Fig 1.
Thiamine and coenzyme A biosynthesis.
(Left) The thiamine biosynthetic pathway uses the branch-point metabolite aminoimidazole ribotide (AIR) from de novo purine biosynthesis. ThiC catalyzes formation of HMP-P from AIR, which is subsequently phosphorylated prior to being condensed with THZ-P to form thiamine-phosphate. Thiamine-phosphate is further phosphorylated to thiamine-pyrophosphate (TPP). (Right) Coenzyme A is synthesized from pantothenate in five steps. The production of the pantoate is predominantly catalyzed from ketopantoate, an intermediate in valine biosynthesis, by PanE. The branched-chain amino acid biosynthetic enzyme acetohydroxyacid isomoreductase (IlvC) has weak ketopantoate reductase activity.
Table 1.
Bacterial strains and primers.
Table 2.
Growth of panE strains containing ThiC variantsa.
Fig 2.
Mutant derivatives of a thiC1129 panE::Cm strain grow on minimal glucose medium.
The parental thiC panE strain fails to grow on minimal glucose medium (filled circles), but a suppressor derivative (DM13897) grows well (filled triangles). Growth of the parental strain was restored by the addition of thiamine (100 nm) (open circles) or pantothenate (100 μM). Growth data show representative experiment repeated with three independent cultures.
Fig 3.
Regulatory region of ilvY and ilvC and IlvY protein sequence.
(A) Genetic context of the regulatory region of ilvY and ilvC in S. enterica genome is schematically represented. Map is zoomed to include only the first 100 bp of ilvY and ilvC genes. Arrows denote transcription start site for the respective gene. Binding of ilvY product at O1 and O2 operator sites, in the presence of IlvC substrate, is required for RNA polymerase binding to the ilvC promoter [38]. (B) Protein alignment of wild-type IlvY with IlvY variants from suppressor mutants. (*M) denotes the start (fMet) of the protein. The solid underlined section indicates the N-terminal helix-turn-helix and the dashed underline section identifies the C-terminal substrate-binding domain. Variant residues in IlvYG92V (ilvY3215), IlvYL235M (ilvY3213), and IlvYC237Y (ilvY3214) are shown above the IlvY protein sequence.
Fig 4.
Mutant ilvY alleles increased expression of ilvC.
Differential expression of ilvC caused by the indicated ilvY alleles is shown. In each case comparison was to the parental strain as described in the text. Strains were grown in minimal glucose medium containing 50 nM thiamine, error bars represent the 95% confidence interval.
Fig 5.
Expression of ilvY3213 or ilvC in trans restores growth to a thiC panE strain.
Strains were grown in glucose minimal medium containing 1.0% arabinose. Growth is shown for a thiC1129 ΔpanE strain containing empty vector, pBAD24 (DM13993; open squares), pBAD24-ilvY (DM13995; solid circles), pBAD24-ilvC (DM13994; open triangles), and pBAD24-ilvY3213 (DM13996; solid squares) as a function of time. Data are representative of three independent cultures.
Fig 6.
ilvY variants feed a pantothenate auxotroph.
DM3547 (panC) was overlaid (in soft agar) onto minimal glucose medium containing 50 nM thiamine. Single colonies of DM13651 (thiC1128 panE), DM13652 (thiC1129 panE), DM13892 (thiC1128 panE ilvY3215), DM13896 (thiC1129 panE ilvY3214), and DM13897 (thiC1129 panE ilvY3213) were stab inoculated into the medium and allowed to grow at 37°C 18 h.
Fig 7.
ilvY variants increase endogenous CoA levels.
Total CoA levels were measured in cells grown in minimal glucose medium with 50 nM thiamine. The data from three independent cultures are represented as the average and standard deviation with an * denoting statistical significance (P < 0.01).
Fig 8.
Model of suppression of thiamine requirement mediated by ilvY alleles.
The IlvY variants described here activate expression of ilvC, enhancing ketopantoate reductase activity in the cell, leading to increased CoA levels that improve ThiC-variant activity to restore thiamine production. The mechanism by which CoA increases ThiC activity remains unclear.