CHARACTERIZATION OF THE CYCHJKL GENES INVOLVED IN CYTOCHROME-C BIOGENESIS AND SYMBIOTIC NITROGEN-FIXATION IN RHIZOBIUM-LEGUMINOSARUM

Mutants of Rhizobium leguminosarum bv. viciae linable to respire via t he cytochrome aa(3) pathway were identified by the inability to oxidiz e N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemente d by cosmid pIJ1942 from an R. leguminosarum clone bank were identifie d. Although pea nodules induced by these mutants contained many bacter oids, no symbiotic nitrogen fixation was detected. Heme staining of ce llular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa(3) and d were present, the latter at a higher-th an-normal level. DNA sequence analysis of complementing plasmids revea led four apparently cotranscribed open reading frames (cycH, cycJ, cyc K, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizob ium japonicum and Rhizobium meliloti proteins thought to be involved i n the attachment of heme to cytochrome c apoproteins; CycK and CycL ar e also homologous to the Rhodobacter capsulatus cell and ccl2 gene pro ducts and the Escherichia coil nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme t o apocytochrome c.