Construction of a Gene Bank of Azorhizobium IRBG-46: Isolation of hup Genes

Genomic DNA from an efficient Hup⁺Sesbania-Azorhizobium strain IRBG-46 was Isolated, partially digested with EcoRI and fractionated on a 10–40% sucrose density gradient to obtain DNA fragments in the size range of 15–23 kb. In order to isolate hup genes from this strain, a gene bank was constructed in Escherichla coll HB101 using a mobilizable plasmid vector pRK290 having a EcoRI cloning site. Approximately 2×10⁴ To-resistant transformants were pooled to constitute the gene bank. Using 12.9 kb EcoRI fragment of cosmid pHU52 as a heterologous hup probe, a total of 2,000 clones were screened by colony hybridization. Five positive clones confirmed by secondary screening and explants uptake hydrogenase activity were identified. An insert size in the range of 15–22 kb was revealed by restriction analysis with EcoRI. These five recombinant plasmids containing Hup-determinants of Azorhizobium IRBG-46 have been designated as pSRH1, pSRH2, pSRH3, pSRH4 and pSRHS. These plasmids were transferred into Hup⁻Cicer-Rhizobium strain Red 301 to check the expression of hup genes in the new genetic background. In the transconjugants so obtained, the hup genes were found to express under ex plants conditions, and uptake hydrogenase activity ranged from 134 to 392 nmol H₂ taken up per h per mg protein.