Mutations in yhiT enable utilization of exogenous pyrimidine intermediates in Salmonella enterica serovar Typhimurium

Abstract

Mutants capable of utilizing the pyrimidine biosynthetic intermediates carbamoylaspartate and dihydroorotate for growth were derived from pyrimidine auxotrophs of Salmonella enterica serovar Typhimurium LT2. The gain-of-function phenotypes both resulted from mutations in a single gene, yhiT, the third gene of a putative four-gene operon, yhiVUTS, for which there is no homologous region in Escherichia coli. Notably, when a mutant yhiT allele was transferred to a pyrimidine-requiring E. coli strain, the transformant was then capable of using carbamoylaspartate or dihydrorotate as a pyrimidine source. The operon arrangement of the yhiVUTS genes was supported by genetic analyses and studies employing RT-PCR, coupled to the determination of the transcriptional start site using 5'-random amplification of cDNA ends (RACE). Computer-generated predictions indicated that YhiT is an integral membrane protein with 12 putative transmembrane domains typical of bacterial transport proteins. Competition experiments showed that mutant YhiT interacts with the C4-dicarboxylates succinate and malate, as well as the amino acids aspartate and asparagine. The native function of wild-type YhiT remains undetermined, but the collective results are consistent with a role as a general transporter of C4-dicarboxylates and other compounds with a similar basic structure.