CLONING, MAPPING AND CHARACTERIZATION OF THE PSEUDOMONAS-AERUGINOSA HEML GENE

The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic acid (ALA). In Pseudomonas 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, the hemL gene was cloned from P. aeruginosa by complementation of an Escherichia coli hemL mutant. An open reading frame of 1284 bp encoding a protein of 427 amino acids with a calculated molecular mass of 45 404 Da was identified. The hemL gene was mapped to the SpeI fragment Z and the DpnI fragment J1 of the P. aeruginosa chromosome corresponding approximately to min 0.3-0.9. One transcription start site was located 280 bp upstream of the translational start site of the hemL gene. No classical sigma(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 in hemL expression as determined by mRNA dot blot assays. Anaerobic denitrifying growth led to a 2-fold stimulation of hemL transcription. Two additional open reading frames were detected downstream of the hemL gene. One open reading frame (orf1) of 549 bp encodes a protein of 182 amino acids with a calculated molecular mass of 19 638 Da. The second open reading frame (orf2) of 1341 bp 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 from Mycobacterium leprae and to a P-methylase from Streptomyces hygroscopicus.