delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli.
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 to 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 reduced 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. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Neill
G P
GP
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Thorbjarnardóttir
S
S
Michelsen
U
U
Pálsson
S
S
Söll
D
D
Eggertsson
G
G
D000622:Aminolevulinic Acid / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D000623:Porphobilinogen Synthase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D004269:DNA, Bacterial; D000622:Aminolevulinic Acid; D005020:Ethyl Methanesulfonate; D000623:Porphobilinogen Synthase