In Salmonella enterica, the thiamine biosynthetic intermediate 5-aminoimidazole ribotide (AIR) can be synthesized de novo independently of the early purine biosynthetic reactions. This secondary route to AIR synthesis is dependent on (i) 5-amino-4-imidazolecarboxamide ribotide (AICAR) accumulation, (ii) a functional phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase (PurC; EC 188.8.131.52), and (iii) methionine and lysine in the growth medium. Studies presented here show that AICAR is a direct precursor to AIR in vivo. PurC-dependent conversion of AICAR to AIR was recreated in vitro. Physiological studies showed that exogenous nutrients (e.g., methionine and lysine) antagonize the inhibitory effects of AICAR on the ThiC reaction and decreased the cellular thiamine requirement. Finally, genetic results identified multiple loci that impacted the effect of AICAR on thiamine synthesis and implicated cellular aspartate levels in AICAR-dependent AIR synthesis. Together, the data here clarify the mechanism that allows conditional growth of a strain lacking the first five biosynthetic enzymes, and they provide additional insights into the complexity of the metabolic network and its plasticity.