## 4 Discussion
Previous studies have demonstrated that the apparent affinity of _N. oligotropha_-like AOB for low ammonium may allow them to outcompete the more copiotrophic _N. europaea_-like AOB strains [33]. In this work, it is clear that the Hao-dependent AOB species differed in the three water samples in parallel with the dramatic changes in ammonia nitrogen (Table 1). The three distinct ammonium-graduated sites QL-H (undetectable ammonium), QL-V (16.34 mg/L), and SR-W (93.43 mg/L) AOB were approximately affiliated to _N. ureae_ with 2%, 13%, and 80%, respectively. The high concentration of ammonia in the influent of SR-W may have contributed to the predominance of clones related to the _N. ureae_ lineage, which was consistent with previous studies that the _N. ureae_ cluster was the predominant AOB, was commonly found in environments rich in ammonium [34], and can tolerate up to 200 mM of ammonium [35]. In contrast, _N. oligotropha_ already dominated in the AOB of the QL-H sample (>90%), and also made up more than 65% in the QL-V sample and small amounts (~8%) in the RS-W sample. The _N. oligotropha_ of AOB species are often found in freshwater due to its lower the _K_s and _Km_ values for ammonia by an order of magnitude more than _N. europaea_, _N. eutropha_, or their close relatives [35], allowing them to grow at low ammonium concentrations [36]. Meanwhile, _N. oligotropha_ can also adapt to grow at higher ammonium availability, but with a longer lag phase [36]. Our data show that in oligotrophic environments, carbon and nitrogen are probably important determinants of bacterial community composition and structure, and possibly shifting toward supporting microbial groups. Therefore, the distinct and characteristic _N. oligotropha_ population structures in the three environments can be attributed to the sharp differences in the nitrogen concentrations, particularly because in oligotrophic environments, representatives of _N. oligotropha_ may outcompete other species. The newly identified NirS sequences were predominantly affiliated with unknown clusters II and III, and occasionally within unknown cluster I, and the distributions of these subclusters clearly differed. The sequences recovered from the oligotrophic environment of QL-H fell in unknown cluster II (Figure 3A), sharing 75-89% protein sequence identity with other reported sequences. This cluster consisted of four unique NirS sequences and 13 clones that were most closely related to the class Gammaproteobacteria, including the family Xanthomonadaceae (80%) and _Marinobacter_ spp. (79%). A previous study demonstrated that members of Xanthomonadaceae could function as autotrophic denitrifiers in a pyrite-fed denitrifying reactor [37], and the _Marinobacter_ NirS protein has an important role as a biological marker of microbial degradation in aquatic environments [38]. The grouping of the QL-H sequences with unique cluster II appears to be closely associated with the oligotrophic status of the lake environment, although they formed a new clade within the dominant group. This is consistent with the 165 rRNA results, which showed that the Gammaproteobacteria had high specificity and were highly abundant in the QL-H sample, which was collected from an environment with low ammonium concentration (Figure 1). This new phylogenetic group has rarely been detected in other high ammonia-nitrogen aquatic environments or lakes, and was never retrieved in previous studies of Lake Bourget. Our high success rate with metagenomic PCR screening indicates that this oligotrophic lake is a unique reservoir of diverse and novel nitrite reductase family members, which may represent novel denitrifying bacteria with key roles in limbic biogeochemical nitrogen cycling. In contrast, the sequences recovered from SR-W were predominantly affiliated with unknown clusters I (30.43%) and III (67.39%) (Figure 3B), sharing 89-100% protein sequence identity with previously reported sequences.