Vitamin B12 Secret Revealed By Science Class Experiment

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Vitamin B12 Secret Revealed By Science Class Experiment

http://www.medicalnewstoday.com/medicalnews.php?newsid=38198

For decades, scientists have wondered how living organisms

manufacture the essential vitamin B12. Now, using laundry whitener

and dirt-dwelling bacteria--the everyday ingredients of an

undergraduate science experiment--researchers may have found the

major clue they need to solve the mystery.

Researchers led by Graham , a Medical Institute

(HHMI) professor and American Cancer Society research professor at

the Massachusetts Institute of Technology, have discovered the first

known mutant bacteria with a specific defect in a gene involved in

the least-understood part of B12 synthesis. They report their

findings in the early online edition of the Proceedings of the

National Academy of Sciences, published February 20, 2006. HHMI

professors are leading research scientists who received $1 million

grants from the Institute to find ways to bring the excitement of the

research lab into undergraduate science classrooms.

In the ancient world, B12 was probably catalyzing reactions before

cells even existed. Now, all animals need B12 to help make the

building blocks of DNA, and children need enough of the vitamin to

help their brain develop normally. Most people consume enough B12

through animal products or fortified foods in their diet. On the

other hand, animals that do not eat other animal products acquire the

nutrient from bacteria in their guts or from bacteria-infected dirt

on their plant food. An estimated one-quarter of people older than 60

in this country have trouble absorbing B12. B12 deficiency can lead

to nerve damage, anemia, and forgetfulness.

's team's genetic discovery was made possible by a gimmick

designed to capture the attention of undergraduate biology

students in the early 1980s. When he added a laundry whitener to a

lab dish, the symbiotic bacteria he studied glowed in ultraviolet

light, just as the additive makes clothes look brighter in the sun.

The teaching trick soon became a popular tool in 's lab for

research that had nothing to do with vitamin B12. There, researchers

have been focusing on how symbiotic bacteria form and invade the

nodules in alfalfa roots that provide the plant with nitrogen and the

bacteria with food. The scientists noticed that some of the bacteria

on the glowing lab dishes did not light up. These stubborn dark spots

revealed bacteria missing key genes needed to construct and enter the

nodules in plant roots, they discovered. By analyzing various

mutations, the researchers were able to track the molecular details

of how the bacteria provide the plant with the nutrients it needs to

grow.

Several years ago, 's graduate student Gordon decided

to look for symbiosis defects in bacterial mutants that, instead of

being dark spots on the glowing lab dish, were even brighter than

their normal counterparts. His findings enabled and his co-

authors to answer a question being asked by many researchers studying

B12 synthesis.

" That is what is so great about basic research, " said. " It

finds answers to things you cannot get at in a direct way. "

isolated the brightest mutants and put them onto the roots

of alfalfa seedlings. Healthy symbiotic partnerships show up on the

plant roots as long pink nodules stuffed with bacteria. In contrast,

seedlings sharing a dish with the most obviously defective bacteria

were stunted and their roots had small white nubs with barely any

bacteria inside. For one of these bright mutants, it turned out that

the root of the problem was the mutant bacteria's inability to

produce B12.

Adopting nomenclature traditional in their field, the researchers

named the mutated gene bluB, after a similar gene found in another

kind of bacteria.

" The important clue came when we noticed bluB was grouped with other

genes important for making vitamin B12 in the other bacterium, "

said. " That's not something we are expert in. " So the

researchers contacted co-author Roth, a professor of molecular

biology at University of California, , who has studied in detail

the intricate series of steps required to assemble B12, the largest

known natural compound that is not made out of repeating units.

" Out of our conversations came the idea that bluB might be required

for an unknown part of the pathway, " said. " B12 is a big,

complicated molecule. Researchers have been unable to crack the

problem of how to make the lower ligand, " a segment of the molecule

known as DMB.

It was a simple experiment, said Michiko Taga, a postdoctoral fellow

and co-first author of the paper. Taga took over the research when

graduated. " If the mutant was broken because it could not

make DMB, " she said, " then if we added DMB back it should be okay. So

we added DMB, and the bacteria went back to acting like ordinary

[symbiotic] bacteria. That was the defining experiment. "

When the researchers provided DMB so that the bacteria did not have

to manufacture it themselves, the bacteria's extraordinary brilliance

subsided to a more uniform fluorescence on the lab dish with the

laundry whitener. And in the lab dish with the seedlings, the

restored bacteria produced a bigger, healthier plant. Chemist and co-

author Kavita Mistry followed up with biochemical experiments to

prove that the bluB mutant could not make B12 without added DMB.

" Our findings just mean bluB is necessary for the reaction, " Taga

said. " We are currently doing experiments to show that it directly

catalyzes the reaction. "

But Roth said the discovery gives him hope for finding all the steps

in the pathway for synthesizing B12. " This is the part that has

resisted genetics and chemistry, " he explained. " We've tried it.

Others have tried it. This appears to be the first enzyme dedicated

to synthesizing the part. "

Other bacteria, such as the Salmonella that Roth studies, appear to

substitute other molecules in place of DMB, stymieing genetic

approaches. But the form of B12 that people need contains DMB.

The discovery of the bluB mutant may overturn a theory that DMB

spontaneously forms without enzymes to speed up the reaction, Roth

said. Before the bluB mutant was identified, that theory made sense

because the reactions that make B12 do not require energy, in

contrast to most biosynthetic reactions.

Taga and are following up to figure out how the bluB mutation

affects the symbiotic relationship between the bacteria and the plant