Complementation of Azotobacter chroococcum hydrogenase mutants by cloned hup determinants from Bradyrhizobium japonicum and Azotobacter chroococcum

Abstract

Mutants of Azotobacter chroococcum defective in H2-uptake (Hup) hydrogenase activity were complemented by plasmids containing hup genes from Bradyrhizobium japonicum and A. chroococcum. Plasmids pHU1 and pHU53 (B. japonicum hup) and pKHT32 (A. chroococcum hup) complemented class A and C mutants. Every pHU53 transconjugant was less active than the corresponding pHU1 transconjugant. Class B mutants were weakly complemented by pHU2 (B. japonicum hup) but strongly complemented by pKHT30 (A. chroococcum hup). pKHT40, which contains all the A. chroococcum DNA which hybridized most strongly to the hup region of B. japonicum, caused overexpression of Hup activity in mutant classes A, B and C and the wild-type. The one class D mutant, which contains a soluble rather than the wild-type membrane-bound hydrogenase, was not complemented by any of these plasmids. The hup genes from A. chroococcum appear to contain at least two transcriptional units and span approximately 15 kb of DNA. Clear similarities and differences occur between the hup gene regions of A. chroococcum and B. japonicum.