脱氮硫杆菌改良的耐热诱变及其诱变特性

Improved mutagenesis and mutagenic characteristics of thermotolerant strains of Thiobacillus denitrificans

摘要:

【背景】菌种诱变尤其是化学诱变已有诸多报道，应用十分广泛，但大多都为常规诱变。这样的诱变通常不对细胞做任何特殊处理，诱变过程随机、诱变不可控，导致诱变效率低下。【目的】筛选出耐热的同步脱硫脱氮菌株，对菌种进行改良的耐热诱变，同时对诱变剂进入细胞内外的情况进行较为详细的探讨。【方法】以脱氮硫杆菌(Thiobacillus denitrificans)为研究对象，对其同时进行常规诱变和改良的耐热诱变。在改良的诱变中，诱变之前需先采用哌嗪-1,4-二乙磺酸(piperazine-1,4-diethylsulfonic acid, PESA)法将对数生长期的细胞处理成易感态，以此改变细胞膜的透过性，然后分别加入不同体积(0.5‰−3.0‰)的诱变剂硫酸二乙酯(diethyl sulfate, DES)，在设定温度45 ℃诱变20 min。在突变株的筛选上采用升温培养的方式在47 ℃测试其耐热能力和脱硫脱氮性能，以得到真正的耐热突变株。另一方面，在菌株诱变的同时，利用S₂O₃²⁻可终止DES诱变的特性对诱变剂在细胞内外的分布状况进行分析，对不同方案下的实验结果进行较为深入的探讨。【结果】在得到耐热的同步脱硫脱氮高性能突变株的同时，通过两种方案的对比实验，证实了改变细胞膜透过性对诱变剂进入细胞内有明显的促进作用。达到同样的诱变效果所需诱变剂的剂量中，改良的耐热诱变约为常规诱变的1/10−1/9；诱变剂进入胞内的比率中，前者约占DES加入总量的41.8%−40.4%左右，而后者仅占5.6%−4.4%；细菌的正突变率可能与进入胞内的诱变剂剂量密切相关，改良的诱变中进入胞内的DES剂量更适合于得到较高的正突变率，但诱变剂剂量不能决定单个突变如突变株的耐热性、硫化物高去除率等在哪种情况下或何时发生；证实了诱变剂进入胞内的最大剂量取决于细胞自身的承载能力，与诱变方案无关。产物分析实验表明，S₂O₃²⁻可用于终止DES诱变。【结论】对细胞先进行易感态处理后诱变，再通过升温复筛，最终得到7株耐热能力提高的脱硫脱氮高性能突变株，为菌种诱变的相关研究提供新思路。

关键词:脱氮硫杆菌;改良的耐热诱变;耐热突变株;诱变剂的分布状况

Abstract:

[Background] Extensive studies have been carried out on strain mutagenesis, especially chemical mutagenesis, in strain breeding. However, most of these studies employ conventional mutagenesis, which usually does not involve any special treatment of target cells and has low mutagenic efficiency due to random and uncontrollable mutagenic process. [Objective] To screen thermotolerant strains for simultaneous sulfide and nitrate removal, we improved the mutagenic scheme on the target strain and studied the entry of mutagen into the cells. [Methods] Both conventional mutagenesis and improved mutagenesis were carried out for Thiobacillus denitrificans. In the improved mutagenesis, piperazine-1,4-diethylsulfonic acid (PESA) was used to treat the exponential-phase cells into a susceptible state before mutagenesis to change the permeability of cytoplasmic membrane, and then different volumes (0.5‰–3.0‰) of the mutagen diethyl sulfate (DES) were separately added into the cell suspension for mutagenesis at a set temperature of 45 ℃ for 20 min. The thermotolerance and sulfide and nitrate removal performance were tested at 47 ℃ to obtain the thermotolerant mutants. During the process of strain mutagenesis, we used S₂O₃²⁻, which could terminate DES mutagenesis, to observe the distribution of mutagen inside and outside the cells and compared the experimental results under different mutagenic schemes. [Results] We obtained the thermotolerant mutants capable of simultaneously removing sulfide and nitrate. The comparison of the two mutagenic schemes proved that changing the permeability of cytoplasmic membrane promoted the entry of mutagen into the cells. To achieve the same mutagenic effect, the improved mutagenesis only needed 1/10–1/9 of mutagen required by conventional mutagenesis. The percentage of mutagen entering the cells was 41.8%–40.4% of the total DES added in the improved mutagenesis and only 5.6%–4.4% in the conventional mutagenesis. The forward mutation efficiency might be closely related to the dosage of mutagen entering the cells, and the dosage in the improved mutagenesis was more suitable for achieving higher forward mutation efficiency. However, the dosage of mutagen did not determine under which circumstance or when individual mutation, such as thermotolerance or higher sulfide removal efficiency of mutants, probably occurred. The maximum dosage of mutagen entering cells depended on cell’s loading capacity and was independent of mutagenic schemes. The product analysis showed that S₂O₃²⁻ could be used to terminate the mutagenesis triggered by DES. [Conclusion] After treating the cells into a susceptible state, mutagenesis was carried out. And then through temperature-rise screening, 7 thermotolerant mutants with high-performance for sulfide and nitrate removal were obtained, providing new ideas for research on strain mutagenesis.

Key words:Thiobacillus denitrificans;improved mutagenesis of thermotolerance;thermotolerant mutant;distribution of mutagen inside and outside cells