Decreased transport restores growth of a Salmonella enterica apbC mutant on tricarballylate

Abstract

Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in iron-sulfur ([Fe-S]) cluster metabolism. An apbC mutant is unable to grow on tricarballylate as a carbon source. Based on the ability of ApbC to transfer an [Fe-S] cluster to an apoprotein, this defect was attributed to poor loading of the [Fe-S] cluster-containing TcuB enzyme. Consistent with these observations, a previous study showed that overexpression of iscU, which encodes an [Fe-S] cluster molecular scaffold, suppressed the tricarballylate growth defect of an apbC mutant (J. M. Boyd, J. A. Lewis, J. C. Escalante-Semerena, and D. M. Downs, J. Bacteriol. 190:4596-4602, 2008). In this study, tcuC mutations that suppress the growth defect of an apbC mutant by decreasing the intracellular concentration of tricarballylate are described. Collectively, the suppressor analyses support a model in which reduced TcuB activity prevents growth on tricarballylate by (i) decreasing catabolism and (ii) allowing levels of tricarballylate that are toxic to the cell to accumulate. The apbC tcuC mutant strains described here reveal that the balance of the metabolic network can be altered by the accumulation of deleterious metabolites.

Fig 1

Tricarballylate catabolism in S. enterica. (A) Model for tricarballylate catabolism based on the work of Lewis et al. (31). TcuC protein transports tricarballylate into the cell, where it is oxidized to cis-aconitate by the flavoprotein TcuA. cis-Aconitate enters the TCA cycle through hydration to isocitrate, catalyzed by the aconitase enzymes (AcnA, AcnB). The reduced flavin of TcuA is recycled by passing the electrons to TcuB, which is thought to ultimately pass the electrons to the ubiquinone pool. ApbC is responsible for the insertion or repair of the two [4Fe-4S] clusters of TcuB. (B) A constraint at TcuB caused by the lack of ApbC has two consequences that can be solved by distinct suppressor mutations. First, decreased TcuAB activity prevents sufficient flux from tricarballylate from reaching the TCA cycle to allow growth. In this case, an increase in non-ApbC loading of clusters in TcuB increases TcuB function (caused by iscR mutations [14]). Second, tricarballylate accumulates, inhibiting isocitrate dehydrogenase and thus preventing growth (described in this study).

Fig 2

tcuC alleles allow the growth of apbC mutants after a lag. Shown is the growth of strains DM10310 (wild type) (●), DM10300 (apbC) (○), DM10452 (apbC tcuC51) (■), and DM10448 (apbC tcuC54) (▴) with 20 mM tricarballylate as the sole carbon and energy source at 37°C in NCE medium supplemented with 100 nM thiamine. Outgrowth after reinoculation had the same lag times as those shown here.

Fig 3

Mutations in tcuC reduce growth on tricarballylate. Shown is the growth of strains DM10310 (wild type) (triangles) and DM11090 (tcuC54) (squares) with 10 mM (filled symbols) or 20 mM (open symbols) tricarballylate as the sole carbon and energy source at 37°C in NCE medium supplemented with 100 nM thiamine.

Fig 4

A tcuB mutant strain is sensitive to tricarballylate when growing with citrate. (A) Strain DM11428 (tcuB17) was grown with citrate (11 mM) with increasing concentrations of tricarballylate in the growth medium. The data presented are from a representative experiment showing growth in the absence of tricarballylate (filled circles) and in the presence of 0.5 mM (open circles), 1 mM (filled inverted triangles), 1.5 mM (open triangles), 2 mM (filled squares), 2.5 mM (open squares), 3 mM (filled diamonds), 3.5 mM (open diamonds), 4 mM (filled triangles), 4.5 mM (open inverted triangles), 5 mM (filled hexagons), and 6 mM (open hexagons) tricarballylate. (B) Plot of the time that strains DM10412 (wt) (□) and DM11428 (tcuB17) (■) took to reach mid-log phase when grown on citrate (11 mM) versus the concentration of tricarballylate present in the growth medium. Growth was monitored at 37°C in NCE medium supplemented with 100 nM thiamine and 20 μM nicotinic acid.

Fig 5

An apbC mutant is sensitive to tricarballylate when grown on citrate, and the growth defect is relieved by the presence of a mutant tcuC allele. Shown is a plot of the time required for strains DM10300 (apbC) ○), DM10448 (apbC tcuC51) (□), and DM10452 (apbC tcuC54) (▴) to reach the mid-log phase of growth when growing on citrate versus the concentration of tricarballylate added to the growth medium. Growth was monitored in NCE medium with 11 mM citrate supplemented with 100 nM thiamine, 20 μM nicotinic acid, and 0 to 30 mM tricarballylate.

Fig 6

The presence of a mutation in iscU exacerbates the growth defect of an apbC mutant. Strains DM10300 (apbC) (○), DM11671 (iscU) (▴), and DM11221 (apbC iscU) (▵) were grown with citrate in the presence of 0 to 30 mM tricarballylate. The time that it took these strains to reach the mid-log phase of growth was plotted against the concentration of tricarballylate present in the growth medium. Growth was monitored in NCE medium with 11 mM citrate supplemented with 100 nM thiamine and 20 μM nicotinic acid.