Going Where No One Has Gone Before

By Daniel Drell

A completed microbial (or any other) genome represents the source code for life. More tangibly, it represents a list of the working and structural parts that a cell or multicellular organism requires to exist and to function. But unlike the instructions that accompany a bookcase or other piece of assemble-it-yourself furniture, the genome doesn't reveal the number of parts nor how to put them together into a working whole. The next steps, given the genome-derived parts list, must be done the "old fashioned" way, by experiment. Only then can a model be built that represents a more sophisticated understanding of the design and functioning of a cell.

On June 13, 2001, DOE's Office of Biological and Environmental Research (OBER) launched the newest and, in the view of some, the boldest initiative since the Human Genome Program: the Microbial Cell Project (MCP). DOE is taking the "parts list" revealed by completed microbial genomes and is beginning the exciting process of working out the users' manual. DOE thus will provide the fundamental understanding that will allow the microbes to be used in addressing the challenges of carbon sequestration, bioremediation, cellulose degradation, energy production, and biotechnology. [See the complete explanation of MCP in HGN 11(3–4).]

With the assistance of DOE's offices of Basic Energy Sciences (BES) and Advanced Scientific Computing Research (ASCR), applications and proposals submitted in response to two solicitations were peer reviewed (www.er.doe.gov/production/grants/Fr01-20.html and www.er.doe.gov/production/grants/Fr01-21.html). Awards were made to initiate research into how microbial cells work and how high-throughput computational technologies can be exploited to model their functioning.

The philosophy of the Microbial Cell Project (MCP) is that with the parts list in hand, studying a microbial cell as a system is unavoidable. It requires looking not only at the individual parts (particularly those whose functions are not obvious) but also at the "machines" that the cell uses the parts to construct. Additionally, we need to know the number of parts or machines in the cell, the effects of cytoplasm (which is known to have a consistency more like dilute gelatin than of pure water) on protein function and protein-protein interactions, the kinetics and rate constants of protein and protein complexes, and the location of proteins and complexes inside the cell. Focused on a few types of fully sequenced microbes, this knowledge can facilitate the rational modeling of a microbial cell's pathways and functions and the exploration of ways to regulate functions in ways that can serve DOE missions.

The MCP is inseparable from the larger new DOE initiative, Genomic Science program (formerly Genomics:GTL). The program aims to explore protein machines at the whole-genome level, characterize the regulatory networks overseeing them, characterize microbial communities and their interactions, and model cell behavior.

The new MCP awards are allied with grants being made by ASCR.

New Awards in MCP

With the assistance of DOE's Offices of Basic Energy Sciences (OBES) and Advanced Scientific Computing Research (OASCR), applications and proposals submitted in response to two Microbial Cell Project solicitations were peer reviewed in June 2001 by the DOE Office of Biological and Environmental Research. Awards were made to the following investigators:

Jim Fredrickson (Pacific Northwest National Laboratory), Jizhong Zhou (Oak Ridge National Laboratory), and Eugene Kolker (Institute for Systems Biology): Initiate the "Shewanella Federation," a multi-investigator and multi-laboratory consortium to characterize the biology of the fully sequenced bacterium Shewanella oneidensis MR-1. This microbe is widespread in the environment and has metabolic capacities allowing it to "handle" (reduce or oxidize) many of the metals of major concern to DOE due to their presence at contaminated DOE sites. The research will use innovative technologies to analyze the proteome and transcriptome using mass spectrometry (MS) and microarray methods; localize the proteins in the cell envelope using various microscope approaches; characterize the biochemistry and physiology, using optical methods to examine protein-to-protein interactions (in collaboration with Shimon Weiss at University of California, Los Angeles); and model cell networks (in collaboration with Bernhard Palsson, University of California, San Diego). OASCR is helping to fund the Kolker and Palsson elements of this federation.

Harley McAdams (Stanford University): Study Caulobacter crescentus, an important organism for bioremediation. The five main goals are to identify genome circuits involved in growth; study the physiology of cells in biofilms; study signal transduction networks; combine these experiments with computational programs to predict metabolic and regulatory pathways and operons and to construct a regulatory map to identify networks that control growth cell cycle, biofilm formation, and response to stress; and model its biology, using this vast array of information generated about the biology of C. crescentus. OBES is helping to fund this project.

Derek Lovley (University of Massachusetts, Amherst): Study the energetics and metabolism of Geobacter sulfurreducens, a model organism for various Geobacter species and other iron-reducing microbes. Geobacter species dominate subsurface sites in which iron reduction occurs. The project will model central metabolism, electron transport, growth under nutrient-limiting conditions, regulatory mechanisms, and environmental responses in G. sulfurreducens. The long-term goal is to develop a predictive in silico model of these processes.

Michael Daly (Uniformed Services University of the Health Sciences): Study the multiple cellular components of the responses of the highly radiation-resistant microbe, Deinococcus radiodurans, to acute and chronic radiation exposure. This project will use computational and experimental approaches including whole cell MS and DNA arrays. D. radiodurans is capable of resisting enormous doses of radiation, and the mechanisms behind this ability are as yet uncharacterized.

Robert Tabita (Ohio State): Study Rhodopseudomonas palustris, a model purple nonsulfur photosynthetic bacterium. This bacterium is remarkably versatile in the ways in which it can grow. The genome sequence is complete, and the annotation is nearing completion. A genetic system is available. This group will carry out research to characterize the biochemical and physiological processes as well as functional proteomic analyses using expression profiling with transcriptional microarray analysis, intracellular localization, and computational cell process modeling. The combination of genomics, proteomics, DNA array technology, bacterial "two hybrid" systems, and directed and random mutagenesis constitutes a global approach to the biology of R. palustris. OBES also is contributing to this project.

Timothy Donohue (University of Wisconsin, Madison): Analyze and model the flux of carbon, nitrogen, and "reducing power" of the versatile microorganism, the alpha proteobacterium Rhodobacter sphaeroides strain 2.4.1. A main goal is to understand the complex regulation system in this versatile bacterium and the expected complexity of the genome. OBES is helping to fund this work.

John Leigh (University of Washington): Study Methanococcus maripaludis, a methane-producing microbe of considerable promise for understanding biomass conversion. The focus will be on understanding its physiology (using microarrays) and its regulatory systems (using mutagenesis of candidate regulatory genes), especially those pertinent to methane production. OBES is taking the lead on funding this project.

Willem Vermaas (Arizona State University): Study Synechocystis, the first photosynthetic organism whose genome was completely sequenced. The main approach will be to exploit the library of targeted gene deletion mutants that are available (with more becoming available) to study photosynthesis and electron transport pathways. This will be combined with other proteomic, metabolic, and intracellular localization information to build a metabolic model. OBES is taking the lead on funding this project.

Three smaller technology-focused activities also are being initiated under the MCP umbrella.

Norman Dovichi (University of Washington): Develop capillary analysis technologies to permit the monitoring of changes in protein expression in single cells using fluorescence and pushing the resolution by an order of magnitude over what it presently is; in short, to build a "better microscope" for tracking gene expression in single cells following environmental challenges (e.g., exposure to radiation).

Kenneth Downing (Lawrence Berkeley National Laboratory): Study the use of electron tomography to image the inside of a microbial cell by freezing intact microbial cells in a way that preserves one layer of liquid water molecules above their membranes (permitting survival and viability). EM images and computer reconstruction are then be used to derive 3-D images of internal cell constituents.

Gary Andersen (Lawrence Livermore National Laboratory): Pursue advanced protein-to-protein interaction systems to characterize the protein machines within Caulobacter crescentus (in collaboration with Harley McAdams, Stanford University). This project will build a cellular protein interaction map using a protein-fragment complementation assay that offers advantages over the yeast two-hybrid system.

Allied with these new awards are grants being made by the DOE OASCR.