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-indolylphosphate-p-toluidine (XP) and p-nitrophenyl phosphate (pNPP). Using these substrates and an E. coli phoA mutant, we have cloned Enterobacter aerogenes genes conferring an XP(+) phenotype. Two types of clones were identified based on phenotypic tests and DNA sequences. One of them is a E. aerogenes phoA gene (XP(+), pNPP(+)) as expected; surprisingly the other one was found to be a ushA gene (XP(+), pNPP(-)), which encodes an UDP (uridine 5'-diphosphate)-sugar hydrolase. The E. aerogenes ushA gene shares high sequence identity with ushA of E. coli and the mutationally silent ushA0 gene of Salmonella typhimurium at both the nucleotide (over 79%) and amino acid (over 93%) levels. Expression of the E. aerogenes ushA gene in E. coli produced high level 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 on a polyacrylamide gel.
Lee
K S
KS
Research Center for Biomedicinal Resources (Bio-Med RRC), Pai-Chai University, Taejon, 302-735, Korea. kslee@www.paichai.ac.kr
Song
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Kim
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Kim
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Kim
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Kho
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BH
Ko
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DK
Choi
Y K
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Lee
Y K
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Kim
C K
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Kim
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Lim
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Kim
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Min
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Wanner
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D010641:Phenotype; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; C406334:ushA protein, E coli; D010727:Phosphoric Diester Hydrolases