Abstract
The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite. When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis. With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment. Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa. In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified. The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN. HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX. Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A. eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector. Likewise, the A. eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth. Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A. eutrophus is nitrate-independent and repressed by oxygen.
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