REGULATION OF THE HEMA GENE DURING 5-AMINOLEVULINIC ACID FORMATION IN PSEUDOMONAS-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 the condensation of succinyl-coenzyme A and glycine, while other bacteria utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-dependent pathway, involving the enzymes glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by hemL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vinelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginosa was cloned by complementation of an Escherichia coli hemA mutant. The hemA gene was mapped to the SpeI A fragment and the DpnIL fragment of 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 calculated molecular mass of 46,234 Da, forms an operon with the gene for protein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre codons. Since the cloned hemA gene did not possess one of the appropriate stop codons, an autoregulatory mechanism such as that postulated for 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 open reading frames located in the 5' region of enterobacterial hemA genes. While orf2 was found to be 59% identical to its enterobacterial counterpart, hemM/orf1 showed only 23% identity. The third open reading frame (orf3), located between the hemA gene and hemM/orf1, encodes a polypeptide with homology to outer membrane proteins. Biochemical and genetic evidence which makes a direct involvement of HemM/Orf1 in the 5-aminolevulinic acid formation of P. aeruginosa very unlikely was obtained. An increase of hemA mRNA was observed under anaerobic denitrifying conditions, while anaerobic growth utilizing the fermentative arginine deiminase pathway led to a drastic decrease of hemA transcription compared with that observed for aerobically grown P. aeruginosa. These results suggest the anaerobic induction of hemA by the presence of nitrate. Two transcription start sites were located in the 5' region of the hemA gene. Utilization of both transcription start sites was changed in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr analog), indicating the involvement of the transcription factor in hemA expression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined transcription start sites.