Vitamin B-12 (cobalamin) helps in the synthesis of DNA and red blood cells and is important in the maintenance of the insulation layer that surrounds nerve cells. Scientists know how virtually every molecule needed for life is made, but vitamin B-12 has remained a puzzle for decades. Researchers may have recently discovered, quite by accident, how cells manufacture the vitamin. After talking to a colleague who had discovered that adding laundry whitener (calcofluor) to a bacterium strain in a lab dish produced fluorescence, Graham C. Walker from the department of biology at MIT in Cambridge, Mass., decided he would try adding it to a strain he was studying. Sinorhizobium meliloti exists in soil and has a symbiotic relationship with plants, in which the bacteria fix nitrogen into ammonia in exchange for food provided by the plant. The bacterium glowed under UV radiation, and his students thought that was pretty cool. Walker and his students used the reaction to see why some of the bacteria seemed to help the plants grow and some did not. They found that mutant bacteria, which could not support plant growth, displayed abnormally bright fluorescence in the petri dish with added calcofluor, while normal bacteria did not. Gordon R.O. Campbell, Walker’s graduate student, isolated one of the brightest mutants, bluB, which was particularly symbiotically defective. He found that the mutant was missing a gene needed for the bacteria’s symbiotic relationship with the plant. The gene happened to resemble one in another bacteria that helps in the biosynthesis of vitamin B-12. When a mutant bacterium that doesn’t contain B-12 is coupled with laundry whitener in a lab dish, it fluoresces under UV radiation (as seen here). Isolating the mutant genes from the bacteria helped the researchers discover an important step in the synthesis of vitamin B-12. The scientists discovered that, when they added 5,6-dimethylbenzimidazole (DMB), a critical component of the vitamin, to the bluB mutant, the bacteria were able to establish symbiosis with the plant. They then involved Kavita Mistry, an analytical chemist from Merck & Co. Inc. in Rahway, N.J., to help them determine what causes defective vitamin B-12 synthesis in the bluB mutant. The researchers first isolated vitamin B-12 and its related components from the mutant and wild-type bacteria. Mistry used a high-performance liquid chromatography system from Agilent Technologies Inc. of Palo Alto, Calif., to separate the material. Because she knew that the differences observed in the samples were probably related to the vitamin, she focused on detection at the 525-nm wavelength, where compounds such as B-12 absorb. “I saw a dramatic difference between the bluB mutant and the wild-type bacteria. The major thing I noticed was that vitamin B-12 was not present in bluB,” Mistry said. She tested the bluB mutant both by itself and with added DMB at 525 nm. With DMB, a peak appeared near 12 minutes, which she knew corresponded to the time where vitamin B-12 was expected. Mistry then coupled the liquid chromatography system with a Finnigan LCQ mass spectrometer from Thermo Electron Corp. of Waltham, Mass., to confirm the presence and identity of B-12 in those samples. When the researchers found another peak adjacent to the B-12 peak that was prominent in the bluB samples, they decided to investigate further. Mistry added a photodiode detector to the liquid chromatography system to observe the full spectrum across that peak. The spectrum revealed an overall similarity to vitamin B-12, but with an additional maxima at 248 nm and a shoulder at 268 nm, characteristic of the presence of guanine. These data suggested that B-12 synthesis in the bluB mutant proceeds until the last step, the replacement of guanine with DMB. Discovering the vitamin B-12 defect in the mutant bacteria allowed the scientists to observe a necessary step in how cells manufacture the vitamin. They hope to eventually break the code for the entire pathway of B-12 synthesis. PNAS, published online Feb. 20, 2006.