Cyanobacteria Membrane Biology Grand Challenge: Systems Analysis of the Dynamics of Membrane Architecture, Composition, and Function- Proteomic, Metabolomic, and Metallomic Characterization
EMSL Project ID
16720
Abstract
The strain Cyanothece sp. ATCC 51142, isolated from benthic waters off the coast near Galveston, Texas, is a unicellular strain approximately 4-7 μm in diameter along the major axis. This organism, which fixes nitrogen at very high rates, has developed a type of temporal regulation in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle, including very high levels of CO2 fixation and storage during light cycles for use in dark cycles. These aspects of carbon and nitrogen storage make Cyanothece almost unique among well-studied cyanobacteria and make it a key candidate for broad characterization. The analysis of gene products and metabolism in Cyanothece can provide tremendous information about how the photosynthetic unicell compensates for this complex multi-tasking effort.This project is directed towards developing approaches and methods for the rapid detection of large numbers of peptides and metabolites in an extremely broad manner that is highly sensitive (e.g., able to detect low level species), quantitative, and is facilitated by high-throughput measurements. In addition, we will develop procedures and techniques for global metallomic characterization of Synechocystis 6803 and Cyanothece 51142 organisms. In this project, we focus on developing methods based upon the use of high-resolution capillary liquid chromatography combined with either Fourier transform ion cyclotron resonance (FTICR) or high mass accuracy time-of flight (TOF) mass spectrometry (MS) to increase the comprehensiveness, quantitation and throughput of the proteomics and metabolomics analyses, and leverage the significant separations and mass spectrometry capabilities originally developed at PNNL in the Biological Systems Analysis and Mass Spectrometry group.
We will apply various MS/MS methods in combination with accurate mass and LC elution time information to initially identify peptides and define “tags” so that subsequent measurements can be made without the need for routine MS/MS analyses. A similar approach will be taken for cataloging and tracking, initially structurally-unidentified, metabolites. Using mass and time (MT) tags, broad proteome and metabolome measurements can be made with much higher throughput. Under the Specific Aims of the project we will: (1) develop approaches using pulse chase stable isotope labeling to quantitatively measure protein turnover rates and dynamics in time-course studies in proteomic methods using both Synechocystis 6803 and Cyanothece 51142 as model systems, (2) Develop procedures and techniques for tracking large numbers of metabolomic features from Synechocystis 6803 and Cyanothece 51142 in time-course studies encompassing the light and dark cycles. This includes improvements to existing software designed to deisotope and display mass spectral data, and development of novel software to address the specific challenges presented by global metabolomics datasets. In addition, we will begin to structurally characterize any features displaying interesting behavior (i.e. dramatic increase or decrease in concentration) through targeted MS/MS studies and other means, if necessary (eg. NMR, chemical derivatization).and (3) develop procedures and techniques for global metallomic characterization of Synechocystis 6803 and Cyanothece 51142 organisms.
Project Details
Project type
Grand Challenge
Start Date
2005-12-16
End Date
2011-12-18
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Aryal UK, J Stockel, EA Welsh, MA Gritsenko, CD Nicora, DW Koppenaal, RD Smith, HB Pakrasi, and JM Jacobs. 2012. "Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light conditions." Journal of Proteome Research 11(2):609-619.
Aryal UK, J Stockel, RK Krovvidi, MA Gritsenko, ME Monroe, RJ Moore, DW Koppenaal, RD Smith, HB Pakrasi, and JM Jacobs. 2011. "Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles." BMC Systems Biology 5:Article No. 194. doi:10.1186/1752-0509-5-194
Ding J, CM Sorensen, Q Zhang, H Jiang, N Jaitly, EA Livesay, Y Shen, RD Smith, and TO Metz. "Capillary LC coupled with high-mass measurement accuracy mass spectrometry for metabolic profiling." 2007. Analytical Chemistry (Electronic publication ahead of print).
Metz TO, Q Zhang, JS Page, Y Shen, SJ Callister, JM Jacobs, and RD Smith. 2007. "Future of liquid chromatography-mass spectrometry in metabolic profiling and metabolomic studies for biomarker discovery." Biomarkers in Medicine 1(1):159-185.
Nagarajan A., M. Zhou, A.Y. Nguyen, M.L. Liberton, K. Kedia, T. Shi, and P.D. Piehowski, et al. 2019. "Proteomic insights into phycobilisome degradation, a selective and tightly controlled process in the fast growing cyanobacterium Synechococcus elongatus UTEX 2973." Biomolecules 9, no. 8:Article Number 374. PNNL-SA-146394. doi:10.3390/biom9080374
Stockel J, EA Welsh, ML Liberton, R Kunnavakkam, R Aurora, and HB Pakrasi. 2008. "Global transcriptomic analysis of Cyanothece 51142 reveals robust diurnal oscillation of central metabolic processes ." Proceedings of the National Academy of Sciences of the United States of America 105(16):6156-6161. doi:10.1073/pnas.0711068105
Stockel J, JM Jacobs, TR Elvitigala, ML Liberton, EA Welsh, AD Polpitiya, MA Gritsenko, CD Nicora, DW Koppenaal, RD Smith, and HB Pakrasi. 2011. "Diurnal Rhythms Result in Significant Changes in the Cellular Protein Complement in the Cyanobacterium Cyanothece 51142." PLoS One 6(2):Article No.: e16680. doi:10.1371/journal.pone.0016680
Toepel J, EA Welsh, T Summerfield, HB Pakrasi, and LA Sherman. 2008. "Differential Transcriptional Analysis of the Cyanobacterium Cyanothece sp. Strain ATCC 51142 during Light-Dark and Continuous-Light Growth." Journal of Bacteriology 190(11):3904-3913. doi:10.1128/JB.00206-08
Wegener KM, EA Welsh, LE Thornton, NS Keren, JM Jacobs, KK Hixson, ME Monroe, DG Camp, II, RD Smith, and HB Pakrasi. 2008. "High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex." Journal of Biological Chemistry 283(41):27829-27837. doi:10.1074/jbc.M803918200
Welsh EA, ML Liberton, J Stockel, T Loh, TR Elvitigala, C Wang, A Wollam, RS Fulton, SW Clifton, JM Jacobs, R Aurora, BK Ghosh, LA Sherman, RD Smith, RK Wilson, and HB Pakrasi. 2008. "The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle." Proceedings of the National Academy of Sciences of the United States of America 105(39):15094-15099. doi:10.1073/pnas.0805418105
Xu M, T. Ogawa, HB Pakrasi, and H Mi. 2008. Identification and localization of the CupB protein involved in constitutive CO2-uptake in the cyanobacterium, Synechocystis sp. strain PCC 6803. Plant and Cell Physiology 49(6): 994-997