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When a new antibiotic isolated from Rhodococcus fascians
is dripped onto a paper disc (white) in the middle of a plate full of other
bacteria (orange), all the bacteria near the filter disc die.
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® Kazuhiko Kurosawa
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Kazuhiko Kurosawa was running out of variables. For eight months he had made
hundreds of cultures of Rhodococcus fascians - manipulating pH,
temperature, salt concentration, media type, oxygen levels, even degree of agitation
- each time attempting to provoke the bacteria into transcribing a set of genes he
knew lay dormant in its genome. But the soil-dwelling bacteria remained
recalcitrant.
Anthony Sinskey's lab at Massachusetts Institute of Technology, which
Kurosawa joined in 2003, first became interested in Rhodococcus while
in collaboration with Merck. The company hoped to use the bacteria to more
efficiently make a precursor for one of its HIV protease inhibitors, Crixivan. While
analyzing the Rhodococcus genome, the MIT researchers were surprised to
find close to 100 genes for nonribosomal peptides, and 30 clusters of genes for
polyketides, two major classes of antibiotics. But unlike most other soil-dwelling
bacteria, Rhodococcus was not known to produce an antibiotic. Finding
the genes "was like Christmas morning," says Philip Lessard, one of the
investigators. Unfortunately, he adds, there appeared to be "some assembly
required."
However, Sinskey's funding was wrapped up in the lab's main projects. So in
2004 Kurosawa embarked on an "under-the-table study" to provoke one species of the
bacteria, Rhodococcus fascians, into using those genes, he recounts in
an E-mail. (Despite the need for new antibiotics, Lessard says funding for new
antibiotics is often hard to come by because of the availability of cheap and
still-effective front line antibiotics like penicillin and ampicillin.) Lessard
recalls every roller in the lab constantly drumming with Kurosawa's cultures. After
the two researchers ran out of variables to test, they took a hint from nature and
decided to introduce R. fascians to an environmental hazard it had yet
to confront in the lab - other bacteria.
In the wild, R. fascians lives in soil populated by thousands of
microbes. The researchers planned a "kindergarten" version of the scenario, Lessard
says, pitting R. fascians against just one other bacterium - a strain
of Streptomyces padanus, an aggressive antibiotic producer that
Kurosawa isolated from the soil in a flowerpot by his lab bench.
Initially, however, they overshot the mark. "Streptomyces really
did kill everything," says Lessard. But one day Kurosawa approached Lessard holding
a plate on which the Streptomyces had been wiped out. Lessard, who now
works for Agrivida, an agricultural biotech (and retains no patents on the
antibiotic), was skeptical - the plate was probably contaminated, he reasoned. But
Kurosawa didn't think so. By sequencing the genes encoding ribosomal RNA of the
bacteria left on the plate, he proved it was the original strain of R.
fascians. Then, Kurosawa used high performance liquid chromatography
(HPLC) to analyze the secreted substance, and became "full of confidence" that the
bacteria were producing an antibiotic, he recalls. The eager biologist reported his
results at a lab meeting in April of 2005. "Prof. Sinskey said, 'No one believes
that. Kazu, you should isolate the antibiotic and elucidate the molecule.'"
It took a year of "painfully tedious" work to purify the secreted antibiotic,
recalls Kurosawa, still based at MIT (where this author studied journalism). He
relied on the other labs to do NMR structural studies of the molecule in their spare
time. The molecule turned out to be an aminoglycoside (J Am Chem Soc,
130:1126-7, 2008). When tested for antimicrobial activity, it proved particularly
effective against Heliobacter pylori, a bacteria that causes stomach
ulcers in humans.
But there was a mystery. Pulse field gel electrophoresis revealed a strange
chunk of DNA in the R. fascians genome, about 150 genes that did not
exist in the parent strain. Analysis of a tiny extract showed it belonged to
Streptomyces. And "in every case we've looked," adds Lessard, "the
loss of the ability to make the antibiotic correlates with the loss of this DNA
element." In the end, R. fascians may never have actually used the
antibiotic-coding genes that prompted the research in the first place. But do the
Streptomyces genes encode the antibiotic, or simply provoke
R. fascians into making it? The project "was a success followed by
ten unknowns," says Lessard.
