The apbE gene encodes a lipoprotein involved in thiamine synthesis in Salmonella typhimurium

Abstract

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First, apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.

FIG. 1

Schematic representation of the purine and thiamine biosynthetic pathways. Some purine gene products are indicated above the reactions they catalyze. Proposed pathways with an unknown number of reactions are indicated (shaded arrows). ApbE, RseC (3), and ApbC (26) are in paratheses to reflect proposed roles in synthesis of HMP. Abbreviations: PRA, phosphoribosylamine; HMP-PP, HMP pyrophosphate; THZ-P, thiazole phosphate.

FIG. 2

Relative positions of insertions that define the apbE gene. The physical organization of insertions in the apbE gene at 49 min is represented. The amino acid sequence of ApbE adjacent to the MudJ insertion is compared to the amino acid sequence of the E. coli homolog. Primers used in PCR and/or sequencing analysis are indicated (small arrows). The positions of the Tn10d(Tc) insertions were determined by amplification with primer 1 or 3 and primers specific to the ends of the insertion elements. Primers 1 and 3 were used to amplify the gene from the chromosome for cloning and determination of its nucleotide sequence.

FIG. 3

apbE encodes a lipoprotein. (A) [³⁵S]methionine-labeled ApbE and inhibition of its processing by globomycin as analyzed by SDS-PAGE (15% polyacrylamide). Lane 1, DM3407(pT7-5); lane 2, DM3196(pApbE3); lane 3, DM3196(pApbE3) plus globomycin (200 μg/ml). (B) Membrane-associated ApbE was specifically labeled with [³H]palmitic acid and analyzed by SDS-PAGE (12% polyacrylamide). Lane 1, DM3196(pApbE3); lane 2, DM3407(pT7-5). Molecular mass standards (in kilodaltons) for each gel are indicated.

FIG. 4

Growth requirements of apbE mutants. Soft-agar overlays were performed on minimal medium supplemented with gluconate and adenine as previously described (12). DM271 (apbE) (A) and DM908 (purF apbE) (B) cells were plated. The following compounds were spotted (20 nmol each): a 2-μl sample of thiamine (B1), a 2-μl sample of HMP, and a 2-μl sample of thiazole (THZ).