replete conditions was assayed by Nile Red fluorescence. Interestingly, for both growth conditions, FACS discriminated two populations with high and low cellular lipid content. These differentiations could be recognized on the population as well as the single-cell levels; proteomics uncovered alterations in carbon fixation and flux, photosynthetic machinery, lipid storage and turnover in the populations. Although heterogeneity patterns have been affected by nitrogen supply and cultivation conditions of the N. oceanica populations, differentiation itself seems to be very robust against these factors. As outlook, improved sorting efficiency of the Raman single cell platforms would give access to cellular features complementary to FACS for sorting, such as 13C-labeled substrate uptake for subsequent proteomics. This would enable real time multi-parametric Raman analysis and sorting in the future.
Projektbezogene Publikationen (Auswahl)
- (2017) Heterogeneous circRNA expression profiles and regulatory functions among HEK293T single cells. Scientific reports 7 (1) 14393
Zhong, Chaofang; Yu, Shaojun; Han, Maozhen; Chen, Jiahuan; Ning, Kang
(Siehe online unter https://doi.org/10.1038/s41598-017-14807-w) - Preprocess and condensation of Raman spectrum for single-cell phenotype analysis. BMC Bioinformatics, 16(S18), S15
Xuetao Wang, Shiwei Sun, Xing Gao, Lihui Ren, Xiaoquan Su, Dongbo Bu and Kang Ning
(Siehe online unter https://doi.org/10.1186/1471-2105-16-S18-S15) - The Tara Oceans Project: New Opportunities and Greater Challenges Ahead. Genomics, Proteomics & Bioinformatics, 2015
Houjin Zhang, Kang Ning
(Siehe online unter https://dx.doi.org/10.1016/j.gpb.2015.08.003) - Single-cell SNP analyses and interpretations based on RNA-Seq data for colon cancer research. Scientific reports, 2016
Jiahuan Chen, Qian Zhou, Yangfan Wang and Kang Ning
(Siehe online unter https://doi.org/10.1038/srep34420) - (2017): Proteomics of FACS-sorted heterogeneous Corynebacterium glutamicum populations, J. of Proteomics 8:160
Harst A., Albaum S., Bojarzyn T., Trötschel C., Poetsch A.
(Siehe online unter https://doi.org/10.1016/j.jprot.2017.03.010)
