Molecular characterization of uptake hydrogenase in Frankia

Abstract

A molecular characterization of uptake hydrogenase in Frankia was performed by using two-dimensional gel electrophoresis, matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry, PCR amplification and Southern blotting. A polypeptide of approx. 60 kDa was recognized in Frankia UGL011102, AVCI1 and KB5 on the two-dimensional gel by blotting with Ralstonia eutropha (Hox G) antibody. Further analysis by MS resulted in a peptide ‘fingerprint’, which was similar to the membrane-bound hydrogenase 2 large subunit (HYD2) in Escherichia coli. In addition, a 127 bp PCR fragment could also be amplified from Frankia AVCI1, which gave a 76% similarity with the large subunit of hydrogenase in, e.g., Azotobacter chrococcum, Bradyrhizobium japonicum and Rhizobium leguminosarum. Although immunological similarity between the small subunit of Frankia hydrogenase and that of other organisms has not yet been found, a PCR product of 500 bp could be amplified from the local source of Frankia, the analysis of which gave 69 and 67% identity with the small subunit of hydrogenases in B. japonicum and R. leguminosarum respectively. A Southern-blot analysis further indicated evidence for the presence of the small hydrogenase subunit in other Frankia strains, i.e. KB5, AvcI1 and CcI3.