Complex metabolic phenotypes caused by a mutation in yjgF, encoding a member of the highly conserved YER057c/YjgF family of proteins

Abstract

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.

FIG. 1

Pathway schematics. (A) Biosynthetic pathway for TPP. The involvement of the purine pathway in HMP-PP synthesis is shown with structural intermediates prior to the AIR branch point. Arrows denoted with dotted lines represent proposed steps. Reactions involved in the conversion of AIR to HMP-PP and in the synthesis of THZ-P have not been clearly defined. Genes whose products are required for selected reactions are indicated next to the relevant arrows. Abbreviations: R-P, ribose-5-phosphate, PRPP, phosphoribosylpyrophosphate. (B) Biosynthetic pathways for the branched-chain amino acids isoleucine and valine. Enzymes that catalyze specific steps are as follows: 1, aspartate transaminase; 2, 3, and 4, aspartate kinases I, II, and III, respectively; 5, aspartate semialdehyde dehydrogenase; 6 and 7, homoserine dehydrogenases I and II, respectively; 8, homoserine kinase; 9, threonine synthase; 10, threonine deaminase; 11 and 12, acetohydroxy acid synthases I and II, respectively; 13, acetohydroxy acid isomeroreductase; 14, dihydroxy acid dehydratase; 15, transaminase B; 16, transaminase C. OAA, oxaloacetic acid.

FIG. 2

Location of the yjgF2::Tn10d(Tc) insertion. A schematic representation of the 96.8-min region of S. typhimurium (St.) is shown. The yjgF2::Tn10d(Tc) insertion was mapped within the yjgF gene based on homology with E. coli (Ec.). DNA between the yjgF2::Tn10d(Tc) insertion and the pyrB2694::MudA insertion was amplified by PCR. Sequences flanking the yjgF2::Tn10d(Tc) insertion were determined, and the predicted amino acids are shown. Sequence analysis of the region flanking the yjgF1::Tn10d(Tc) insertion placed it at the same location. Amino acids conserved between the two organisms are in boldface, and numbers represent amino acids in E. coli YjgF protein. The inserts contained in the pT₇-5YjgF1 and pSU19YjgFa plasmids are indicated by the hatched lines. The mgtA gene is not drawn to scale.

FIG. 3

The YjgF protein is highly conserved. The DNASTAR alignment program was used to align several proteins homologous to YjgF of S. typhimurium. Regions shaded in black represent amino acids that are conserved with respect to YjgF. Proteins in the alignment include E. coli YjgF, B. subtilis YabJ, Synechocystis hypothetical 13.8-kDa protein, S. cerevisiae YER057c, rat 14.5-kDa translational inhibitor protein, and human 14.5-kDa translational inhibitor protein.

FIG. 4

Overexpression of YjgF. Overexpression of YjgF was performed by using the T7 overexpression system as described in Materials and Methods. Proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. Extracts from DM5032 and DM5031, which contain plasmids pT₇-6YjgFa and pT₇-5YjgFa, respectively, are shown in lanes A and B, respectively. pT₇-6YjgFa contains the insert in the proper orientation for YjgF expression from the T7 promoter. Numbers at left indicate molecular mass in kilodaltons.

FIG. 5

Phenotypes caused by a yjgF mutation. Growth curves were obtained from strains grown at 37°C as described in Materials and Methods. (A) Suppression of the requirement for Gnd in thiamine synthesis. Growth of DM574 [purF2085 gnd175::Tn10d(Tc)] in gluconate-adenine medium and gluconate-adenine-thiamine medium is indicated by open and solid squares, respectively. Growth of DM4772 [purF2085 gnd175::Tn10d(Tc) yjgF3::MudJ] in the same medium is indicated by open and solid inverted triangles, respectively. (B and C) Sensitivity to exogenous serine. Shown are growth of LT2 (wild type) (B) and growth of DM3480 (yjgF3::MudJ) (C) in minimal glucose medium (solid circles), glucose medium supplemented with serine (open triangles), and glucose medium supplemented with serine and isoleucine (solid triangles).

FIG. 6

Flux through the isoleucine biosynthetic pathway is important for restoration of thiamine synthesis by a yjgF mutation. Growth curves were obtained from strains grown at 37°C as described in Materials and Methods. Shown are growth of DM4767 [purF2085 gnd174::MudJ yjgF2::Tn10d(Tc)] (A) and growth of DM4769 [purF2085 gnd174::MudJ yjgF2::Tn10d(Tc) ilvA219] (B) in minimal gluconate medium supplemented with adenine (solid squares); adenine and isoleucine (open squares); adenine and thiamine (solid circles); and adenine, thiamine, and isoleucine (open circles).