A heme-deficient strain of Escherichia coli has a three-base pair deletion in a "hotspot" in hemA.

The key regulatory step in heme biosynthesis in Escherichia coli is at the level of glutamyl-tRNA reductase (GTR), an enzyme which is encoded by hemA. A strain, HU227, with a spontaneous in-frame mutation in hemA has no GTR activity. The mutation is shown to be a three-base deletion at a "hotspot" in the gene.

Expression of glutamyl-tRNA reductase in Escherichia coli.

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA.

Characterization of a hemA/hemE mutant of E. coli and regulation of hemE.

Uroporphyrinogen III is the committed intermediate common to heme and siroheme biosynthesis in E. coli. Uroporphyrinogen III decarboxylase is the first enzyme at the branch point which commits to heme synthesis.

5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA.

hemA and hemM, which are 213 bp apart and divergently transcribed, were separately cloned. We found that hemA is required for 5-aminolevulinic acid (ALA) synthesis in two ALA- auxotrophs. Overexpression of hemM alone did not produce ALA.

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21.

We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange.

Cloning and structure of the hem A gene of Escherichia coli K-12.

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h.

Evidence for the essential function of 2,4-dienoyl-coenzyme A reductase in the beta-oxidation of unsaturated fatty acids in vivo. Isolation and characterization of an Escherichia coli mutant with a defective 2,4-dienoyl-coenzyme A reductase.

For the purpose of assessing in vivo the importance of 2,4-dienoyl-CoA reductase (EC 1.3.1.

5-Aminolevulinic acid synthesis in Escherichia coli.

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate.

The structure of the Escherichia coli hemB gene.

The Escherichia coli hemB gene, which encodes 5-aminolevulinic acid dehydratase, and was cloned into pTZ18U, a multicopy plasmid, was sequenced. The hemB insert was double-digested with restriction enzymes and recloned back into pTZ18U and pTZ19U to allow for sequencing in two directions. In a second procedure, used to fill in gaps and to confirm the sequence derived from the first procedure, the whole insert was cloned into M13 phages.

Availability of porphobilinogen controls appearance of porphobilinogen deaminase activity in Escherichia coli K-12.

A hemin-permeable hemB mutant had no 5-aminolevulinate dehydratase (ALA D) and extremely low porphobilinogen deaminase (PBG D) activity. When the structural gene for hemB was introduced into this strain on a single-copy plasmid, both activities were observed. When the mutant was grown on PBG, normal PBG D activity was observed.

Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase.

Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392.

Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.

Effect of transcription on RecBC- and RecF-mediated recombination within the tryptophan operon of Escherichia coli K-12.

Recombination between tryptophan gene mutations within the trp operon was determined among transductants for an outside linked cysB marker under conditions of repression and derepression. These studies, carried out with recipient strains utilizing the RecBC or RecF pathway, or a combination of these pathways of recombination, demonstrate that transcription of trp genes as regulated by the trp repressor has no significant effect on RecBC- or RecF-mediated recombination within the trp operon.

Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition.

Without significant killing, d-serine at concentrations greater than 50 mug/ml inhibits growth in minimal media of mutants of Escherichia coli K-12 unable to form d-serine deaminase. The mutants eventually recover at lower concentrations. There is no evidence of d-serine toxicity in rich media.

D-serine transport system in Escherichia coli K-12.

The d-serine transport system in Escherichia coli K-12 was studied by use of a mutant unable to form d-serine deaminase, yet resistant to d-serine. The mutant is greatly impaired in its ability to accumulate d-serine, d-alanine, and glycine. Transport of l-alanine is partially affected but transport of l-serine is unaffected.

Genetic transformation in Escherichia coli K12.

An auxotrophic strain of E. coli K12 treated with CaCl(2) was transformed for several markers at a frequency of up to 10(-6) per recipient cell by a DNA preparation isolated from a prototrophic strain. The transforming activity of the DNA preparation was eliminated by treatment with DNase, heat, or sonication, whereas RNase or Pronase treatment had little effect.

L-Serine-sensitive mutants of Escherichia coli K-12.

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).