## 3 Result
### Phylogenetic Diversity of Hao-Dependent Nitrifying Bacteria in Sediments  
The nitrifying bacterial _hao_ genes in the three samples were amplified from the metagenomic DNA (template) with primers JBHAO-170F and JBHAO-939R to detect the presence of ammonia-oxidizing bacteria (AOB). The PCR products revealed the presence of AOB in these samples (Figure 2). The amplification products were cloned and sequenced, and three clone libraries were constructed. An analysis of randomly selected clones indicated that all the clones of approximately the expected size (817 b.p.) were _hao_ gene fragments, and no non-_hao_ genes were amplified. A total of 132 clones from the three clone libraries were randomly selected and sequenced. The BLASTP results indicated that all the clones contained highly similar _hao_ sequences, with approximately 90-99% identity to other _hao_ gene sequences in the database. Only 79 novel gene sequences were detected among the sequenced clones, except for the redundant clone sequences. The deduced amino acid sequences of these gene fragments  
Figure 1: Analysis of microbial communities based on 16S rRNA partial gene sequences retrieved from three environmental collections and other representative organisms’ species. Clones H16S, V16S, and W16S were from a high-quality Qingshan Lake water site (QL-H), the Qingshan Lake site closest to the village (QL-V), and domestic pollution in the Sludge River (SR-W), respectively. matched the conserved regions in the previously characterized functional domains within other Hao proteins. These results demonstrate that the PCR primers that were newly designed to detect _hao_ genes in various sediment samples, JBHAO-170F and JBHAO-939R, are useful for identifying the diversity of AOB nitriftiers with high resolution (100% coverage) and specificity. A multiple alignment of all these sequences showed that the percentage identity between pairs of Hao protein sequences ranged from 82.96% to 99.63%. The nucleic acid sequences corresponding to these protein sequences showed between 77.90-99.63% identity. The 79 Hao gene sequences were classified based on previous studies by comparing the amino acid sequences of AOB Hao enzymes [20]. As shown in Figure 2A, a phylogenetic analysis of the Hao sequences with the neighbor-joining method showed that the QL-H sample bacterial Hao proteins were affiliated to the _Nitrosomonas_ cluster, whereas no sequence from the _Nitrosospira_ lineage, which dominates high-nitrogen environments, was observed [21]. All of the retrieved QL-H Hao sequences strongly belonged to the _N. oligotrophia_ cluster and shared between 89.34-99.63% identity. Only one sequence (Hhao-A45) clustered with Hao of _N. ureae_, with 91.54% amino acid sequence identity. However, 17 of the 25 Hao proteins from the QL-V sample were most affiliated with the _N. oligotrophia_ cluster and shared 91.18-96.63% amino acid identity; five proteins fell between the _N. communis_ and _N. halophila_ clusters; and three proteins (Vhao-1, Vhao-02, and Vhao-C8) did not match the Hao proteins of these species (Figure 2C). Interestingly, SR-W sample sequences from Sewage River sludge were sporadically affiliated with the subphyla _N. communis_, _N. halophila_, and _N. oligotrophia_ clusters, but a new cluster accounted for nearly 80% of the cloned sequences and shared 90.07-99.63% identity with the closest environmental cluster, _N. ureae_ (Figure 2B). Figure 2: Trees of hydroxylamine oxidoreductase (HAO) constructed based on protein sequences using the neighbor-joining method. Sequences from this study are shown in red **(A)** sample QL-H; **(B)** sample SR-W; **(C)** sample QL-V).