UTILIZATION OF OROTATE AS A PYRIMIDINE SOURCE BY SALMONELLA-TYPHIMURIUM AND ESCHERICHIA-COLI REQUIRES THE DICARBOXYLATE TRANSPORT PROTEIN ENCODED BY DCTA

Mutants deficient in orotate utilization (initially termed out mutants ) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome. A gene c omplementing the mutations was cloned and sequenced and found to posse ss extensive sequence identity to characterized genes for C4-dicarboxy late transport (dctA) in Rhizobium species and to the sequence inferre d to be the dctA gene of Escherichia coli, The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expec ted phenotype of dctA mutants, and introduction of the cloned DNA resu lted in restoration of both C4-dicarboxylate and orotate utilization, Further, succinate was found to compete,vith orotate for entry into th e cell, The S. typhimurium dctA gene encodes a highly hydrophobic poly peptide of 45.4 kDa, and the polypeptide was found to be enriched in t he membrane fraction of minicells harboring a dctA(+) plasmid. The DNA immediately upstream of the deduced -35 region contains a putative cy clic AMP-cyclic AMP receptor protein complex binding site, thus afford ing an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source, The E. coli dctA gene was cloned from a lambda vector and shown to complement C l-dicarboxylate and orotate utilization in FOA-resistant mutants of both E. coli and S. typhimurium. The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.