The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.     

Secondary transport of amino acids in Nitrosomonas europaea

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity ( molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H⁺/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.     

Ammonia and O2 uptake in relation to proton translocation in cells of Nitrosomonas europaea

The uptake of ammonia and O₂ by washed cells of Nitrosomonas has been followed simultaneously and continuously using electrode techniques. The stoichiometry of NH ₄ ⁺ oxidation, O₂ uptake and NO ₂ ^- production was 1 : 1.5 : 1.0 and for NH₂OH oxidation a ratio of 1 for O₂ : NO ₂ ^- . A variety of inhibitors of electron transport and metals as well as uncouplers restricted ammonia uptake more markedly than O₂ utilization. There is good evidence for the involvement of copper in the NH ₄ ⁺ uptake process.A quinacrine fluorescence technique has been used to study the proton extrusion by washed cells on adding NH₄Cl and NH₂OH respectively as substrates. The uptake of NH ₄ ⁺ was followed by the extrusion of H⁺ and this process was depressed by those inhibitors which were also effective in the electrode experiments. A requirement for copper is also established for the translocation of protons into the medium, resulting from the uptake of NH ₄ ⁺ by cells.Abbreviations mCCCP carbonyl cyanide m-chlorophenylhydrazone - DBP 2,4 dibromophenol - DCCD N-N-dicyclohexylcarbodimide - DIECA Sodium diethyldithiocarbamate - DNP 2,4 dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - NBD chloride 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole - N-serve 2-chloro-6-trichloromethyl-pyridine - PCP pentachlorophenol - 2-TMP 2-trichloromethyl-pyridine - TPB tetraphenylboron - TTFA 1-[thenoyl-(2)]-3,3,3-trifluoracetone - KSCN Potassium thiocyanate     

A mathematical description of the behaviour of mixed chemostat cultures of an autotrophic nitrifier and a heterotrophic nitrifier/aerobic denitrifier; a comparison with experimental data

Abstract A general, unstructed mathematical model has been used to describe the behaviour of nutrient-limited growth of two bacteria in a continuous co-culture. The experimental system consisted of a two-membered mixed culture of the heterotrophic nitrifier/aerobic denitrifier, Thiosphaera pantotropha , and the autotrophic nitrifier, Nitrosomonas europaea , competing for ammonia in chemostat culture. The outcome of competition was only dependent on the Monod constants and the growth yields of the two bacteria. The model shows that both bacteria will oxidize equal amounts of ammonia when the cell ratio of T. pantotropha/N. europaea is 260.     

Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41–3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.     

重组菌Ralstonia eutropha W50-EAB D-木糖代谢相关的限速靶点

【目的】通过代谢工程改造真养罗氏菌(Ralstonia eutropha)W50-EAB木糖代谢的相关限速靶点,进一步提高R.eutropha W50-EAB的D-木糖利用效率,为获得高效利用纤维素水解液的菌株奠定基础。【方法】利用PCR技术扩增R.eutropha转酮酶基因tkt A,cbb T2和转醛酶基因tal,将扩增的tkt A,cbb T2和tal基因分别构建到表达载体p BBR1MCS-3上,获得重组质粒p WL1-TKT,p WL1-CBBT2,p WL1-TAL。通过电转的方式将质粒分别转化W50-EAB获得重组菌W50-KAB,W50-CAB和W50-TAB。利用基因敲除的方法,获得醛还原酶基因h16_A3186敲除株W50’-EAB。通过电转的方式将重组质粒p WL1-TAL导入敲除株W50’-EAB获得重组菌株W50’-TAB。通过摇瓶发酵研究重组菌株W50-KAB,W50-CAB,W50-TAB,W50’-EAB以及W50’-TAB的发酵特性。【结果】酶活分析结果表明,转酮酶和转醛酶基因实现表达。摇瓶发酵结果表明,转酮酶基因过表达菌株W50-KAB和W50-CAB相比于对照菌株W50-EAB/p3,表现出降低的木糖利用能力;而转醛酶基因过表达重组菌株W50-TAB以及敲除菌株W50’-EAB对木糖的利用得到一定的提高。在0.1 mol/L木糖的发酵培养基中,W50-EAB的最大比生长速率为0.035 h-1,PHB干重比为16.2±1.01%;而W50-TAB的最大比生长速率提高到0.039 h-1,PHB干重比达到20.5±0.76%;醛还原酶基因敲除菌株W50’-EAB最大比生长速率提高到0.040 h-1,PHB含量提高到19.8±1.05%。结果显示转醛酶基因的过表达与醛还原酶基因的敲除对木糖利用均表现出一定的优势,将这两种优势组合获得菌株W50’-TAB,摇瓶发酵分析结果为最大比生长速率达到0.042 h-1,PHB积累达到27.9±0.47%,相比于对照菌株提高了72.2%。另外,在含有葡萄糖(0.01 mol/L)和木糖(0.09 mol/L)的混合糖培养下,重组菌株W50-TAB,W50’-EAB和W50’-TAB相比于在纯木糖培养下都表现出更高的生物量和胞内PHB积累量。【结论】磷酸戊糖途径关键酶转醛酶基因的过表达加速了木糖代谢流,从而可以高效利用木糖积累一定量的PHB。醛还原酶对木糖代谢有阻碍作用,敲除该酶基因后木糖代谢能力有了一定的提高,而两者协同作用可以进一步提高重组菌株的木糖利用效率和PHB积累能力。     

亚硝化单胞菌（Nitrosomonas sp．）THD-1分离鉴定及高密度培养

从浙江舟山桃花岛近海海域分离筛选到一株高活性氨氧化细菌,命名为THD-1。菌株THD-1为革兰氏阴性,球状至椭球状,大小约为（0．5～0．8）μm×（0．7～1．3）μm;菌落呈无色透明状,针尖大小,边缘光滑;可在盐度为0～50％0的培养基中生长。16SrDNA序列比对表明,菌株THD—1与Nitrosomonas europaea ATCC25978^T的相似性为96．3％。培养液的pH值对菌体生长和氨氧化活性影响明显,当pH值降至6．0以下时,菌株的生长和氨氧化活性几乎完全被抑制,但这种抑制可通过回调pH值的方法解除;在所试的4种碱液中,碳酸氢铵调节pH值对菌体生长和氨活性改善效果最好。建立了菌株THD-1的分批补料式高密度培养方法,最终OD600可达0．214,最大活菌数可达7．5×10^8cells／mL。