Making do with the bare minimum

Mycobacterium tuberculosis is the bacterium responsible for tuberculosis. Exploring the organism’s so-called essential genes could reveal ways to battle drug-resistant strains. Several groups have touted the information that could be gleaned from a minimal set of genetic instructions in building organisms from the ground up or stripping away all nonessential genes to create a minimal organism. More practically perhaps, all antibiotics target essential genes. Finding those ingredients that an infection can’t live without might prove useful. Researchers have been looking for the essential genome for at least a decade, using tactics such as comparative genomics to computationally identify genes that organisms have in common1 and brute force mutagenesis to whittle away the chaff. The papers featured here represent two influential attempts to achieve the same goal. In one, Eric Rubin’s lab at Harvard University used probes generated from randomly transposon-mutagenized Mycobacteria tuberculosis, spotted onto microarrays, to determine where the transposons had landed, and thus which genes could be disrupted. In the other, a large consortium from Europe and Japan systematically inactivated more than 4,000 Bacillus subtilis genes to determine which mutant organisms were viable.

Knowing the essential complement of genes, like knowing the sequence, is all part of characterizing a bacterium these days, notes Nina Salama of the Fred Hutchinson Cancer Research Center. Her own Helicobacter studies use methods similar to those of Rubin. “We have a sequence, but what does it mean?” asks Howard Ochman, who studies the molecular evolution of bacterial genes and genomes at the University of Arizona. Determining a gene’s function may be important, he says, but one also needs to assess whether a particular gene is critical to an organism’s growth. By providing an answer to this question, these papers “open all sorts of avenues to develop drugs that can inhibit microorganisms,” says Dusko Ehrlich of the National Institute for Agricultural Research (INRA) in France, who led the Bacillus effort. The lists have provided a valuable reference, says James Sacchattini, director of the TB Structural Genomics Consortium from Texas A&M University. Rubin’s work has been like “a divining rod” to the consortium, says Sacchattini, helping it to identify proteins and pathways to study as potential targets for chemotherapy. These papers examine “experimentally, what is the minimal gene set required to make a living cell,” says Ehrlich. About 80% of the approximately 250 genes found essential for B. subtilis are present in all bacteria that have a genome of “a decent size, about 2.5 to 3 megabases or above,” he says.

In contrast, Salama’s group has found surprisingly little overlap, only 11%, among the essential genomes of Helicobacter pylori and the other bacteria they examined, with 55% of the genes shared by only some species.5 “I think that reflects all these subtly different niches for which these different bacteria are adapted,” she says. “Targeted disruption is a gold standard, but it’s very time consuming.”

Other distinctions are important. Rubin tries to avoid the word ‘essential,’ and prefers instead to talk about genes required for optimal in-vitro growth. Ehrlich points out that essential genes really need to be defined by the conditions under which they were tested: “We used rich medium for our test. If we used different medium, many more genes would be required, because [the bacteria may] have to synthesize all the amino acids and vitamins.” Assays such as Rubin’s screen for optimal growth in randomly mutagenized cultures. Thus, a mutation conveying drastically slower growth would probably be scored as lethal (because the bacteria harboring it would be out-competed), and the gene would be seen as essential. “Our method is just a screen, but it’s fast and easy,” Rubin says.

“The Bacillus method is very precise – there’s no arguing with it – but it’s a huge amount of work,” he says. “Targeted disruption is kind of a gold standard, in that you’re specifically targeting each of the open reading frames,” Salama agrees. Yet both random and targeted approaches still rely on negative results: The genes considered to be essential are those that cannot be, or are not, mutagenized. “If a gene is essential, you can’t knock it out,” says Ehrlich.

Researchers employ a variety of strategies to assure that the inability to obtain a mutant is not merely an artifact of the methodology. Ehrlich’s consortium placed recalcitrant genes behind an inducible promoter, allowing them to demonstrate that the bacteria were viable when the gene was induced, and nonviable when it was not. Rubin’s group takes cosmids containing the gene, inserts them into Escherichia coli, and then mutagenizes them, showing that the gene can be inactivated. Salama adds a second copy of the gene to her bacteria, and then knocks out one of the copies. But even these methods do not assure that every open reading frame gets queried. “It’s hard to measure, but I think in general, the list of genes has held up pretty well,” says Rubin. “It’s held up to the analysis that we applied, and subsequently it has held up pretty well in other people’s experiments.”

The Scientist
January 17, 2006

Original web page at The Scientist