已知高NH4+负载抑制细菌甲烷氧化。这是由于CH4和NH3之间的竞争颗粒甲烷单加氧酶(pMMO)的活性位点,将CH4转化为CH3OH。这里,我们将全球蛋白质组学与氨基酸谱分析和氮氧化物测量相结合,以阐明Methylocystissp。的细胞适应反应。菌株SC2达到高NH4+水平。相对于1mMNH4+,在CH4充足的条件下,高(50mM和75mM)NH4负荷显着增加了蛋白质组调整所需的滞后期持续时间。差异调节蛋白的数量与NH4负荷的增加高度显着相关。细胞对增加的离子和渗透胁迫的反应涉及应激响应蛋白的显着上调,K+“盐入”策略,相容溶质(谷氨酸和脯氨酸)的合成,和谷胱甘肽代谢途径的诱导。在生长期期间CH4氧化的表观Km值的显着增加表明基于pMMO的NH3氧化为有毒羟胺的增加。羟胺氧化还原酶(HAO)的解毒活性导致NO2-的大量积累,随着O2张力的降低,N2O。一氧化氮还原酶和杂合簇蛋白(Hcps)是生产N2O的候选酶。总之,菌株SC2具有响应于NH4暴露增加而精确地重新平衡酶和渗透物组成的能力,但是需要同时对抗离子渗透胁迫和羟胺的毒性作用可能是其适应能力仅限于75mMNH4的原因。重要性除了减少湿地和垃圾填埋场的CH4排放外,Methylocystis属的α变形杆菌甲烷氧化剂的活性有助于森林和草地土壤对大气甲烷的吸收能力。甲基囊藻属的甲烷氧化活性。是,然而,对高NH4+浓度敏感。这是由于CH4和NH3争夺颗粒甲烷单加氧酶的活性位点,从而导致产生具有增加的NH4+负载的有毒羟胺。对甲基囊虫的生理和分子反应机制的理解。因此非常重要。这里,我们将全球蛋白质组学与氨基酸分析和NOx测量相结合,以理清Methylocystissp。适应的细胞机制。应变SC2增加NH4+负荷。
A high NH4+ load is known to inhibit bacterial methane oxidation. This is due to a competition between CH4 and NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of Methylocystis sp. strain SC2 to high NH4+ levels. Relative to 1 mM NH4+, a high (50 mM and 75 mM) NH4+ load under CH4-replete conditions significantly increased the lag phase duration required for proteome adjustment. The number of differentially regulated proteins was highly significantly correlated with an increasing NH4+ load. The cellular responses to increasing ionic and osmotic stress involved a significant upregulation of stress-responsive proteins, the K+ \"salt-in\" strategy, the synthesis of compatible solutes (glutamate and proline), and the induction of the glutathione metabolism pathway. A significant increase in the apparent Km value for CH4 oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2- and, upon decreasing O2 tension, N2O. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH4+ exposure, but the need to simultaneously combat both ionic-osmotic stress and the toxic effects of hydroxylamine may be the reason why its acclimatization capacity is limited to 75 mM NH4+. IMPORTANCE In addition to reducing CH4 emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus Methylocystis contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of Methylocystis spp. is, however, sensitive to high NH4+ concentrations. This is due to the competition of CH4 and NH3 for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH4+ load. An understanding of the physiological and molecular response mechanisms of Methylocystis spp. is therefore of great importance. Here, we combined global proteomics with amino acid profiling and NOx measurements to disentangle the cellular mechanisms underlying the acclimatization of Methylocystis sp. strain SC2 to an increasing NH4+ load.