Dr.S.KARTHIKUMAR Associate Professor Department of Biotechnology Kamaraj College of Engineering and Technology, K.Vellakulam-625701, TN, India Email: skarthikumar@gmail.com

1. HUP genes
Hydrogen uptake genes

2. • An undesirable side reaction of nitrogen fixation is the reduction of
H+ to H2 gas.
• Energy in form of ATP is wasted on production of hydrogen which is
eventually lost to atmosphere
• This lower the efficiency of nitrogen fixing process.
• Theoretically if H2 would be recycled to H+ the extend of energy loss
could be minimized and the nitrogen fixation process could become
more effective

3. ROLE OF HUP GENES

5. • Energy is wasted
• Hydrogen is eventually lost in to atmosphere
• Lower the efficiency of nitrogen fixation
• Bradyrhizobium japonicum
• In Bradyrhizobium japonicum hydrogen is used as a energy source for
growth in microaerophillic conditions.
• They possess an enzyme hydrogenase which takes H2 from
atmosphere and convert to H+
• The gene is approsimately 60Kda, organized in one to three
transcriptional unit covering 15Kb of genome.

6. • The first hydrogenase gene to be isolated (E.coli membrane bound
hydrogenase) was selected by complementation of an E.coli mutant that
no longer expressed this activity with a clone bank of wild type E.coli
DNA in plasmid pBR322.
• The mutants were unable to grow on minimal medium containing
formate
• The transformants that were able to grow on this medium were assayed
for the presence of hydrogenase activity

7. • Similarly, hup genes from B.japonicum using a clone bank of wild type
DNA in broad host range vector pLAFR 1 to complement B.japonicum
Hup- mutants.
• The transformants were selected by their capacity to reduce
methylene blue dye in hydrogen atmosphere.
• Thus the isolated hup genes would be used as hybridization probe to
select homologous genes from the clone bank.