Healing agents from the soil of Pulau Ubin

Antitumour chemotherapeutic agents from the soil of Pulau Ubin
Source: National University of Singapore

We heard of meliodosis -- caused by bacteria in the soil which enters the body through cuts or wounds – a disease which could be deadly. But the soil also yields "healing" bacteria. A team of NUS researchers started searching for these bacteria in 1996. Led by Associate Professor Nga Been Hen, Department of Microbiology the team scoured the forest floor of Pulau Ubin, an island off Singapore -- in search of soil containing bacteria belonging to the actinomycete group which produces antitumour chemotherapeutic agents.

After years of painstaking research, the team has managed to isolate a new strain from the soil. This is the first time researchers in Southeast Asia had literally "unearthed" a new strain of actinomycete that produces antitumour polyketide compounds. There are not many such recorded discoveries in Asia and among these are those in Japan and Korea. The team has recently applied for a patent for this new strain.

Actinomycete strains are able to synthesise antitumour compounds belonging to aromatic polyketides. These are characterised by their chemical structure of four "aromatic" rings, with a sugar group attached to one of the rings. They are capable of intercalating into DNA and thus inhibiting the synthesis of macromolecules.

Associate Professor Nga recalled: "Four of us went to Pulau Ubin. We needed to look for places where the soil is pristine and not disturbed. We brought with us sterilised bottles and spatulas. We scraped one to three centimetres of the soil below the surface of leaf litter and scooped this into the bottles.

"We were so absorbed in our work that we didn't even know that mosquitoes were out in full force and attacking us until we felt the itch. I slapped on the itch and was surprised to find that I had actually killed three mosquitoes at one go!"

Bringing the soil back to the lab was the first step. The team needed to make sure that the bacteria in the soil were what they were looking for – the types which can stop tumours in their track.

Screening

One gramme of soil was mixed with three millilitres of sterile water and shaken gently for three minutes. Small quantity of this mixture was inoculated onto a thin layer of agar in a Petri dish. The culture was left to grow for four days at 28 degrees Centigrade.

The bacteria grew as tiny spots on the agar. The antibiotic component, daunorubicin in the bacteria is identifiable as it is red in colour. They were then screened for antimicrobial activity against Bacillus subtilis.

"When we found that the actinomycetes were actually working to inhibit the growth of the Bacillus, we know we were on the right track," said Associate Professor Nga.

The chromosomal DNA of the actinomycete strains were further screened for the presence of the ketoacyl synthase gene (an early and essential gene responsible for the biosynthesis of the aromatic polyketide compounds).

The team proceeded to build a cosmid genomic library – a collection of clones made from the strain's genomic DNA fragments (20-25 kilobases). 2,000 clones were characterised. They were analysed for containing daunorubicin-resistance genes (a gene present in the daunorubicin gene cluster) by employing the PCR technique using appropriate primers.

The polyketide compounds produced by this strain have the potential to be made into antitumour drugs.

Thanks for the alert from Marcus Tay