UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme

The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  C. Price,et al.  Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase and is controlled by stationary-phase transcription factor sigma B , 1993, Journal of bacteriology.

[3]  E. P. Kennedy,et al.  Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides , 1992, Journal of bacteriology.

[4]  C. Ueguchi,et al.  Multicopy suppression: an approach to understanding intracellular functioning of the protein export system , 1992, Journal of bacteriology.

[5]  G. Brede,et al.  Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene , 1991, Journal of bacteriology.

[6]  P. Reeves,et al.  Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2) , 1991, Molecular microbiology.

[7]  M. Black,et al.  Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure , 1991, Molecular microbiology.

[8]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[9]  P. Postma,et al.  Trehalose transport and metabolism in Escherichia coli , 1990, Journal of bacteriology.

[10]  P. Tempst,et al.  Examination of automated polypeptide sequencing using standard phenyl isothiocyanate reagent and subpicomole high-performance liquid chromatographic analysis. , 1989, Analytical biochemistry.

[11]  E. Pradel,et al.  Mapping of the Escherichia coli acid glucose-1-phosphatase gene agp and analysis of its expression in vivo by use of an agp-phoA protein fusion , 1989, Journal of bacteriology.

[12]  A. Strøm,et al.  Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli , 1988, Journal of bacteriology.

[13]  C. Marck,et al.  'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. , 1988, Nucleic acids research.

[14]  P. Matsudaira,et al.  Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. , 1987, The Journal of biological chemistry.

[15]  C. Richardson,et al.  DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Elledge,et al.  Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli , 1985, Journal of bacteriology.

[17]  J. Preiss,et al.  Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glg C gene. , 1983, The Journal of biological chemistry.

[18]  E. Thomassen,et al.  Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene , 1976, Journal of bacteriology.

[19]  M. Sunshine,et al.  Extent of Host Deletions Associated with Bacteriophage P2-Mediated Education , 1971, Journal of bacteriology.

[20]  T. Nakae,et al.  Multiple molecular forms of uridine diphosphate glucose pyrophosphorylase from Salmonella typhimurium. II. Genetic determination of multiple forms. , 1971, The Journal of biological chemistry.

[21]  T. Nakae,et al.  Multiple molecular forms of uridine diphosphate glucose pyrophosphorylase from Salmonella typhimurium. I. Catalytic properties of various forms. , 1971, The Journal of biological chemistry.

[22]  T. Nakae.  Multiple molecular forms of uridine diphosphate glucose pyrophosphorylase from Salmonella typhimurium. 3. Interconversion between various forms. , 1971, The Journal of biological chemistry.

[23]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[24]  K. Nikaido,et al.  Extended deletions in the histidine-rough-B region of the Salmonella chromosome. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. Shapiro.  Chromosomal location of the gene determining uridine diphoglucose formation in Escherichia coli K-12 , 1966, Journal of bacteriology.

[26]  H. Kalckar,et al.  Biochemical observations on E. coli mutants defective in uridine diphosphoglucose. , 1962, Proceedings of the National Academy of Sciences of the United States of America.

[27]  K. Kurahashi,et al.  A new enzymic defect of galactose metabolism in Escherichia coli K-12 mutants. , 1962, Biochemical and biophysical research communications.

[28]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[29]  H. Kalckar.  Uridinediphospho galactose; metabolism, enzymology, and biology. , 1958, Advances in enzymology and related subjects of biochemistry.