ASSEMBLY OF IRON-SULFUR CLUSTERS - IDENTIFICATION OF AN ISCSUA-HSCBA-FDX GENE-CLUSTER FROM AZOTOBACTER-VINELANDII

An enzyme having the same L-cysteine desulfurization activity previous ly described for the NifS protein was purified from a strain of Azotob acter vinelandii deleted for the nifS gene. This protein was designate d IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer, Informa tion gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair & vinelandii genomic s egment that includes the iscS gene. The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contain ed within the major nif cluster of A. vinelandii previously designated orf6. These genes have been designated iscU and iscA, respectively. I nformation available from complete genome sequences of Escherichia col i and Hemophilus influenzae reveals that they also encode iscSUA gene clusters. A wide conservation of iscSUA genes in nature and evidence t hat NifU and NifS participate in the mobilization of iron and sulfur f or nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formati on or repair of iron-sulfur clusters. The proposal that IscS is involv ed in mobilization of sulfur for iron-sulfur cluster formation in A. v inelandii is supported by the presence of a cysE-like homolog in anoth er gene cluster located immediately upstream from the one containing t he iscSUA genes. O-Acetylserine synthase is the product of the cysE ge ne, and it catalyzes the rate-limiting step in cysteine biosynthesis, A similar cysE-like gene is also located within the nif gene cluster o f A. vinelandii. The Likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation. Another feature of the iscSUA gene cluster region from it vinelandii i s that E. coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed w ith, the iscSUA genes. The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E. coli and H. influenzae. The E. coli hscA and hscB gene products have previously been shown to bear p rimary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shoc k cognate molecular chaperone system of unknown function. The close pr oximity and apparent co expression of iscSUA and hscBA in A. vinelandi i indicate that the proposed chaperone function of the hscBA gene prod ucts could be related to the maturation of iron-sulfur cluster-contain ing proteins. Attempts to place non polar insertion mutations within e ither A. vinelandii iscS or hscA revealed that such mutations could no t be stably maintained in the absence of the corresponding wild-type a llele, These results reveal a very strong selective pressure against t he maintenance of A. vinelandii iscS or hscA knock-out mutations and s uggest that such mutations are either lethal or highly deleterious, In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably mai ntained. These results show that the cysE-like gene located upstream f rom iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcrip tion units.