| Abstrakt: |
An enzyme having the samel-cysteine desulfurization activity previously described for the NifS protein was purified from a strain ofAzotobacter vinelandiideleted for the nifSgene. This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer. Information 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 A. vinelandiigenomic segment that includes the iscSgene. The iscSgene is contained within a gene cluster that includes homologs to nifUand another gene contained within the major nifcluster ofA. vinelandiipreviously designated orf6. These genes have been designated iscUand iscA,respectively. Information available from complete genome sequences ofEscherichia coliand Hemophilus influenzaereveals that they also encode iscSUAgene clusters. A wide conservation of iscSUAgenes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUAgenes could play a general role in the formation or repair of iron-sulfur clusters. The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A. vinelandiiis supported by the presence of acysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUAgenes. O-Acetylserine synthase is the product of thecysEgene, and it catalyzes the rate-limiting step in cysteine biosynthesis. A similar cysE-like gene is also located within the nifgene cluster of 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 iscSUAgene cluster region from A. vinelandiiis that E. coligenes previously designated as hscB,hscA, and fdxare located immediately downstream from, and are probably co-transcribed with, theiscSUAgenes. The hscB, hscA, andfdxgenes are also located adjacent to theiscSUAgenes in both E. coliand H. influenzae. The E. coli hscAand hscBgene products have previously been shown to bear primary sequence identity when respectively compared with the dnaKanddnaJgene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function. The close proximity and apparent co-expression of iscSUAand hscBAin A. vinelandiiindicate that the proposed chaperone function of the hscBAgene products could be related to the maturation of iron-sulfur cluster-containing proteins. Attempts to place non-polar insertion mutations within eitherA. vinelandii iscSor hscArevealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele. These results reveal a very strong selective pressure against the maintenance of A. vinelandii iscSor hscAknock-out mutations and suggest that such mutations are either lethal or highly deleterious. In contrast toiscSor hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained. These results show that thecysE-like gene located upstream from iscSis not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdxgenes are contained within separate transcription units. |
