What do you consider the primary lessons that have emerged so far from the microbial sequences?

The first would probably be the large number of genes that encode proteins of currently unknown function. I very distinctly remember some of our earliest discussions on the Haemophilus project, where we speculated that once we had the genome completed and the whole list of genes written down, it would be clear how all these genes worked together to create a living cell. It's now astounding to me that we could have been either so naïve or arrogant, or both—we couldn’t even come close to figuring out what the genes in Haemophilus did. One of the biggest surprises was that about 35% of the predicted genes had no match to any other genes in our databases. That observation was really the driving force in our choosing Mycoplasma genitalium for our second organism. Its genome size was estimated to be less than 500 genes. So we were thinking that although we might not have been able to understand the genome of Haemophilus at 1,750 genes, we'd certainly manage to understand the Mycoplasma genome with less than 500. But when we finished that sequence, we found once again that 30 to 35% of the genes were unknown. At that point, after having done our second genome, one from the most minimal self-replicating organisms, we all realized that it was going to take us a long time before we figured out the biology of even the simplest prokaryotic cell. And that 30% number has held up even though we're now at 60-plus completed genomes.

A second lesson has been the observation that horizontal gene transfer in the microbial world seems to play a much bigger role in generating species diversity than has ever been appreciated before. There's now a very long list from sequencing projects at TIGR and elsewhere and very strong evidence to suggest that, in some of these genomes, as much as a quarter of the genes look as if they've been acquired horizontally from different species. In fact, Vibrio cholerae, which we completed last year, has a large and small chromosome, and it looks as if the smaller chromosome was probably acquired in its entirety from another species at some point in the history of the organism.

How does a species acquire genes horizontally, let alone an entire chromosome?

There are a number of species—Haemophilus happens to be one of them—that are known to have the ability to take up naked DNA from solution. That's one obvious mechanism. In other cases, it may be that bacteriophage are involved. And in some of these cases it's not clear at all how this lateral gene transfer may have occurred.

Most-Cited Papers by Claire M. Fraser Published Since 199 (Ranked by total citations) Rank Paper Total Citations 1 R.D. Fleischmann, et al., "Whole-genome random sequencing and assembly of Haemophilus influenzae Rd," Science Science, 269(5223):496-512, 1995. 2,118 2 C.J. Bult, et al., "Complete genome sequence of the methanogenic archaeon, Methanococcus jannachii," Science, 273(5278):1058-73, 1996. 1,337 3 N. Papadopoulos, et al., "Mutation of a mutL homolog in hereditary colon cancer," Science, 263(5153):1625-9, 1994. 1,063 4 C.M. Fraser, et al., "The minimal gene complement of Mycoplasma genitalium, Science, 270(5235):397-403, 1995. 1,003 5 J.F. Tomb, et al., "The complete genome sequence of the gastric pathogen Helicobacter pylori," Nature, 388(6642):539-47, 1997. 983 SOURCE: ISI's Personal Citation Report, 1981 - 2001

How have these sequenced microbial genomes changed our understanding of the evolution of microorganisms?

Well, this is the point I was making about the fallacy of a model microbial organism. One of the more interesting ideas that’s come out of all of this genome analysis—and the idea of lateral gene transfer feeds into this somewhat—is that we may need to redefine, if we can, our definition of what constitutes a prokaryotic species. For the past 25 years, the definition has been based on Carl Woese's classification of species from their 16s ribosomal RNA sequences. Open any biology textbook and look at the tree of life, and it's based on comparing sequences from a single gene. That phylogenetic scheme presupposes that species arise essentially by the transfer of DNA, of traits from ancestors to progeny. But there is no way that you can get insight into how much lateral gene transfer has occurred and what the overall biology of a particular species is going to be by comparing sequences from a single gene. Take E. coli, for example. There's the K12 strain, which has been well-studied for many, many decades, and which has a genome size just over 4 million base pairs. Recently Fred Blattner's lab finished sequencing E. coli O:157, the strain making trouble in undercooked hamburgers. That organism has a genome size 20% larger than the K12 strain. So these are both strains of E. coli and yet their genomes differ by close to 1,000 genes. Are they truly the same species? All of this has forced many researchers, certainly a lot of evolutionary biologists, to start to rethink how to define prokaryotic species. It's just not as simple any more as looking at the sequence of a single gene and being able to discern from that everything you need to know about that particular organism.

So where do you go from here? What are the next five years going to bring?

It's important to keep doing what we're doing, but I also think it's time to make a shift in terms of the types of organisms that we sequence. So far we've been sequencing organisms that could be grown in the lab. We have to start looking at organisms that come from different environments but which cannot be grown in culture. If we're going to understand biodiversity on the planet, and a great deal of biodiversity is represented by microbial species, we're going to have to start looking beyond what's targeted so far. Some of the more important insights on the role of microbial species in the bio-geo-chemical cycles of the planet will come from looking at some of these environmental organisms that grow in communities and which, because they are absolutely dependent on other members of the community for nutrients, probably cannot be easily grown in isolation in the lab.

The possibilities that have now opened up before all of us are just enormous. In fact, I currently sense some frustration on the part of the various funding agencies. There is simply so much good science going forward already, and opportunities have increased exponentially with the number of base pairs we've sequenced. But the funding isn't even close to keeping pace. They're now having to set some difficult priorities between continued sequencing versus starting to use the information we now have to do all sorts of functional studies. At the same time, we also need to put sufficient funding into building databases to handle the information that will come from functional genomics studies and be able to link that back to the gene and protein sequences that already exist. It's both a good and a bad time. There's so much good science to be done, but we have to deal with the frustration of setting very difficult priorities.