Cloning and analysis of the shiA gene, which encodes the shikimate transport system of Escherichia coli K-12

• Published in: Gene Vol. 209; no. 1; pp. 185 - 192
• Main Authors: Whipp, M.J, Camakaris, H, Pittard, A.J
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 16.03.1998
• Subjects: Amino Acid Sequence; amn gene; Aromatic intermediate; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Carrier Proteins - biosynthesis; Carrier Proteins - chemistry; Carrier Proteins - genetics; Chromosome Mapping; Cloning, Molecular; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Genes, Bacterial; Major facilitator superfamily; Membrane Transport Proteins; Mini-Mu replicon; Models, Molecular; Molecular Sequence Data; Open Reading Frames; Protein Structure, Secondary; Sequence Alignment; Sequence Homology, Amino Acid; Shikimic Acid - metabolism; amn, gene encoding AMP nucleosidase; Mini-Mu replicon; Xgal, 5-bromo-4-chloro-3-indolyl β- d-galactopyranoside; Km, kanamycin; ORF, open reading frame; RBS, ribosome-binding site; bp, base pair(s); R, (superscript) resistance; amn gene; MFS, major facilitator superfamily; nt, nucleotide(s); ShiA, shikimate transport protein; kb, kilobase(s); Major facilitator superfamily; aroD, gene encoding 3-dehydroquinate dehydratase; aroE, gene encoding shikimate dehydrogenase; DHS, dehydroshikimate; shiA, gene encoding shikimate transport protein; a r o B, gene encoding 3-dehydroquinate synthase; lacZ, gene encoding β-galactosidase; Aromatic intermediate
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(98)00043-2

In Escherichia coli K-12, the shiA gene is involved in the uptake of shikimate. This gene has been cloned and its nucleotide sequence determined. The gene is predicted to encode a protein of 438 amino acids and lies adjacent to the amn gene. The hydropathy profile and the amino acid sequence indicate that the ShiA protein is a polytopic membrane protein that shows a homology with members of the major facilitator superfamily of transport proteins. Recombining an inactive form of the cloned gene into the chromosome creates mutants unable to transport shikimate. Introducing a wild-type gene on a multicopy plasmid into a shiA mutant restores the ability to transport shikimate. When this multicopy shiA plasmid is introduced into an aroE strain, this strain is now able to grow with shikimate as the aromatic supplement, consistent with the notion that dehydroshikimate (DHS) accumulated in an aroE strain prevents uptake of shikimate by competition. Expression of the shiA gene does not appear to be regulated by the TyrR protein, a repressor/activator that controls the expression of other genes involved with the biosynthesis or transport of the aromatic amino acids.