Escherichia coli K-12 clones that overproduce dam methylase are hypermutable.

A strain of Escherichia coli K-12 that overproduces dam methylase 50-fold was found to be hypermutable, and mutations which resulted in loss of excess methylase activity restored mutation frequencies to wild-type levels. These results are consistent with involvement of this deoxyribonucleic acid methylase in mismatch correction.     

Cloning and sequence of the crp gene of Escherichia coli K 12.

We have determined the nucleotide sequence of the crp gene of Escherichia coli K 12. From a lambda transducing phage, the crp region was subcloned into pBR322. The gene was localized on the cloned fragment by determining the length of deletions which affect its expression. Its nucleotide sequence was established by using the technique of Maxam and Gilbert. The deduced amino-acid sequence is in agreement with the previously published amino acid composition of the protein (1, 2). Analysis of the sequence confirms that the DNA binding domain is located in the C-terminal portion of the protein.     

Isolation and characterization of a mutant of Escherichia coli K12 synthesizing DNA polymerase I and endonuclease I constitutively

A mutant of Escherichia coli K12, highly resistant to ultraviolet radiation, has been isolated. Preliminary tests show that this mutant is also resistant to mitomycin C, nalidixic acid, fluorouracil and thymineless death. The mutant strain apparently repairs its damaged DNA more efficiently than wild-type E. coli K12 strains and, to do so, constitutively produces 35 times more DNA polymerase I and 12 times more endonuclease I than the wild-type strain.     

The DNA sequence of argI from Escherichia coli K12.

The argI gene from E. coli K12 has been sequenced. It contains an open reading frame of 1002 bases which encodes a polypeptide of 334 amino acids. Three such polypeptides are required to form the functional catalytic trimer (c3) of ornithine transcarbamoylase (OTCase-1, EC 2.1.3.3). The molecular mass of the mature trimer deduced from the amino acid sequence is 114,465 daltons. An altered form of argI was produced when a 1.6 kilobase DdeI fragment was subcloned into the HincII site of plasmid pUC8 extending the open reading frame an additional 20 nucleotides. It has been previously reported that the amino-terminal region of the respective polypeptides of argI, argF, and pyrB of E. coli possessed significant homology. In contrast, the homologous promoter/operator regions of argI and argF did not appear to share any homologies with pyrB. However, a closer scrutiny of the nucleotide sequence immediately preceding the pyrBI attenuator revealed a remarkable similarity to the argI and argF control region.     

Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli

Summary The host-controlled EcoK-restriction of unmodified phage .O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold. In addition, the EcoK modification activity is substantially decreased in dam ^- strains. We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants. However, no relief of EcoRI restriction (Type II) occurs in dam ^- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed. We interpret the alleviation of the type I restriction in dam ^- strains to be a consequence of induction of the function which interferes with type I restriction systems.     

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants.

RecBCD protein, necessary for Escherichia coli dam mutant viability, is directly required for DNA repair. Recombination genes recF+, recN+, recO+, and recQ+ are not essential for dam mutant viability; they are required for recBC sbcBC dam mutant survival. mutH, mutL, or mutS mutations do not suppress subinduction of SOS genes in dam mutants.     

The complete nucleotide sequence of the glnALG operon of Escherichia coli K12.

The nucleotide sequence of the E. coli glnALG operon has been determined. The glnL (ntrB) and glnG (ntrC) genes present a high homology, at the nucleotide and aminoacid levels, with the corresponding genes of Klebsiella pneumoniae. The predicted aminoacid sequence for glutamine synthetase allowed us to locate some of the enzyme domains. The structure of this operon is discussed.     

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.     

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.