Cloning and characterization of the UDP-sugar hydrolase gene (ushA) of Enterobacter aerogenes IFO 12010

A bacterial alkaline phosphatase (BAP, the phoA gene product) is primarily responsible for the hydrolysis of the substrates 5-bromo-4-chloro-3-indolyl -phosphate-p-toluidine (XP) and p-nitrophenyl phosphate (pNPP). Using these substrates and an E. coli phoA mutant, we have cloned Enferobacter aerogen es genes conferring an XP+ phenotype. Two types of clones were identified b ased on phenotypic tests and DNA sequences. One of them is a E. aerogenes p hoA gene (XP+, pNPP(+)) as expected; surprisingly the other one was found t o be a ushA gene (XP+, pNPP(-)), which encodes an UDP (uridine 5'-diphospha te)-sugar hydrolase. The E. aerogenes ushA gene shares high sequence identi ty with ushA off. coli and the mutation-ally silent ushAO gene of Salmonell a typhimurium at both the nucleotide (over 79% and amino acid lover 93%) le vels. Expression of the E. aerogenes ushA gene in E, cell produced high lev el of UDP-sugar hydrolase, as confirmed by TLC (thin layer chromatography) analysis together with a presence of a strong band due to a XP hydrolysis o n a polyacrylamide gel. (C) 2000 Academic Press.