GroEL chaperonins are required for the formation of a functional nitrogenase in Bradyrhizobium japonicum

At least five highly conserved, but disparately regulated groESL operons ar e present in Bradyrhizobium japonicum. Expression of groESL(3) is coregulat ed with symbiotic nitrogen fixation genes, implying a role of GroESL chaper onins in the nitrogen fixation process. Null mutants of individual groEL ge nes, however, were not impaired in symbiotic nitrogen fixation activity. By contrast, the groEL(3)-plus-groEL(4) double mutant strain D4, which is mut ated in those groEL genes that contribute most to the GroEL pool under symb iotic conditions, exhibited less than 5% Fix activity as compared to the wi ldtype. Expression of lacZ fusions made to several representative nif and f ix genes was not, or only marginally, reduced in mutant D4, indicating that the requirement of chaperonins for nitrogen fixation does not occur at the level of RegSR-NifA-sigma(54)- or FixLJ-FixK(2)-dependent gene regulation. Instead, immunoblot analyses revealed that the level of Nif)I and NifDK ni trogenase proteins was drastically decreased in extracts prepared from D4 b acteroids and from free-living cells grown anaerobically. Transcriptional f usions of the anaerobically induced groESL(3) promoter (P3) to all five B. japonicum groESL operons and also to groESL from Escherichia coli were inte grated into the chromosome of mutant D4. Strains harboring P3 fused to groE SL(1), groESL(2), groESL(5), or E. coli groESL partially complemented the s ymbiotic defect of mutant D4, whereas the wild-type phenotype was completel y restored in strains complemented with P3 fused to groESL(3) (control) or groESL(4). Likewise, the growth defect of an E. coli groEL mutant could be corrected at least partially by individual B, japonicum groESL operons. Tn conclusion, both series of complementation analyses were not indicative of a strict substrate specificity of any of the B. japonicum groESL gene produ cts, which is in good agreement with their high degree of sequence conserva tion.