Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium

Abstract

The isc genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the isc operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the isc operon. Here we demonstrate that null mutations in two genes of unknown function, apbC and apbE, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the apbC and apbE mutants, distinguishing them from previously described isc mutants. The deficiencies caused by the apbC and isc mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.Fe-S clusters are cofactors for many proteins and aid in a variety of functions, including electron transfer, protein structure maintenance, substrate binding, and regulation in response to small molecules (reviewed in references 3, 4, 13, 14, 26, and 46). In the last several years, significant strides have been made in our understanding of Fe-S cluster assembly. Expanded efforts in this area were prompted by the discovery of an operon in Azotobacter vinelandii that was involved in Fe-S metabolism (62). Homologues to the genes in this operon (isc) were subsequently demonstrated in Escherichia coli (49,56,57), Salmonella enterica (52), cyanobacteria (51), Pseudomonas aeruginosa (9), Thermotoga maritima (35), mitochondrial eukaryotes (reviewed in reference 34), and amitochondrial anaerobic eukaryotes (55). Genomic sequencing efforts continue to reinforce the conservation of these genes.In A. vinelandii, the isc operon is essential for cell viability (62), but in others, E. coli, S. enterica, and P. aeruginosa, for instance, multiple isc genes or the entire isc gene cluster can be deleted. In each case, the resulting strains are viable but have various growth defects (9,20,49,52,57). In addition, elimination of the isc genes results in reduction but not complete loss of activity of several Fe-S proteins (49, 52, 57), suggesting that additional proteins or systems are involved in the assembly and repair of Fe-S clusters in vivo. One such system is likely to include the Suf proteins, which were hypothesized to have a role in Fe-S cluster metabolism based on their similarity to Isc proteins (41). The suf genes are located in an operon that is regulated by iron and oxidative stress (18,39,41,63) and includes sufA, which has similarity to iscA; sufB, whose gene pr...

The YggX Protein of Salmonella enterica Is Involved in Fe(II) Trafficking and Minimizes the DNA Damage Caused by Hydroxyl Radicals

“…Work from our laboratory has shown that mutations in apbC, apbE, and gshA result in [Fe-S] cluster defects in strains with low levels of YggX (19,49,50). Accumulation of YggX is sufficient to suppress both the nutritional and biochemical defects associated with these mutations (19).…”

The eukaryotic P loop NTPase Nbp35: An essential component of the cytosolic and nuclear iron–sulfur protein assembly machinery

“…The yeast P loop NTPase Cfd1p is required for maintenance of Fe͞S protein activities in the cytosol (10). Likewise, the Salmonella protein ApbC was reported to perform an auxiliary role in Fe͞S protein maturation, cooperating with ApbE (17).…”

Mutational Analysis of ThiH, a Member of the Radical S-Adenosylmethionine (AdoMet) Protein Superfamily

“…The induction of metK in plasmid pMETK2 increases AdoMet synthase activity Ͼ5-fold. 2 ThiH* Phenotype Caused by Other Metabolic Functions Is Distinct from That of thiH Alleles-Mutations in loci involved in Fe-S cluster metabolism (gshA, apbC, apbE, iscA) result a ThiH* phenotype (12,18,19). The induction of metK in gshA, apbC, or apbE mutant strain backgrounds failed to allow significant growth in the absence of tyrosine or THZ (data not shown).…”

“…Mutants defective in apbC, apbE, or gshA functions are compromised in the assembly and/or repair of Fe-S clusters (12,18), and thus we have proposed that the ThiH* phenotype in strains lacking these functions reflects a compromised Fe-S cluster (12 the ThiH* phenotype do so by disrupting the structure of the Fe-S cluster. The effect of such mutations would not be corrected by increased levels of AdoMet as is the case for mutations affecting AdoMet binding.…”

“…The characterized proteins of this family generate radical species by reductive cleavage of AdoMet at the [Fe-S] cluster, and structural and biochemical data have shown that the AdoMet molecule binds either the iron that is not liganded by the protein or one of the 3 -bridging sulfides of the cluster (14 -17). Evidence for the presence of an [Fe-S] cluster in ThiH has been provided by genetic (18,19) and biochemical (20) studies. However, data directly tying the [Fe-S] cluster to enzymatic activity have not been reported.…”