Question

A colleague has identified in her model bacterium (M. wolverium) a gene (cbxC) involved in the synthesis of vitamin B12. This gene is essential for M. wolverium growth when no B12 is supplemented in the medium. A bioinformatics analysis revealed that your model organism M. spartacus possesses a homologous gene, so you decide to inactivate it to confirm its function. To your surprise, M. spartacus lacking cbxC can grow without B12 and synthesize its own. Therefore, you hypothesize that M. Spartacus has evolved another metabolic pathway to synthesize B12. Using microbial genomics techniques, how would you test your hypothesis and identify genes involved in the alternative pathway? Please provide a long answer with relevant examples and evidence

Answer 1

As mentioned above, when no B12 is supplemented in medium, cbxC still promotes growth and have homologous gene on cromosome. If one form of this gene is inactivated it can adopt to alternative pathway to synthesize B12. In this case genes from alternative pathway can be identified by expressed transcriptomic profile.

You should extract and purify m-RNA from M. wolverium growned with cbxC as control and without cbxC as test sample. Bacterium with cbxC gene will follow regular pathway for synthesizing B12 where as bacterium without cbxC gene will follow alternative pathway and will express m-RNA for the alternative pathway. By comparing both the samples of expressed m-RNA you will identify and quantify that which m-RNA is more expressed and which one are down regulated or suppressed.

m-RNA (responsible for respected proteins) which are not expressed or down regulated compared with control will follows the alternative pathway. Hence activated and non activated pathways can be identified. Non activated pathway will show expression of other protein’s m-RNA which are from alternative pathway. Hence we can prove our hypothesis of expression of alternative pathway and the responsible genes from respective expressed m-RNA.