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Nucleotide sequence of the gene encoding the GMP reductase of Escherichia coli K12.

ABSTRACT: (1) The nucleotide sequence of a 1991 bp segment of DNA that expresses the GMP reductase (guaC) gene of Escherichia coli K12 was determined. (2) This gene comprises 1038 bp, 346 codons (including the initiation codon but excluding the termination codon), and it encodes a polypeptide of Mr 37,437 which is in good agreement with previous maxicell studies. (3) The sequence contains a putative promoter 102 bp upstream of the translational start codon, and this is immediately followed by a (G + C)-rich discriminator sequence suggesting that guaC expression may be under stringent control (4) The GMP reductase exhibits a high degree of sequence identity (34%) with IMP dehydrogenase (the guaB gene product) indicative of a close evolutionary relationship between the salvage pathway and the biosynthetic enzymes, GMP reductase and IMP dehydrogenase, respectively. (5) A single conserved cysteine residue, possibly involved in IMP binding to IMP dehydrogenase, was located within a region that possesses some of the features of a nucleotide binding site. (6) The IMP dehydrogenase polypeptide contains an internal segment of 123 amino acid residues that has no counterpart in GMP reductase and may represent an independent folding domain flanked by (alanine + glycine)-rich interdomain linkers.

SUBMITTER: Andrews SC

PROVIDER: S-EPMC1135187 | biostudies-other | 1988 Oct

REPOSITORIES: biostudies-other

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Molecular cloning and characterization of Escherichia coli K12 ygjG gene.

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Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

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Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

Project description:The DNA sequence of a 3.23-kilobase fragment of the Escherichia coli chromosome encoding biosynthetic arginine decarboxylase (ADC) was determined. This sequence contained the speA open reading frame (ORF) as well as partial speB and metK ORFs. The ADC ORF is 1,974 nucleotides long; the deduced polypeptide contains 658 amino acids with a molecular size of 73,980 daltons. The molecular weight and predicted ADC amino acid composition are nearly identical to the amino acid analysis of purified ADC performed by Wu and Morris (J. Biol. Chem. 248:1687-1695, 1973). A translational speA-lacZ fusion, pRM65, including 1,389 base pairs (463 amino acids) of the 5' end of speA was constructed. Western blots (immunoblots) with beta-galactosidase antisera revealed two ADC::beta-galactosidase fusion proteins in E. coli bearing pRM65: 160,000 and 156,000 daltons representing precursor and mature hybrid proteins, respectively. The predicted amino acid sequence of ADC contains a region of six amino acid residues found in two bacterial diaminopimelic acid decarboxylases and three eucaryotic ornithine decarboxylases. This conserved sequence is located approximately eight amino acids from the putative pyridoxal phosphate-binding site of ADC and is predicted to be involved in substrate binding.

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Control of gene expression by PdeL, a transcription regulator and c-di-GMP phosphodiesterase in Escherichia coli K12

Project description:PdeL is a transcription regulator and c-di-GMP specific phosphodiesterase in Escherichia coli. To address the transcription regulator function of PdeL we analyzed the transcriptomes of four E. coli K12 strains during the exponential growth phase by RNA-sequencing. These four strains included (1) wild-type E. coli K12 strain BW30270 carrying an empty vector control plasmid, (2) an isogenic pdeL deletion mutant carrying the control plasmid, as well as the pdeL mutant that was complemented with (3) a plasmid carrying pdeL under control of the IPTG-inducible tac promoter or (4) a plasmid encoding a fusion protein of the PdeL’s DNA-binding domain and the C-terminal dimerization domain of phage Lambda cI repressor (PdeL-DBD_cI-C). Expression of plasmid-encoded pdeL and pdeL-DBD_cI-C, respectively, was induced by addition of IPTG for 15 minutes prior to RNA isolation. Analyses of the RNA-seq data revealed that plasmid-provided PdeL (and PdeL-DBD_cI-C) repress transcription of class II flagellar genes and presumably regulate the transcription of additional loci, while only little differences were observed between the transcriptomes of wild-type strain BW30270 and its isogenic pdeL mutant.

2020-07-31 | GSE136273 | GEO

Electron-paramagnetic-resonance studies on nitrate reductase from Escherichia coli K12.

Project description:Nitrate reductase was purified from anaerobically grown Escherichia coli K12 by a method based on the Triton X-100 extraction procedure of Clegg[(1976) Biochem. J.153, 533-541], but hydrophobic interaction chromatography was used in the final stage. E.p.r. spectra obtained from the enzyme under a variety of conditions are well resolved and were interpreted with the help of the computer-simulation procedures of Lowe [(1978) Biochem. J.171, 649-651]. Parameters for five molybdenum(V) species from the enzyme are given. The low-pH species (g(av.) 1.9827) is in pH-dependent equilibrium with the high-pH species (g(av.) 1.9762), the pK for interconversion of the species being 8.26. Of a variety of anions tested, only nitrate and nitrite formed complexes with the enzyme (in the low-pH form), giving modified molybdenum(V) e.p.r. spectra. These complexes, as well as the low-pH form of the free enzyme, showed interaction of molybdenum with a single exchangeable proton. The fifth molybdenum(V) species, sometimes detected in small amounts, appears not to be due to functional nitrate reductase. After full reduction of the enzyme with dithionite, addition of nitrate caused reoxidation of molybdenum to the quinquivalent state, in a time less than the enzyme turnover. Activity of the enzyme in the pH range 6-10 is controlled by a pK of 8.2. It is suggested that the low-pH signal-giving species is the form of the enzyme involved in the catalytic cycle. Iron-sulphur and other e.p.r. signals from the enzyme are briefly described and the enzymic reaction mechanism is discussed.

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A possible glycine radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD gene.

Project description:During anaerobic growth of Escherichia coli an oxygen-sensitive ribonucleoside-triphosphate reductase, different from the aerobic ribonucleoside diphosphate-reductase (EC 1.17.4.1), produces the deoxyribonucleoside triphosphates required for DNA replication. The gene for the anaerobic enzyme has now been cloned and was found to contain a 2136-nucleotide coding region, corresponding to 712 amino acid residues, and an Fnr binding site 228 base pairs upstream of the initiator ATG. The deduced amino acid sequence shows 72% identity to a gene of coliphage T4, sunY, hitherto of unknown function, suggesting that the virus codes for its own anaerobic reductase. The location of an organic free radical formed during activation of the bacterial anaerobic reductase is proposed to be on Gly-681, since the pentapeptide RVCGY at positions 678-682 shows a striking similarity to the C-terminal sequence. RVSGY, of pyruvate formate-lyase. During activation of the anaerobically induced pyruvate formate-lyase, the glycine residue of the pentapeptide becomes an organic radical [Wagner, A. F. V., Frey, M., Neugebauer, F. A., Schäfer, W. & Knappe, J. (1992) Proc. Natl. Acad. Sci. USA 89, 996-1000]. The gene for the anaerobic reductase is located at a position around 96 min on the E. coli genomic map.

| S-EPMC45706 | biostudies-other

Transcriptional responses of Escherichia coli k12 TPEN

Project description:DNA microarrays were conducted on E. coli K12 cells stressed with 10 μM in N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Overall, 260 genes varied in expression, 114 up-regulated and 146 down-regulated by Zn deprivation Keywords: TPEN stress

2008-12-31 | GSE11303 | GEO

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