Microbiology
Method Designed for Finding Genes Essential to Survival in Bacteria
A central strategy in the fight against disease-causing bacteria is to knock out those proteins that the pathogen needs to survive. The biggest guns in the antibiotic world obey this principle: streptomycin binds to ribosomal proteins, disrupting protein synthesis. Rifampin hits the beta subunit of the enzyme that makes mRNA, producing a similar effect. Yet only a fraction of the products produced by bacterial genes play such essential roles, and finding these genes has not been easy. Recently, a team of Harvard Medical School researchers has discovered a quick and easy method for identifying these essential genes--one that could unjam the door to a new generation of potent antibiotics.
John Mekalanos and his colleagues Brian Akerley and Eric
Rubin (not shown) have devised a new method for identifying essential genes
that could lead to the development of new antibiotics. "The future
is to try and understand what essential gene products do and devise drugs
that might inhibit that activity," says Mekalanos.
"The pharmaceutical industry has been working on making new drugs for a long, long time. And they've had trouble solving the problem of knowing which genes are essential for the normal functioning of bacteria," says John Mekalanos, the Adele Lehman professor of microbiology and molecular genetics, and head of the research team. He presented the new method on May 18 at the annual meeting of the American Society for Microbiology in Atlanta.
Pinning Down Genes
One problem is that until recently, methods for identifying essential genes have been laborious, requiring the isolation and study of individual mutants. Another is that these methods have been limited to species with well-developed genetic systems--in which genes and gene products are known--and cannot be readily applied to microorganisms whose genomes have recently been sequenced.
A newer approach, called comparative genomics, compares two different genomes, such as that of Mycoplasma genitalium and Haemophilus influenzae, the rationale being that any gene found in both is likely to be essential. But even this approach appears to be flawed. For example, using their new technique, called GAMBIT, Mekalanos and his colleagues found they could disrupt some of the genes predicted to be essential by comparative genomics without killing H. influenzae.
Essentially, GAMBIT allows them to refine the list of target genes and indentify new ones.
GAMBIT identifies essential genes through an easily performed combination of techniques, including polymerase chain reaction (PCR), transposition, and genetic footprinting. Specific chromosomal segments are amplified by PCR and then mutated by the insertion of transposons (which are segments of DNA capable of introducing themselves into other DNA sites). The mutated chromosomal segments are then introduced into normal bacteria (such as H. influenzae), where they recombine with normal chromosomes. The position of each of these mutations is assessed by genetic footprinting. Mutations producing the kinds of growth defects that might be caused by inserting a transposon into an essential gene are then detected and mapped. "So you generate data sets--you find out which genes you can hit without killing the organism and which genes you can't," says Mekalanos.
Mekalanos and his colleagues have tested the new method in two human pathogens, H. influenzae and Streptococcus pneumoniae. GAMBIT successfully detected known essential genes in each, including the SecA gene, isolated in Jon Beckwith's lab. SecA is a conserved gene, found in several species.
"If you're going to make a new antibiotic, it's better to use this type of gene, first, because you know it's essential and, second, because it's conserved--all bacteria have it. So it sounds like a drug target to me--that's really the payoff." GAMBIT also assigned essentiality to several previously uncharacterized chromosomal regions.
"Based on what we think will be another year's worth of work, we'll be able to define every essential gene in Haemophilus and Streptococcus," says Mekalanos. Still, he cautions, it will be a long road to designing new antibiotics. Once the essential genes are identified, the next step will be to discover what their protein products actually do. "That's hard work, but it's doable," says Mekalanos. He then speculates it might be possible, using two-hybrid methods and other techniques, to see what proteins interact with the essential gene products.
Ultimately, the hope is to identify and characterize specific enzymatic reactions. "We can then use our knowledge of the detailed chemistry of the enzymatic reactions to design small molecules to interfere with those reactions," he says.
This work was supported by grants from the National Institutes of Health, the Pew Scholars Program, and the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation.
--Misia Landau