EMSL Scientific Grand Challenge: Membrane Biology
Membrane Biology GC Resources
Project Achievements
EMSL's Membrane Biology Scientific Grand Challenge researchers have grown Cyanothece in defined culture conditions and entrained it to a 12-hour light/12-hour darkness cycle with samples being collected every two hours. Using those samples, researchers sequenced and annotated the Cyanothece 51142 genome, purified various membrane fractions from cyanobacterial cells, and determined the structure of several of its proteins. In addition, researchers have built a bioreactor in which the Cyanothece is cultured, developed software tools to aid in the analysis of data gathered, been able to visualize in detail the cell membrane using TEM imaging, and produced a number of publications.
Cyanothece 51142 genome
The Pakrasi lab, in collaboration with the Genome Sequencing Center at the Medical School of Washington University in St. Louis, and Louis Sherman at Purdue University, is currently completing the sequencing and annotation of the genome of the unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. Oxygenic photosynthesis and N2 fixation are important metabolic processes that are at odds with each other, since the N2-fixing enzyme, nitrogenase, is highly sensitive to oxygen. We are interested in the strategies devised by Cyanothece to permit N2 fixation and photosynthesis to coexist in the same cell. This organism has developed a type of temporal regulation in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle with very high levels of CO2 fixation during the light and high levels of N2 fixation in the dark. The analysis of gene structure and regulation in this organism can provide tremendous information about how this photosynthetic unicell deals with one of the biosphere’s greatest juggling jobs. Therefore, we are sequencing the Cyanothece genome so that we can better use this organism to study processes such as circadian rhythms, CO2 fixation and sequestration, N2 fixation, hydrogen production, compartmentalization of metabolites (such as carbohydrate and cyanophycin) and the relationship of redox control to photosynthesis and respiration.
Structures
Determined the structures of:
- NrtA
- CmpA
- Rfr23
- Rfr32
Morphology & Physiology
To study morphology and physiology of Cyanothece, the Membrane Biology Grand Challendge team:
- Developed the use of fluorescent antibodies for tagging specific proteins in intact Cyanothece
- Quantified changes in phycobilisome and chlorophyll expression along the cycle
- Identified circadian rhythm in cell size and complexity
- Developed electron tomography and cryostage, HPF and AFS for high resolution structural studies
Software
Developed software to support analyzing interactions of membrane protein sand membrane systems, energy production, and the circadian clock apparatus. This includes:
- PQuad
- ScalaBLAST
- SEBINI
- Similarity Box
Systems for Culturing Cyanothece
- Designed, fabricated and deployed photobioreactor systems for culturing cyanobacteria.
- Established pure Cyanothece cultures
- Enabled data integration across laboratories
Contact: Dave Koppenaal | david.koppenaal@pnl.gov | (509) 371-6549
