Construction of genetically defined aroA mutant of a native E. coli O78:K80 isolated from avian colibacillosis, in Iran

A precisely defined, single gene deletion of aroA was made systematically by Datsenko and Wanner based approach in native Escherichia coli O78: K80, and its nature was examined. The aroA gene encodes 5-enolpyrovylshikimate3-phosphate synthase and participates in the aromatic amino acids and folic acid, which are universal metabolic pathways of bacteria. For construction of mutant strain, the gene was replaced by recombination with a chloramphenicol cassette, flanked by FLP recognition target site. Primers designed to create in-frame deletion upon excision of the resistance cassette. The aroA deletion was confirmed by both polymerase chain reaction and failure of the mutant to replicate and grow on minimum medium lacking aromatic amino acids. On the other hand, the wild-type parent strain grows well in this medium. The sequence of the aroA gene in native strain was different, and knowing this subject was important to produce a mutant strain with deletion-based methods of mutation. Furthermore, use of antibiotic cassette replacement facilitates mutant construction in a more cost-effective manner in comparison with other techniques such as use of suicide vectors.