Genetics of Tetrapyrrole Synthesis in Gram-Positive Bacteria

Sammanfattning: The organization and regulation of genes for tetrapyrrole synthesis were studied in low G+C Gram-positive bacteria. Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans. The structure of the hem gene clusters was compared to that of other Gram-positive bacteria. The hemEHY operon required for protoheme IX synthesis from uroporphyrinogen III (UroIII) is conserved among low G+C Gram-positive bacteria. Mainly aerobic Bacillus, Brevibacillus and Stapylococcus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of UroIII from glutamyl-tRNA. In P. macerans and Clostridium species, the hem genes for UroIII synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysGB and cysGA-hemD, encoding the enzymes required for synthesis of siroheme from UroIII. B. subtilis contains genes for three proteins, NasF, YlnD and YlnF, with sequence similarity to Escherichia coli CysG which is a multi-functional protein catalyzing siroheme synthesis from UroIII. It is shown that YlnF is required for siroheme synthesis and probably catalyses the precorrin-2 to siroheme conversion. YlnD probably catalyses precorrin-2 synthesis from UroIII and NasF seems to be specific for nitrite reduction. The function of the hemX gene product was studied. The deduced amino acid sequence suggests HemX to be an integral 32 kDa membrane protein. This was confirmed by experiments using E. coli minicells and hemX-phoA gene fusions. Deletion of the hemX gene from the B. subtilis chromosome demonstrated that this gene is not required for heme synthesis or spore formation. However, the deletion strain was found to overexpress the hemA gene product, glutamyl-tRNA reductase. Expression of HemX did, however, not affect hemA transcription or the activity of a HemA-LacZ fusion protein, nor did HemX affect HemB activity. A combination of results obtained with B. subtilis hemA and hemX in E. coli and B. subtilis shows that HemX negatively affects the steady-state cellular concentration of HemA protein. The mechanism by which HemX regulates HemA is still not clear but most probably involves some type of posttranscriptional regulation. The B. subtilis hemA promoter of the hemAXCDBL operon was mapped by primer extension and a single transcriptional start point was found 66 nucleotides upstream of the proposed ATG start codon. Transcription of the hemAXCDBL operon was increased during ALA limiting conditions and addition of ALA decreased the transcription.