Two identical copies of the hydrazine synthase gene clusters found in the genomes of anammox bacteria

Hydrazine synthase (HZS), encoded by the hzsABC gene cluster, is the unique and irreplaceable enzyme for the anammox reaction in this group of bacteria. This gene is widely used as a functional gene biomarker to retrieve the diversity, abundance and activity of anammox bacteria in different ecological niches. Genomes of anammox bacteria are constantly acquired, but the copy number of hzsABC gene cluster in the genome of anammox bacteria remains unsettled. In this study, three high quality anammox bacterial genomes were acquired from metagenomic datasets of activated sludge samples containing anammox bacteria. Interestingly, the coverage of hzsABC gene cluster in all of the three genomes was approximately two folds, comparing to the coverage of the corresponding genomic bin. However, only one assembled scaffold contained the full length of hzsABC in each genome, therefore it is likely that two identical copies of the hzsABC gene cluster are present in each of the three genomes. To confirm this, specific PCR primers were designed based on the assembled scaffolds containing the full-length hzsABC and partial hzsC genes. Two complete sequences connecting with the 5' terminal of hzsC were successfully amplified using genomic DNA as templates, which further supported our hypothesis of the two identical copies of the gene. Moreover, the twofold coverage relationships of hzs genes and genomes were also found in the published genomes of“Ca. Brocadia sinica”, “Ca. Brocadia_caroliniensis”, “Ca. Brocadia_fulgida”. Overall, we would conclude that the genome of anammox bacteria contains two identical copies of hzsABC gene cluster, but functionality of the two identical genes needs further confirmations.