Scientists supported by the National Institute of Allergy and
Infectious Diseases (NIAID) have developed a technique that
theoretically will allow researchers to study the function of every gene
in the bacterium that causes tuberculosis (TB). The finding, reported
in the Sept. 30, 1997 issue of the Proceedings of the National
Academy of Sciences (PNAS), has significant implications for the
development of new TB drugs and vaccines and for advancing our
understanding of how TB bacteria cause disease.
"This is a major advance for the TB research community and it
represents an important step in NIAID's efforts to develop research-
based solutions to one of our foremost global health problems," says
NIAID Director Anthony S. Fauci, M.D. "One-third of the world's
population is infected with Mycobacterium tuberculosis (M.tb), the
organism that causes TB. Each year, an estimated 3 million people
die from TB, more than from any other infectious disease."
TB bacteria are notoriously difficult organisms to study in the
laboratory. Many of the molecular techniques that scientists use
routinely to analyze other microorganisms have until recently been of
little use in TB research. Consequently, systematic methods for
creating mutations in M.tb genes have eluded scientists. Such
methods are extremely valuable in the study of disease pathogens, as
they allow scientists to examine the effect of individual gene
mutations on an organism's ability to grow or cause disease. These
studies can reveal new drug targets or identify potential vaccine
"Analyses of M.tb have been hampered by the lack of efficient
systems for transferring new genetic material into this pathogen,"
explains William R. Jacobs, Jr., Ph.D., senior author of the study.
"Furthermore, because M.tb is such a slow-growing bacterium,
methods of creating and analyzing mutations that involve exposing
cells to DNA-damaging agents, and then characterizing colonies
arising from single cells, are of limited value."
Dr. Jacobs, an NIAID grantee at the Howard Hughes Medical
Institute (HHMI) at Albert Einstein College of Medicine in the Bronx,
N.Y., led a research team that found an efficient way to create
mutations in TB genes using fragments of DNA known as
transposons. Transposons insert themselves at random into bacterial
DNA, inactivating any gene in which they take up residence.
Scientists have used transposon mutagenesis, as this process is
called, to generate vast numbers of mutations in many other kinds of
bacteria. These so-called mutation libraries allow scientists to study
the function of individual bacterial genes. Until now, transposon
mutagenesis has not been feasible in TB bacteria.
Dr. Jacobs, NIAID grantees Barry R. Bloom, Ph.D., also of
HHMI at Albert Einstein College of Medicine, and Graham F. Hatfull,
Ph.D., of the University of Pittsburgh, and their colleagues
constructed special delivery vectors to carry transposons inside TB
cells. Known as conditionally replicating shuttle phasmids, these
vectors essentially are genetically engineered mycobacteriophages --
viruses that infect M.tb and related bacteria. For this study, the
researchers developed phasmids with mutations that prevented them
from replicating at a temperature of 37 degrees Celsius (C). The
transposons carried by these shuttle phasmids contained a gene
conferring resistance to the antibiotic kanamycin.
The researchers mixed M.tb cells with the transposon-bearing
shuttle phasmids and incubated them at 37 C. Instead of replicating
repeatedly and destroying the M.tb cells, at this "non-permissive"
temperature the phasmids simply stuck to and entered the bacterial
cells, providing an opportunity for the transposons to insert
themselves into the bacterial DNA.
To determine the success of their efforts, the researchers
transferred the TB-phasmid mixture to culture media containing
kanamycin. Only those TB bacteria carrying the kanamycin
resistance gene, by virtue of having undergone transposon
mutagenesis, will grow on this media. Dr. Jacobs and his colleagues
recovered thousands of kanamycin-resistant M.tb mutants in three
"Analyses of DNA from randomly selected kanamycin-resistant
M.tb colonies revealed a random distribution of the
transposon insertions," says Dr. Jacobs, "suggesting that the mutants
obtained in our experiments represent libraries of independent
mutants of M.tb."
In the same issue of PNAS, researchers from France's
Pasteur Institute describe an alternative method for performing
transposon mutagenesis in M.tb, which also results in the production
of thousands of mutants. Dr. Jacobs also is a co-author of that study.
"With these techniques, researchers theoretically should be
able to create mutations in virtually every gene of Mycobacterium
tuberculosis," adds Ann Ginsberg, M.D., Ph.D., NIAID's TB program
officer. "This should provide an unprecedented opportunity to make
rapid and substantial progress in the understanding of pathogenesis
and development of novel therapeutics and vaccines for TB."
In addition to Drs. Jacobs, Bloom and Hatfull, collaborators on
the NIAID-funded study include Stoyan Bardarov, M.D., Ph.D., Jordan
Kriakov, M.D., Ph.D., Christian Carriere, M.D., Ph.D., Shengwei Yu,
and Carlos Vaamonde, M.D., of Albert Einstein College of Medicine;
and Ruth A. McAdam, Ph.D., of the Central Veterinary Laboratory in
Surrey, Great Britain.
NIAID, a component of the National Institutes of Health (NIH),
supports research on AIDS and other sexually transmitted diseases,
tuberculosis and malaria, as well as allergies and asthma. NIH is an
agency of the U.S. Department of Health and Human Services.
Press releases, fact sheets and other NIAID-related materials are
available on the Internet via the NIAID home page at