The scientists then infected mice with the mutant strains of bacteria, each of which contained a unique DNA tag. Three weeks later, they harvested the bacteria from the lungs of the mice and looked for strains of bacteria that failed to thrive. They quickly zeroed in on three mutant strains of M. tuberculosis that did not flourish in the lungs of mice despite being able to grow in the liver and spleen. Looking closely at the mutations, they noticed that all three strains contained mutations in a region of the tuberculosis genome where genes responsible for making the lipid phthiocerol dimycocerosate (PDIM), a component of the bacterial cell wall, were known to be located.

"At that point, though, we had shown only that the mutants failed to grow in the lungs," said Jacobs. "But when we grew the bacteria on agar, we found that they formed quite distinctive colonies that indicated defective cell wall biosynthesis."

Wild-type TB bacilli form flat and corded colonies on agar, but the mutant colonies looked strikingly different. "They looked like the Pompidou Center in Paris, with the pipes running all over the outside of the building," explained Jacobs.

Further experiments revealed that two of the mutant strains of bacteria could not synthesize PDIM. The third mutant produced normal levels of the lipid. Additional studies revealed, however, that this mutant contained a defective gene called mmpL7 and could not transport PDIM outside of the bacterial cell, where the lipid apparently aids the infection process.

"This finding established that PDIM is required for virulence, and has to be exported from the cell," said Jacobs. "What’s particularly exciting about the discovery of the mmpL7 mutant
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Contact: Jim Keeley
keeleyj@hhmi.org
301-215-8858
Howard Hughes Medical Institute
3-Nov-1999