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

(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.     

Nucleotide sequence and gene-polypeptide relationships of the glpABC operon encoding the anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12.

The nucleotide sequence of a 4.8-kilobase SacII-PstI fragment encoding the anaerobic glycerol-3-phosphate dehydrogenase operon of Escherichia coli has been determined. The operon consists of three open reading frames, glpABC, encoding polypeptides of molecular weight 62,000, 43,000, and 44,000, respectively. The 62,000- and 43,000-dalton subunits corresponded to the catalytic GlpAB dimer. The larger GlpA subunit contained a putative flavin adenine dinucleotide-binding site, and the smaller GlpB subunit contained a possible flavin mononucleotide-binding domain. The GlpC subunit contained two cysteine clusters typical of iron-sulfur-binding domains. This subunit was tightly associated with the envelope fraction and may function as the membrane anchor for the GlpAB dimer. Analysis of the GlpC primary structure indicated that the protein lacked extended hydrophobic sequences with the potential to form alpha-helices but did contain several long segments capable of forming transmembrane amphipathic helices.     

Autoregulation of the nar operon encoding nitrate reductase in Escherichia coli

Summary Nitrate reductase is demonstrated to exert an autogenous control on its own synthesis. This effect requires the participation of the molybdenum cofactor. Use of strains in which the control region of the nar operon is mutated reveals two loci in this region: one, affected in strain LCB94, is common to both autoregulation and induction by nitrate while the other, mutated in strain LCB188, is specific for the induction by nitrate. It is proposed that the autogenous control prevents the unnecessary accumulation of the nitrate reductase subunits in the cytoplasm.     

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.     

Nucleotide sequence of the mannitol (mtl) operon in Escherichia coli

The nucleotide sequence of the known portions of the mannitol operon in Escherichia coli (mtlOPAD) has been determined. Both the operator-promoter region and the intercistronic region between the mtlA and mtlD genes (encoding the mannitol-specific Enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase, respectively) show parallels with corresponding regions of the glucitol (gut) operon, but neither the mtlA nor the mtlD gene products show obvious homology with the corresponding gene products of the glucitol operon. Five potential cyclic AMP receptor protein binding sites were identified in the mtlOP region, all showing near identity with the consensus sequence. Four regions of dyad symmetry (four to seven bases in length), serving as potential repressor binding sites, overlap with the potential cyclic AMP receptor protein binding sites. Repetitive extragenic palindromic (REP) sequences, forming stem-loop structures in the intercistronic region between mtlA and mtlD and following the mtlD gene were identified. Probable terminator sequences were not found in any of these three regulatory regions. Mannitol-1-phosphate dehydrogenase exhibits two overlapping, potential NAD+ binding sites near the N-terminus of the protein. Computer techniques were used to analyse the mtlD gene and its product.     

Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli

The nucleotide sequence of the promoter region of the melibiose operon of E. coli was determined. Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region. The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon. A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region. The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.     

Nucleotide sequence analysis of the purEK operon encoding 5''-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.     

Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli.

We determined the DNA sequence of the hag gene of Escherichia coli K-12 and deduced the primary structure of the flagellin consisting of 497 amino acid residues. Comparison of the amino acid sequence with those of other bacterial flagellins revealed a high homology in the NH2- and COOH-terminal regions.     

Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli

We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.     

Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli.

The gene katG, encoding catalase HPI of Escherichia coli, was sequenced, predicting a 726-amino-acid protein. The sequence was confirmed by identification of potential regulatory elements and amino acid sequencing of peptides. HPI shows no homology to other catalases. The distances between katG, metF, and ppc were defined.     

Nucleotide sequence and expression of the gutQ gene within the glucitol operon of Escherichia coli

The glucitol operon in Escherichia coli is known to consist of five structural genes with the order: gut O P A B D M R. We have sequenced downstream from gutR and have identified an open reading frame encoding a water-soluble protein (223 amino acids; molecular weight = 23,562) with a putative ATP binding site. Expression of this protein in a maxicell system has been demonstrated. A repetitive extragenic palindromic (REP) sequence capable of forming stem-loop structures follows gutQ in the downstream, presumptive intercistronic region. The function of the Gut Q protein is not known.