Metabolic defects caused by mutations in the isc gene cluster in Salmonella enterica serovar typhimurium: implications for thiamine synthesis

Abstract

The metabolic consequences of two insertions, iscR1::MudJ and iscA2::MudJ, in the isc gene cluster of Salmonella enterica serovar Typhimurium were studied. Each of these insertions had polar effects and caused a nutritional requirement for the thiazole moiety of thiamine. Data showed that IscS was required for the synthesis of nicotinic acid and the thiazole moiety of thiamine and that one or more additional isc gene products were required for a distinct step in the thiazole biosynthetic pathway. Strains with isc lesions had reduced succinate dehydrogenase and aconitase activities. Furthermore, isc mutants accumulated increased levels of pyruvate in the growth medium in response to exogenously added iron (FeCl(3)), and this response required a functional ferric uptake regulator, Fur.

FIG. 1

Biosynthetic pathway for thiamine synthesis. Enzymes for the catalysis of each reaction are indicated above the relevant arrow. Enzymes that have not been demonstrated but are predicted to function at certain steps in thiamine biosynthesis are in parentheses. Putative [Fe-S]-containing enzymes are marked by asterisks. The dotted lines represent predicted reactions in the PurF-independent formation of phosphoribosyl amine (PRA). PRPP, phosphoribosylpyrophosphate; AIR, 5-aminoimidazole ribotide; TPP, thiamine pyrophosphate.

FIG. 2

Organization of the isc gene cluster in S. enterica serovar Typhi. The relative locations of two MudJ insertions are indicated by solid triangles, and the assigned allele numbers are shown. The proposed functions of the products are listed below each gene. Note that orf2 in the annotated E. coli genome has been redesignated iscR (D. R. Dean, personal communication).

FIG. 3

isc mutants are altered in pyruvate accumulation. Pyruvate was monitored over time through the A₄₄₃ after derivatization with DNPH. Strains were grown at 37°C in minimal glucose medium supplemented with 100 nM thiamine and 20 μM NA with 50 nM (A) or 50 μM (B) FeCl₃ as described in Materials and Methods. Medium samples were taken every 1.5 h during growth. The strains tested were LT2 (□), DM5419 (◊), and DM5420 (○).

FIG. 4

Regulation of pyruvate accumulation involves Fur. Pyruvate accumulation in the growth medium was monitored every 1.5 h as described in Materials and Methods. Strains were grown at 37°C in minimal medium supplemented with 100 nM thiamine and 20 μM NA with 50 nM (open symbols) or 50 μM (solid symbols) FeCl₃. The circles represent strains containing the fur-1 null mutation, and the squares represent the isogenic fur⁺ strains. The strains tested were the wild type (A), iscR1::MudJ (B), and iscA2::MudJ (C).