Suppressor analyses identify threonine as a modulator of ridA mutant phenotypes in Salmonella enterica

Abstract

The RidA (YjgF/YER057c/UK114) family of proteins is broadly conserved in the three domains of life yet the functional understanding of these proteins is at an early stage. Physiological studies of ridA mutant strains of Salmonella enterica provided a framework to inform in vitro studies and led to the description of a conserved biochemical activity for this family. ridA mutant strains of S. enterica have characteristic phenotypes including new synthesis of thiamine biosynthetic intermediate phosphoribosylamine (PRA), inability to grow on pyruvate as a sole carbon and energy source or when serine is present in the minimal growth medium, and a decreased specific activity of transaminase B (IlvE). Secondary mutations restoring growth to a ridA mutant in the presence of serine were in dapA (encoding dihydrodipicolinate synthase) and thrA (encoding homoserine dehydrogenase). These mutations suppressed multiple ridA mutant phenotypes by increasing the synthesis of threonine. The ability of threonine to suppress the metabolic defects of a ridA mutant is discussed in the context of recent biochemical data and in vivo results presented here.

Figure 1. Mutations in dapA restore growth to ridA mutants in the presence of serine.

Growth was monitored over time as optical density at 650 nm. Strains were grown at 37°C in minimal glucose medium with no additions (closed symbols) or 5 mM serine (open symbols). Shown are strains ridA (DM3480), squares; ridA dapA356 (DM11637), triangles; and ridA dapA360 (DM11640), circles. Curves displayed were representative of 3 biological replicates.

Figure 2. Pathway for synthesis of aspartate-derived amino acids.

Aspartate is a precursor to lysine, methionine, threonine, and isoleucine, as depicted here. Aspartate 4-semialdehyde (ASA) is a branchpoint metabolite controlled by the activities of DapA, ThrA, and MetL.

Figure 3. Suppressor mutations increase growth in purF ridA strain background.

Strains were grown at 37°C in minimal glucose medium with adenine (open symbols) or further supplemented with 0.3 mM threonine (closed symbols). Growth was monitored over time as optical density at 650 nm. Shown are strains purF ridA (DM3871), triangles; purF ridA thrA1371 (DM6309), diamonds; and purF ridA dapA356 (DM11412), circles. Error bars represent standard deviations of three biological replicates.