REGULATION OF THE HEMA GENE DURING 5-AMINOLEVULINIC ACID FORMATION INPSEUDOMONAS-AERUGINOSA

The general tetrapyrrole precursor 5-aminolevulinic acid is formed in bacteria via two different biosynthetic pathways. Members of the alpha group of the proteobacteria use 5-aminolevulinic acid synthase for th e condensation of succinyl-coenzyme A and glycine, while other bacteri a utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-de pendent pathway, involving the enzymes glutamyl-tRNA reductase (encode d by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by he mL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomona s putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vi nelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginos a was cloned by complementation of an Escherichia coli hemA mutant. Th e hemA gene was mapped to the SpeI A fragment and the DpnIL fragment o f the P. aeruginosa chromosome corresponding to min 24.1 to 26.8. The cloned hemA gene, coding for a protein of 423 amino acids with a calcu lated molecular mass of 46,234 Da, forms an operon with the gene for p rotein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre c odons. Since the cloned hemA gene did not possess one of the appropria te stop codons, an autoregulatory mechanism such as that postulated fo r the enterobacterial system was ruled out. Three open reading frames of unknown function transcribed in the opposite direction to the hemA gene were found. hemM/orf1 and orf2 were found to be homologous to ope n reading frames located in the 5' region of enterobacterial hemA gene s. While orf2 was found to be 59% identical to its enterobacterial cou nterpart, hemM/orf1 showed only 23% identity. The third open reading f rame (orf3), located between the hemA gene and hemM/orf1, encodes a po lypeptide with homology to outer membrane proteins. Biochemical and ge netic evidence which makes a direct involvement of HemM/Orf1 in the 5- aminolevulinic acid formation of P. aeruginosa very unlikely was obtai ned. An increase of hemA mRNA was observed under anaerobic denitrifyin g conditions, while anaerobic growth utilizing the fermentative argini ne deiminase pathway led to a drastic decrease of hemA transcription c ompared with that observed for aerobically grown P. aeruginosa. These results suggest the anaerobic induction of hemA by the presence of nit rate. Two transcription start sites were located in the 5' region of t he hemA gene. Utilization of both transcription start sites was change d in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr anal og), indicating the involvement of the transcription factor in hemA ex pression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined transcription start sites.