Table_8_Comparative Proteomics of Three Species of Ammonia-Oxidizing Bacteria.XLSX

Ammonia-oxidizing bacteria (AOB) are important members of terrestrial, marine, and industrial microbial communities and play a fundamental role in the Nitrogen cycle within these systems. They are responsible for the first step of nitrification, ammonia oxidation to nitrite. Although AOB are widespread and essential to environmental and industrial systems, where they regularly experience fluctuations in ammonia availability, no comparative studies of the physiological response of diverse AOB species at the protein level exist. In the present study, we used 1D-LC-MS/MS proteomics to compare the metabolism and physiology of three species of ammonia AOB, Nitrosomonas europaea, Nitrosospira multiformis, and Nitrosomonas ureae, under ammonia replete and ammonia starved conditions. Additionally, we compared the expression of orthologous genes to determine the major differences in the proteome composition of the three species. We found that approximately one-third of the predicted proteome was expressed in each species and that proteins for the key metabolic processes, ammonia oxidation and carbon fixation, were among the most abundant. The red copper protein, nitrosocyanin was highly abundant in all three species hinting toward its possible role as a central metabolic enzyme in AOB. The proteomic data also allowed us to identify pyrophosphate-dependent 6-phosphofructokinase as the potential enzyme replacing the Calvin-Benson-Bassham cycle enzyme Fructose-1,6-bisphosphatase missing in N. multiformis and N. ureae. Additionally, between species, there were statistically significant differences in the expression of many abundant proteins, including those related to nitrogen metabolism (nitrite reductase), motility (flagellin), cell growth and division (FtsH), and stress response (rubrerythrin). The three species did not exhibit a starvation response at the proteome level after 24 h of ammonia starvation, however, the levels of the RuBisCO enzyme were consistently reduced after the starvation period, suggesting a decrease in capacity for biomass accumulation. This study presents the first published proteomes of N. ureae and N. multiformis, and the first comparative proteomics study of ammonia-oxidizing bacteria, which gives new insights into consistent metabolic features and differences between members of this environmentally and industrially important group.

氨氧化细菌（Ammonia-oxidizing bacteria, AOB）是陆地、海洋及工业微生物群落的重要组成类群，在对应生态系统的氮循环中发挥核心作用。它们负责硝化作用的第一步——将氨氧化为亚硝酸盐。尽管AOB分布广泛且对环境与工业系统至关重要，且它们常面临氨可用性波动的压力，但目前尚无针对多种AOB物种在蛋白质组水平上的生理响应开展的比较研究。
本研究采用一维液相色谱-串联质谱（1D-LC-MS/MS）蛋白质组学技术，对比了三种氨氧化细菌——欧洲亚硝化单胞菌（Nitrosomonas europaea）、多形亚硝化螺菌（Nitrosospira multiformis）以及脲基亚硝化单胞菌（Nitrosomonas ureae）——在氨充足与氨饥饿条件下的代谢与生理特征。此外，本研究通过比对同源基因的表达情况，解析了这三个物种在蛋白质组组成上的主要差异。
研究发现，每个物种中约有三分之一的预测蛋白质组得到表达，而参与核心代谢过程（氨氧化与碳固定）的蛋白质丰度位居前列。红色铜蛋白亚硝化蓝素（nitrosocyanin）在三个物种中均呈现高丰度表达，提示其可能作为AOB中的核心代谢酶发挥功能。蛋白质组数据还帮助我们鉴定出焦磷酸依赖型6-磷酸果糖激酶，可作为替代卡尔文-本森-巴斯哈姆循环中果糖-1,6-二磷酸酶的潜在酶类，而该酶在多形亚硝化螺菌与脲基亚硝化单胞菌中缺失。
此外，不同物种间多种高丰度蛋白质的表达存在统计学显著性差异，包括与氮代谢（亚硝酸还原酶）、运动性（鞭毛蛋白）、细胞生长与分裂（FtsH蛋白）以及应激响应（红系氧还蛋白）相关的蛋白质。在氨饥饿处理24小时后，三个物种在蛋白质组水平上未表现出饥饿响应；但核酮糖-1,5-二磷酸羧化酶/加氧酶（RuBisCO）的丰度在饥饿处理后持续降低，提示其生物质积累能力有所下降。
本研究首次公开了脲基亚硝化单胞菌与多形亚硝化螺菌的蛋白质组数据，同时也是首个针对氨氧化细菌的比较蛋白质组学研究，为解析这一兼具环境与工业重要性的类群内部成员共有的代谢特征及物种间差异提供了新的认知。