Increased salinity triggers significant changes in the functional proteins of ANAMMOX bacteria within a biofilm community

Anaerobic ammonium oxidation (ANAMMOX) processes can potentially be influenced by salinity related to variable salinity in water environment. Here, we used 16S rRNA sequencing analysis combining with iTRAQ-based quantitative proteomic approach to reveal the response of microbial community and functional proteins to salinity, which was increased from 0 to 20 g L⁻¹ with a step of 5 g L⁻¹ (designed as S5, S10, S15 and S20) compared to control reactor (without salinity stress desined as S0). The 16S rRNA sequencing analysis showed that a high salinity (20 g L⁻¹, S20) decreased the abundance of genus Candidatus Jettenia but increased that of Candidatus Kuenenia. A total of 1609 differentially expressed proteins were acquired in the three comparison groups (S5:S0, S20:S0 and S20:S5). Of these, 39 proteins co-occurred in the three salt-exposed samples. Hydrazine dehydrogenase (HDH; Q1PW30) and nitrate reductase (Q1PZD8) were up-regulated more than 3-folds in the exposure of 20 g-NaCl/L. The functional enrichment analysis further showed that some proteins responsible for ion binding, catalysis and oxidation-reduction reaction were up-regulated, which explained the physiological resilience of ANAMMOX bacteria under salinity stress. Additionally, ANAMMOX bacteria responded to salinity by modulating the electron transport systems, indicating that the cells retained a high potential for proton pumping, as well as the ATP production. Furthermore, the over-expression of HDH which associated with ANAMMOX metabolism, was potentially related to the increased abundance of halophilic Candidatus Kuenenia. These findings provide a comprehensive baseline for understanding the roles of salinity stresses in shaping the functional proteins of ANAMMOX bacteria.