Cloning, mapping and characterization of thePseudomonas aeruginosa hemL gene

Abstract

The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic acid (ALA). InPseudomonas aeruginosa ALA is synthesized via a two-step reaction from aminoacylated tRNA^Glu by the action of glutamyl-tRNA reductase and glutamate-1-semialdehyde-2,1-amino mutase. To initiate an investigation of the regulation of the second step in ALA formation, thehemL gene was cloned fromP. aeruginosa by complementation of anEscherichia coli hemL mutant. An open reading frame of 1284 by encoding a protein of 427 amino acids with a calculated molecular mass of 45 404 Da was identified. ThehemL gene was mapped to theSpeI fragment Z and theDpnI fragment J1 of theP. aeruginosa chromosome corresponding approximately to min 0.3–0.9. One transcription start site was located 280 by upstream of the translational start site of thehemL gene. No classicalσ ⁷⁰-dependent promoter was detected. Oxygen stress induced by the addition of H₂O₂ to the growth medium led to an approximately 3.5-fold increase inhemL expression as determined by mRNA dot blot assays. Anaerobic denitrifying growth led to a 2-fold stimulation ofhemL transcription. Two additional open reading frames were detected downstream of thehemL gene. One open reading frame (orf1) of 549 by encodes a protein of 182 amino acids with a calculated molecular mass of 19 638 Da. The second open reading frame (orf2) of 1341 by encodes a protein of 446 amino acids with a calculated molecular mass of 49 967 Da and showed similarity to a protein of unknown function fromMycobacterium leprae and to a P-methylase fromStreptomyces hygroscopicus.