Projects

Systems Biology of Photosynthetic Organisms

The central approach in most of the biological sciences during the last half of the twentieth century has been based on reductionism, and has resulted in a massive increase in our knowledge of individual cellular components.  However, we have also realized that while we know how the individual ‘nuts and bolts’ function in isolation, our understanding of how these components interact with each other to define the cellular, organismal, and population level behaviors of living beings has remained far less sophisticated.  A ‘Systems Biology’ approach should provide enabling technologies to examine complex biological processes, which should in turn result in an integrated and predictive understanding of how an organism behaves and responds to environmental changes.  The successful implementation of a systems approach requires collaborative interactions between biologists, computer scientists, mathematicians and model builders, engineers, physicists, chemists, and perhaps specialists in other disciplines. 

The goal of this project is to use systems biology approaches to determine the underlying network that governs photosynthetic processes in cyanobacteria, vascular plants (Arabidopsis) and mosses.  In photosynthetic organisms, the cellular components are always in flux, and molecular machines assemble, function and disassemble as a function of time and environmental alterations such as light intensity and nutrient availability.  It is imperative to utilize a systems biology approach and integrate temporal information from microarray and proteomic studies into a predictive, dynamic model to understand the functioning of a photosynthetic organism.  We have recently initiated two large scale, multi-institutional, systems biology projects (NSF-FIBR and PNNL Grand Challenge).

 

Photosystems

We study two pigment-protein complexes, photosystem I (PSI) and photosystem II (PSII), in the thylakoid membranes in cyanobacteria and plants.  Both PSI and PSII convert solar energy into biologically usable chemical energy.  PSII catalyzes the production of molecular oxygen from water. We use Synechocystis 6803, a unicellular cyanobacterium as a microbial model system to study the photosystems. Synechocystis 6803 cells are naturally transformable with exogenous DNA.  Moreover, it is the first photosynthetic organism, the genome of which has been completely sequenced.  

The two photosystems are each composed of a large number of protein subunits and cofactors.  Their complex structure requires precise and regulated assembly.  Furthermore, PSII is turned over frequently because the complex is damaged during its normal function.  We are using various genetic, biochemical and biophysical approaches to study PSII function and biogenesis. To examine the sequence of events leading to the biogenesis of the photosystems in chloroplasts, we are using Arabidopsis thaliana, a green plant, as well as the moss, Physcomitrella patens, as experimental organisms.

 

Membrane Biology

Cyanobacteria are unique prokaryotes, since they contain a differentiated membrane system.  In addition to the envelope layer consisting of outer membrane (OM), periplasm and plasma membrane (PM), these Gram-negative bacteria have the intracellular chlorophyll-containing thylakoid membrane (TM) involved in oxygenic photosynthesis.  Recent work from our lab emphasizes the close relationship of cyanobacterial membrane architecture and photosystem biogenesis.  

In cyanobacteria, the TM is the site for both photosynthetic and respiratory electron transfer reactions.  Thus, the two photosystem complexes, PSI and PSII, function in the TM.  However, one recent unexpected finding was that many of the central components of both PSI and PSII are also present in the purified PM.  Furthermore, these proteins are assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, and are capable of undergoing light-induced charge separation.   Based on these data, we have proposed that the PM, and not the TM, is the site for the early steps of biogenesis of these two complexes in cyanobacteria. 

Our photosystem biogenesis model predicts movements of partially assembled protein complexes from one membrane system to the other.  Such translocations can occur if the thylakoid membranes are contiguous with the plasma membrane or if there is directional flow of membrane vesicles from one membrane to the other.  We are currently employing a variety of ultrastructural and proteomic techniques to unravel the translocation mechanism.

 

Metal Homeostasis

The metal content within cells are under strict homeostatic control that involve acquisition, storage and disposal of metals, since either too much or too little of these metals lead to deleterious effects.  Transition metals are especially important in energy transduction pathways in photosynthetic organisms.  The photosynthetic apparatus in cyanobacteria and chloroplasts has at least 24 different metal cofactors.  Key elements of our investigations on photosynthesis are specifically focused on how Synechocystis 6803 maintains the intracellular balance of manganese.  We have identified and studied transporters for manganese (MntABC) and zinc (ZnuABC) in Synechocystis 6803, as well the transcriptional regulators for these transporter complexes (MnrSR and Zur).  Our collaborator Tom Smith (Donald Danforth Plant Science Center) has recently solved the structure of ZnuA by x-ray crystallography.

 

Redox Processes

Redox homeostasis is central to the overall functions of oxygenic photosynthetic organisms such as cyanobacteria and plants.  We are using a systems approach to analyze the impact of cellular redox status on the overall function of these organisms.  We focus on the cyanobacterium Synechocystis 6803, Arabidopsis, a vascular plant, and Physcomitrella, a non-vascular plant.  Although the detailed inventory of the genes, transcripts and proteins are available for Synechocystis, it is inadequate to comprehend the organizational hierarchy of the complex functions of this organism.  Our approach to resolve this gap in our fundamental knowledge is multidisciplinary involving molecular genetics, biochemistry, proteomics, metabolomics, and computational biology.  An iterative process based on experimental results and predictive model-building will generate fundamental insights into the organization and function of the redox control network (RCN) in these organisms.  Furthermore, such an approach, first to model an RCN in cyanobacteria, and then to apply it to plants, will highlight the conserved nature of these processes during the evolution of land plants.

 

Genome Sequencing (Genomics)

Our lab, in collaboration with the Genome Sequencing Center at the Medical School, and Louis Sherman at Purdue University, is currently sequencing and annotating 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.