The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli

Abstract

The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established. To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized. Using this iscS deletion strain, we have observed decreased specific activities for proteins containing [4Fe-4S] clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase). A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS. These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis. We also have characterized a growth phenotype associated with the loss of IscS. Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required. The lack of IscS also had a general effect on the growth of E. coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied.

Figure 1

Genomic organization of the isc region. Plasmids used in this study are indicated. The genotype of the iscS mutant is indicated with the iscS coding region having been replaced by the Kn^R gene.

Figure 2

The iscS mutant has a small colony phenotype. Colony phenotype of the iscS mutant (PK4331; Top) after 48 h of growth on LB plates at 37°C compared with the isogenic IscS⁺ strain (RZ4500; Bottom). Each spot represents a 10-fold serial dilution. The iscS mutant complemented with iscS in trans on plasmid pPK4196 (Middle).

Figure 3

Growth phenotype of the iscS mutant. (A) Growth curves of the iscS mutant (PK4331; ■) and the isogenic IscS⁺ parent strain (RZ4500; □) grown aerobically in LB medium. (B) Growth curves of the iscS mutant (●) and the parent strain (○) grown aerobically in glucose minimal medium, and the iscS mutant (■) and the parent strain (□) grown in glucose minimal medium supplemented with thiamine and nicotinic acid.

Figure 4

The iscS mutant has reduced FNR activity that can be complemented by the addition of iscS in trans. FNR activity was measured by assaying the amount of β-galactosidase (Miller units) produced in strains grown under anaerobic conditions containing the FNR-dependent dmsA′∷lacZ promoter fusion. The genotypes are indicated below the figure. Complemented strains contained either pPK4196 (iscS) or pPK4194 (iscSUA) and as with complemented strains in Figs. 2 and 3, expression of the isc gene(s) depended on fortuitous promoter elements from the vector (pET11a). The strains are all derivatives of either PK3292 (FNR⁺) or PK3293 (FNR^-; Table 1).

Figure 5

The effect of the iscS mutation on the activity of FNR mutants that exhibit altered Fe-S cluster requirements. β-galactosidase activity produced from the dmsA′∷lacZ promoter fusion was assayed from strains (IscS⁺; PK3293, filled columns, IscS^-; PK4359, open columns) grown under aerobic conditions (Upper) and anaerobic conditions (Lower). WT FNR, FNR-D154A, and FNR-L28H were present on the multicopy plasmid pACYC184 (58).