Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene

The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.     

Purification of the NusB gene product of Escherichia coli K12

Summary The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al. 1984; Swindle et al. 1984). The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite. Purification of the protein was monitored using ¹⁴C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker. The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3. Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.Abbreviations SDS sodium dodecyl sulphate - kDa kilodaltons - bp base pair(s) - kbp kilobase pair(s)     

Sequence of the lacZ gene of Escherichia coli.

The nucleotide sequence of the lacZ gene coding for beta-galactosidase (EC 3.2.1.23) in Escherichia coli has been determined. Beta-Galactosidase is predicted to consist of 1023 residues, resulting in a protein with a mol. wt. of 116 353 per subunit. The protein sequence originally determined by Fowler and Zabin was shown to be essentially correct and in an Appendix these authors comment on the discrepancies.     

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.     

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower.     

Identification of the purC gene product of Escherichia coli.

The purC region of the Escherichia coli chromosome was isolated from in vivo-derived lambda transducing bacteriophages and cloned in high-copy-number plasmids. The product of the purC gene, phosphoribosylaminoimidazolesuccinocarboxamide synthetase, was identified as a protein with an Mr of ca. 27,000. The level of the protein is increased by more than 60-fold in strains carrying the gene on a high-copy-number plasmid. Purine addition represses the enzyme level in both plasmid- and non-plasmid-containing strains.     

Sequence and overexpression of the menD gene from Escherichia coli.

The menD gene of Escherichia coli codes for the first enzyme of menaquinone biosynthesis, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase. DNA sequence analysis of menD shows an open reading frame encoding a 52-kilodalton protein. Possible promoter and ribosome binding sites are present. Insertion of the menD gene into a tac promoter expression vector leads to nearly a 100-fold increase in the level of SHCHC synthase activity upon induction with isopropyl-beta-D-thiogalactoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins shows a 61-kilodalton protein produced upon induction of the menD-containing expression vector. This is the first reported sequence analysis of a men gene and the first significant amplification of any of the menaquinone biosynthetic enzymes.     

Effects of altered rho gene product on the expression of the Escherichia coli histidine operon.

An altered rho gene product affects expression of the his operon of Escherichia coli. The effect is greater for the operator proximal portion of the his operon than for the operator distal portion. This "rho effect" appears to be independent of the site of action of hisT-altered histidyl-tRNA.     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.     

The lrp gene product regulates expression of lysU in Escherichia coli K-12.

In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion. A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein. The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA.     

Regulation of the Escherichia coli glyA gene by the purR gene product.

The purine regulon repressor protein, PurR, was shown to be a purine component involved in glyA regulation in Escherichia coli. Expression of glyA, encoding serine hydroxymethyltransferase activity, was elevated in a purR mutant compared with a wild-type strain. When the purR mutant was transformed with a plasmid carrying the purR gene, the serine hydroxymethyltransferase levels returned to the wild-type level. The PurR protein bound specifically to a DNA fragment carrying the glyA control region, as determined by gel retardation. In a DNase I protection assay, a 24-base-pair region was protected from DNase I digestion by PurR. The glyA operator sequence for PurR binding is similar to that reported for several pur regulon genes.     

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.