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Minimal Genomes: Life in a Nutshell

With the advent of genome sequencing, the age-old question of what constitutes life has taken on a whole new meaning. As we learn more about the functions of individual genes, we are asking more and more how much of whom we are is determined by our genetic code. And now, for the first time in human history, we are close to having the tools to not just decipher the function of genes, but to stitch those genes together to create the world's first synthetic organism. But are we really ready for made-to-order creatures?

Craig Venter, the scientist who gained renown by founding Celera, the private effort that sequenced the human genome in a race with the government-sponsored Human Genome Project. But even as he undertook the daunting task of sequencing the 3 billion bases of the human DNA, Venter was thinking about the bigger questions.

As early as 1997, years before the human genome project was completed, Venter was already speaking publicly about his interest in what constitutes life. At that time, Venter had targeted Mycoplasma genitalium, a bacterium with only about 500 genes, as the model organism to answer that question. With one circular chromosome of only 580,000 base pairs, M. genitalium is considered to be the simplest self-replicating organism known. Venter reasoned that if we ever hope to understand a complex genome like that of human beings (which consists of about 40,000 genes), we first ought to be able to understand how a much simpler set of genes functions to form the basis of life.

To answer the question of whether life can really be defined by an essential set of genes, Venter and his colleagues at The Institute for Genomic Research (TIGR), a non-profit institute that provides sequence data to the scientific public, proposed to create a synthetic organism. They would identify the genes essential for life in M. genitalium, and then chemically build an artificial genome in the hopes of generating a living microbe. The idea, in other words, was to create life from scratch in a test tube.

Mycoplasma genitalium: A Model Organism

In 1995, after sequencing the M. genitalium genome in just three months, the TIGR group began by studying Mycoplasma to decide how many of its 500 genes were absolutely essential for survival of the microbe. The plan was then to chain those essential genes together in hopes of creating the world's first synthetic organism.

Box 1: A Different Kind of Minimalism

In another of the steady stream of surprises emerging from the world of Archaea, the hyperthermophile Nanoarchaeum equitans reveals that one way to support a truly minimal genome is to depend on the largesse of a friendly neighbor. N. equitans has the smallest microbial genome sequenced to date--only 490,885 base pairs--and almost all of that DNA is chock full of genes. A full 95% of the genome is devoted to coding for proteins or RNA. This organism has genes which code for a full complement of ribosomal and transfer RNAs, together with a large set of enzymes for replication, transcription, and translation, including DNA repair enzymes. Surprisingly, however, N. equitans lacks almost all the genes for carbon metabolism such as the glycolytic pathway, citric acid cycle, and pentose phosphate pathway. It is also missing essentially all the elements required for the biosynthesis of lipids, amino acids, and nucleotides. While it has the genes for a very minimal ATP synthase, it is not at all clear how the organism could generate its own energy supply.

Given this paucity of metabolic functionality, it should come as no great surprise that N. equitans is an obligate parasite on Ignicoccus species, crenarchaea with which it grows in co-culture. By all accounts, N. equitans makes up for its lack of biosynthetic and metabolic activities by importing the needed products from its host. By doing so, it needs only the tiniest of genomes to survive, albeit always with the requirement for a generous lifetime partner.

E. Waters et al, Proc. Nat. Acad. Sci. in press October 2003

To determine which genes were truly essential, researchers performed a painstaking set of experiments where one gene was knocked out at a time. Any gene whose destruction resulted in the death of the bacterium was considered essential. By 1999, this strategy had identified about 300 genes that the organism absolutely could not live without. But even this impressive achievement did not definitively solve the riddle of the minimal genome.

Since the life-or-death screening technique used by the TIGR group identifies only genes whose absence causes death, genes that are not essential for Mycoplasma to live in the pampered environs of the laboratory (but might be essential in nature) could be missed. Thus, it might be that those 300 essential genes, if strung together, would actually result in a viable organism. Even if it did, the resulting microbe might be so sickly it would be hard to call it alive. It would be much like creating a laboratory mouse that could not breathe without a ventilator, or whose heart would not beat without a pacemaker. That mouse might be able to live as long as technological life support was available, but it would be unclear if such a creature would truly fit the definition of a self-sufficient, self-replicating life form. Mycoplasma is bound to have such genes; genes that might only play a supporting role in the sheltered environment of a laboratory flask, but that would indeed be essential for the bacterium to survive in a natural environment.

It thus turns out to be fiendishly difficult to actually answer the question of what genes are truly essential to life. Nonetheless, such fundamental research will improve our understanding of how genes work together to make organisms tick. And it is this understanding that is likely to spawn the most useful applications of synthetic organism research, including the treatment of bacterial infections or the understanding of human diseases.

Can Microbes Save Us from Fossil Fuels?

Venter's latest endeavor is a somewhat more ambitious offshoot of the original "minimal organism" project he started with Mycoplasma genitalium at TIGR. His new venture, the Institute for Biological Energy Alternatives (IBEA), received a $3 million dollar grant from the U.S. Department of Energy in late 2002 to develop a synthetic organism that will produce fuel or absorb pollutants. Picking up on the original project started at TIGR, the IBEA group will first focus on creating a synthetic organism with a minimal genome. To accomplish this, Venter's group will first paste together the essential Mycoplasma genes identified in 1999. This will create an artificial M. genitalium chromosome. They hope to then take this man-made loop of DNA and place it in a M. genitalium cell whose own DNA has been destroyed by UV radiation. If they succeed is creating a self-replicating organism, Venter and his colleagues will be the first to create synthetic life.

Animation: Venter's group plans to chemically synthesize each desired gene from the genetic building blocks A, G, T, and C. The genes will then be assembled end-to-end to create a circular genome of about 300 genes. The human-made chromosome is then injected into Mycoplasma cells whose own DNA has been destroyed by UV irradiation. Scientists hope this technique will result in a viable organism able to divide and replicate its new, artificial genome.

While success in this project may be years in the making, Venter has already plotted his next move. Once the synthetic bacterium has been created, the IBEA team hopes to add genes that will allow the new microbe to perform useful work. One idea is to create an organism that will generate clean-burning fuel, such as hydrogen gas, as a by-product of its metabolism. Or perhaps a bacterium could be engineered to absorb carbon dioxide, the "greenhouse gas" pollutant believed to be a major cause of global warming.

The idea that we might be able to generate organisms that will solve our most vexing fuel and pollution problems is tantalizing. Hydrogen fuel, which burns in oxygen to produce only clean water as a by-product, is being seriously studied as a fuel of the future. Extensive use of hydrogen fuel could drastically reduce acid rain, greenhouse gas emissions, and air pollution, but right now hydrogen fuel is too costly to produce. Creating an organism that would eat garbage and produce large amounts of hydrogen gas could make the Earth a much healthier place to live. Fossil fuels won't last forever, and it is becoming increasingly clear that massive worldwide use of carbon-based fuels is causing widespread, perhaps irreparable damage to our environment.

Thus, synthetic microbes that could absorb excess carbon dioxide from the atmosphere, or that could be made to eat whatever toxic chemical might need to be destroyed, would be able to reverse damage that humans have done to ecosystems around the globe. It is these exciting possibilities that drive Venter and his team of researchers at IBEA.

Microbes Made to Order: Is the World Really Ready for "McBug?"

Potential medical miracles and environmental breakthroughs notwithstanding, synthetic life is not without its drawbacks. It is not difficult to imagine sinister applications of such research. For example, M. genitalium, which makes its home in the human urogentinal tract, is well suited for living inside the human body. Such a trait could be exploited by someone designing a biological warfare agent. And even if the first synthetic organism created by the IBEA team turns out to be barely able to live in a Petri dish, the knowledge gained from the research is unlikely to be easily contained.

Box 2: Researchers Create the World's First Synthetic Virus

In July of 2002, a team from the University of New York at Stony Brook announced that they had constructed the world's first synthetic virus, a replica of the pathogen that causes polio. According to the Stony Brook team, creating the artificial polio virus, which is not quite as potent as its natural cousin, turned out to be relatively easy. The viral genome is small compared to more complicated viruses like smallpox, and the researchers bought gene sequences by mail order. They even used a protocol they found on the internet to glue the genes together. But, the scientists note, creating a bacterium, or even a more complicated virus (with their much larger genomes) would be an entirely different story. Still, Dr. Eckhard Wimmer, who headed the Stony Brook team, cautioned that it is only a matter of time before it becomes technically feasible for terrorists to assemble viruses of their choice, such as Ebola, in a lab. Although such a scenario is still many years away, Wimmer suggested that "the world had better be prepared."

Image of a polio virus (courtesy CDC/Public Health Image Library)

Since the first synthetic virus has already been created (see box 1), and we appear to be well on our way toward creating the first synthetic bacterium, it may be moot to ask whether pursuing artificial life is something we, as a society, ought to be doing. But as it turns out, the scientists at TIGR did ask a panel of scientific ethicists to study the implications of the project back in 1999, when the group first began working towards creating a minimal microbe from essential Mycoplasma genes. The panel deliberated for over a year, and in the end decided that it was not inherently unethical to create such an organism for the purposes of scientific study. They noted, however, that it would depend on how such knowledge was used. And that may be exactly the problem.

Once the roles of individual genes in organizing life are better understood, and the gene-swapping technologies become available, the proverbial genie may be out of the bottle. While the construction of even a feeble synthetic bug is likely years away, even Venter admits that his research might inevitably create a national security risk. If microbes can be made-to-order by labs working for the good of mankind, they can also be made by others with less-than-altruistic motives. But, Venter suggests, his work could also lead to better detection methods for existing biological weapons. One thing is clear: such research will continue to be closely watched by scientists, ethicists, governments, and citizens the world over for years to come.

Pinning Down the Meaning of "Life"

Ironically, even the best lab may never be able to determine the set of genes that determines life. That is because the lab setting is inherently artificial, and how organisms grow in the lab won't necessarily tell scientists what genes are needed in the constantly changing conditions of the natural world. And even if scientists can eventually agree on the basic set of genes needed for life, such research doesn't begin to address how those genes come together to create human self-awareness, emotions, and thoughts. Making microbes may be one thing, but humans tend to consider themselves more than just the sum of their parts. Will there ever be a time that life will be reducible to a list of genes and their physiological functions? That may be a question only philosophers can answer, but even Venter admits that pinning down a molecular definition of life could be difficult. "Nature," he notes, "just refuses to be so easily quantified."