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Title: Identification of the enzymatic basis for. delta. -aminolevulinic acid auxotrophy in a hemA mutant of escherichia coli

Abstract

The hemA mutation of Escherichia coli K-12 confers a requirement for {delta}-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem{sup +} strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-({sup 14}C)glutamate and 3,4-({sup 3}H)glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNA{sup Glu}. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem{sup +} cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem{sup +} cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.

Authors:
;  [1]
  1. (Brown Univ., Providence, RI (USA))
Publication Date:
OSTI Identifier:
7191671
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Bacteriology; (USA); Journal Volume: 171:6
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ESCHERICHIA COLI; MOLECULAR BIOLOGY; OXIDOREDUCTASES; GENES; AMINOLEVULINIC ACID; CARBON 14 COMPOUNDS; CHLORELLA; ENZYME ACTIVITY; MUTANTS; TRACER TECHNIQUES; TRANSFER RNA; ALGAE; AMINO ACIDS; BACTERIA; CARBOXYLIC ACIDS; CHLOROPHYCOTA; ENZYMES; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MICROORGANISMS; NUCLEIC ACIDS; ORGANIC ACIDS; ORGANIC COMPOUNDS; PLANTS; RNA; UNICELLULAR ALGAE; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Avissar, Y.J., and Beale, S.I. Identification of the enzymatic basis for. delta. -aminolevulinic acid auxotrophy in a hemA mutant of escherichia coli. United States: N. p., 1989. Web.
Avissar, Y.J., & Beale, S.I. Identification of the enzymatic basis for. delta. -aminolevulinic acid auxotrophy in a hemA mutant of escherichia coli. United States.
Avissar, Y.J., and Beale, S.I. 1989. "Identification of the enzymatic basis for. delta. -aminolevulinic acid auxotrophy in a hemA mutant of escherichia coli". United States. doi:.
@article{osti_7191671,
title = {Identification of the enzymatic basis for. delta. -aminolevulinic acid auxotrophy in a hemA mutant of escherichia coli},
author = {Avissar, Y.J. and Beale, S.I.},
abstractNote = {The hemA mutation of Escherichia coli K-12 confers a requirement for {delta}-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem{sup +} strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-({sup 14}C)glutamate and 3,4-({sup 3}H)glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNA{sup Glu}. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem{sup +} cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem{sup +} cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.},
doi = {},
journal = {Journal of Bacteriology; (USA)},
number = ,
volume = 171:6,
place = {United States},
year = 1989,
month = 6
}
  • Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required {delta}-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar tomore » a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduce ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. Analysis of another class of ALA-requiring mutants showed that the auxotrophy of the hem-205 mutant could be relieved by either methionine or cysteine and that the mutation maps in the cysG gene, which encodes uroporphyrinogen III methylase. The properties of these nonleaky ALA-requiring strains suggest that ALA is involved more extensively in E. coli intermediary metabolism than has been appreciated to date.« less
  • We previously reported the usefulness of a fluorometric method to determine urinary delta-aminolevulinic acid (ALA) concentrations by using post-column derivatization to monitor the effect of lead exposure. We have further improved the method by introducing pre-column derivatization by using reaction of ALA with acetylacetone and formaldehyde. Response of the hematopoietic system to lead exposure can now be easily detected at blood lead concentrations as low as 162 micrograms/L. The fluorescent ALA derivative, a new aromatic product, 2-methylideneamino-3,5-diacetyl-4,6-dimethylphenylpropionic acid, is separated on octadecyl silica column by high-performance liquid chromatography and the fluorescence intensity is detected with a fluorophotometer. Sample recoveries formore » 12 urine samples from workers exposed to lead and unexposed controls were 91.9-110.2%. The results obtained by the pre-column derivatization method agreed with those by the post-column derivatization method. The new method increases the sensitivity to a detection limit to 10 micrograms of delta-aminolevulinic acid per milliliter of urine and is simple enough to be used for routine monitoring of the biological effect of exposure to low concentrations of lead.« less