The main goal of this project is to understand how organisms simultaneously sense and appropriately process changes in a wide array of environmental factors to mount an optimal response.

Summary.

The degree of sensitivity exhibited by an organism to protein and DNA damaging stress is related not only to the efficiency of repair systems, and avoidance strategies such as phototaxis and production of photo-protective substances, but also to regulatory mechanisms that elicit the optimal response. As a consequence of natural selection pressure in the environment, organisms have evolved sophisticated mechanisms to minimize and correct cellular damage resulting from stressful conditions such as desiccation, increased metal ions and ultraviolet (UV) radiation. These include: physiological mechanisms that recognize and repair damage (e.g., photolyase repair of UV damage to DNA); the production of antioxidants to neutralize reactive oxygen species; and physical movement away from damaging circumstances or synthesis of UV-absorbing compounds. Therefore, identifying and understanding these regulatory mechanisms at a genetic and biochemical level is fundamental to understanding the system-wide response of an organism to individual environmental factors and combinations thereof.

Primary methodologies/approaches/strategies used to accomplish the goals

We are using systems approaches to investigate responses in Halobacterium to extremes in a wide variety of environmental factors. Halobacterium is an extremely halophilic archaeon that thrives in an environment of saturated salinity (~4.5M) that is fraught with unusually high amounts of DNA and protein damaging agents. This naturally makes Halobacterium an ideal organism in which to interrogate environmental response mechanisms. Some of the environmental factors being investigated include metal salts, UV radiation, oxygen stress and gamma radiation.

For propagating cells in controlled environments, we have shaking incubators with light banks, a chemostat, and a sun box to deliver controlled doses of perturbations in closely monitored and tightly-controlled culture conditions. To isolate cells of similar defined characteristics such as size, DNA content, etc., we utilize cell sorting strategies in collaboration with the van den Engh laboratory at the ISB. Our investigations into phototaxis utilize microfluidic devices (in collaboration with Carl Hansen) as they are an ideal environment for studying phototaxis due to favorable fluid dynamics where viscose forces dominate and there is a complete absence of turbulence. Further, light intensity and spectral quality can be controlled by creating micron scale light gradients. This level of fine scale experimental control and manipulation is only possible within microfluidic devices. Another study we are conducting in collaboration with Dr. Carl Johnson at Vanderbilt University is focused on halobacterial behavior in response to daily fluctuations in quality and quantity of light. For this we also make use of culturing incubators that can simulate day light conditions.

We also have devised a flexible array of tools for high throughput and genome-wide interrogation of changes that occur at all of the information levels in the cell while mounting a robust response to perturbations in one or more environmental factors. These tools include a high-density expression microarray for measuring mRNA level changes, a whole genome high density array for monitoring protein-DNA interactions using ChIP-chip, and ICAT- and iTRAQ-based proteomic technologies for measuring protein levels and protein-protein interactions. To deconvolute genetic pathways involved in the response, we also have optimized an in-frame gene replacement strategy for rapid construction of gene deletions and targeted chromosomal mutations, as well as protein expression systems for over-expression and epitope tagging of candidate proteins. The complex systems biology datasets that result from these high-throughput inquiry strategies are integrated and analyzed with semi-unsupervised statistical learning procedures that are guided by existing knowledge in literature.

Key results obtained for the project as of June 2005.

Systems Analysis of Phototrophy:
Our proof-of-principle systems analysis of purple membrane biogenesis focused on three genetic perturbations in two genes encoding bacteriorhodopsin (bR) synthesis, bop, the structural gene for bacterio-opsin (Bop), and bat, the transcriptional activator which regulates bacterio-opsin as well as retinal biosynthesis; (bat knockout: bat-; bat over-producer: bat+; bop knockout: bop-). Bop and retinal form the light driven proton pump, bR, multiple copies of which assemble in the membrane to form the purple membrane. The purple membrane drives ATP synthesis under anaerobic conditions in the presence of light.

Perturbation in the transcriptional regulator bat resulted in differential expression of nearly seven percent of all of the 2,400 genes interrogated by microarray analysis. In contrast the perturbation in bop was associated with differential expression of a mere one percent of all genes, implying that Bat plays a bigger role in the regulatory network than Bop. Next, we used the ICAT approach to analyze the patterns of protein expression in the bat knockout (bat-) and bat over-producer (bat+) strains. A total of 1,120 tryptic peptides, containing cysteine-attached light and heavy ICAT isotopes, were analyzed from 272 different proteins. At least 50 of these proteins were differentially expressed with only 17 (35 percent) corresponding changes at the mRNA level emphasizing the importance of an integrative approach to systems analysis.

Using the data integration and visualization software tool Cytoscape, the mRNA and protein level data were analyzed in the context of function categories and functional associations among proteins derived through evolutionary comparisons. The integration of quantitative changes in the mRNA, and protein levels in the Halobacterium NRC-1 wild type, and the three mutants, revealed a coherent picture of the regulation of phototrophy and its interactions with other aspects of metabolism. The over-expression of bat induces all four regulon genes for carotenoid and bR biosynthesis to generate the purple membrane. It is striking that the genes of arginine fermentation, arcA, B, and C are down regulated in keeping with the fact that bR is generating significant ATP and, presumably, a balance in ATP production is maintained by down-regulating the second major source of ATP under anaerobic conditions of arginine fermentation. The bat,- strain, in contrast, suppresses the bR biogenesis, and the arginine synthesis and the carotenoid synthesis genes. It up-regulates the genes of arginine fermentation presumably to complement the loss of bR mediated ATP production. It also up regulates the arg-tRNA synthetase gene argS, presumably to capture sufficient arginine to maintain protein synthesis and an amino acid transporter yhdG, which may transport more arginine into the cell.

Systems Analysis of UV damage:
Another proof-of-principle for our systems approach is provided by the systems analysis of responses of Halobacterium sp. To damaging UV-C radiation. Besides demonstrating a role for Phr2 (photolyase) in light repair, the UV-C response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2,400 genes during light and dark repair. In addition to photo-reactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes and two proteases for eliminating damaged proteins. Moreover, a UV-induced down regulation of many important metabolic functions was observed during light repair. Global repression of metabolism during DNA repair has been reported in E. coli, D. radiodurans and S. cerevisiae, suggesting that this might be a generalized stress response mechanism shared by all three domains of life.

Global down-regulation of genes results from the necessity of the cells to maintain internal homeostasis in response to abrupt changes in the environment. This global down-regulation might also help conserve energy required for costly DNA repair processes, as has been previously proposed for down-regulation of ribosomal protein genes during stress response in yeast.

The systems analysis also facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. In addition to DNA repair mechanisms, the systems analysis provides insights into regulatory mechanisms for several processes, including ribosome biogenesis, transcription and cofactor biosynthesis.

Future directions of this project. Current research is focused on the molecular mapping and dynamic simulation of gene regulatory and signaling circuits combined with experimental testing of simulated dynamics. Upon verifying the regulatory networks we then plan to explore exciting avenues such as designing novel circuits to enhance physiological capabilities of this microorganism. This will require development of new algorithms for regulatory circuit design and also new tools such as tunable promoters that can be turned "on" or "off" in a controlled manner.

How does the project address the issues of predictive, preventive or personalized medicine?

The outcome of this project will be a model for perturbation-induced rewiring of the gene-regulatory network of Halobacterium sp. so we can correlate input (environmental perturbations) to output (phenotype), i.e., a predictive model for regulatory mechanisms for environmental response systems. This basic model can then serve as a template for designing systems approaches to model higher complexities such as those associated with eukaryotic systems. Another long term vision is to use these predictive mathematical models for gene regulatory networks in this organism to engineer designer circuits for a variety of biotechnological applications such as environmental clean up and predictive and preventive medicine.

Group members involved with Project

Kenia Whitehead, Amy Schmid, Min Pan, Amardeep Kaur, Nitin Baliga (PI)

Internal ISB groups/people involved with project.

Ger van den Engh
Carl Hansen
Ruedi Aebersold

External collaborators

Carl Johnson — Vanderbilt University, Nashville, TN
Jocelyne DiRuggiero — University of Maryland, College Park, MD
Raafat El-Geweley — University of Tromso, Norway

Representative publication(s):

Baliga, N.S., Pan, M., Goo, Y.A., Yi, E.C., Goodlett, D.R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W.V., and Hood, L. (2002) Coordinate regulation of energy transduction modules in Halobacterium sp. Analyzed by a global systems approach. Proc Natl Acad Sci U S A 99: 14913-14918.

Baliga, N.S., Bjork, S.J., Bonneau, R., Pan, M., Iloanusi, C., Kottemann, M.C.H., Hood, L., and DiRuggiero, J. (2004) Systems Level Insights Into the Stress Response to UV Radiation in the Halophilic Archaeon Halobacterium NRC-1. Genome Res. 14: 1025-1035.