The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the University Of East cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX, the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene, /ipA, which is predicted to encode an outer-membrane lipoprotein.Further upstream of /@A, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes, feuP and feu0, did not affect the transcription of /@A, or of the cycHJKL operon. However, a mutation in feu0 also led to the loss of the high affinity iron uptake system, although siderophores were still produced. John lnnes Centre, ColneyLane, Norwich NR4 7UH, UK 1

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa 3 pathway were identified by the inability to oxidize N,N-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular 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 higherthan-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli 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 to apocytochrome c.

Covalently bound haem proteins and cytochromes were analysed in Rhizobium leguminosamm biovar viciae free-living cells and nitrogen-f ixing bacteroids isolated from pea nodules. Increased levels of spectroscopically detectable cytochrome c in bacteroids were correlated with the appearance of two proteins of M, 30000 and 28000 that contained covalently bound haem. Conversely, bacteroids had undetectable levels of a periplasmic cytochrome c of M, 14000 that is normally present in free-living bacteria. Difference spectra confirmed that the terminal oxidases, cytochromes aa, and d, were absent, and photodissociation spectra revealed novel components that may be due to a bacteroid terminal oxidase.