Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli.

AUTOR(ES)

Frustaci, J M

RESUMO

Complementation analysis showed that the Bradyrhizobium japonicum hemH gene was both necessary and sufficient to rescue mutant strains I110ek4 and I110bk2 in trans with respect to hemin auxotrophy, protoporphyrin accumulation, and the deficiency in ferrochelatase activity. The B. japonicum hemH gene was expressed in an Escherichia coli T7 expression system and yielded a 39-kDa protein, which was consistent with the predicted size of the deduced product. The overexpressed protein was purified and shown to contain ferrochelatase activity, thereby demonstrating that the hemH gene encodes ferrochelatase. When expressed from the lac promoter, the B. japonicum hemH gene was able to complement the enzyme activity of a ferrochelatase-defective E. coli mutant, and it also conferred hemin prototrophy on those cells. These latter findings confirm the identity of the hemH gene product and demonstrate that B. japonicum ferrochelatase can interact with the E. coli heme synthesis enzymes for heme formation in complemented cells.

Documentos Relacionados

• Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene.
• Isolation and expression of the Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia coli.
• Cloning and overexpression of the Rhodobacter capsulatus hemH gene.
• Cloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli.
• Effect of iron availability on expression of the Bradyrhizobium japonicum hemA gene.