一种新的细菌间基因水平转移途径? - - 枯草芽孢杆菌细胞间遗传重组过程的基本特性研究及机理揭示

In bacteria, horizontal gene transfer takes place by natural transformation, conjugation, or transduction. They can be distinguished by two simple criteria: sensitivity to the presence of deoxyribonuclease (DNase) and dependence on cell contact. We had observed that Bacillus subtilis could conduct cell to cell genetic exchange with the absence of additional free DNA. The process was once believed to be transformation since its sensitivity to DNase treatment. The source of the transforming DNA had been believed being made by PBSX, a kind of defecive prophage existing universally in B. subtilis genome. Nevertheless, the cell to cell genetic exchange frequencey of the related PBSX gene knockout mutants kept almost no change compared to that of wild type strains, and the intercellular genetic recombination frequency was much higer than that of "DNA to cell" transformation process conducted in the same condition, suggesting that specific extracellular DNA freguments resulted from the activity of PBSX may not be the key factor in this cell to cell horizontal gene tansfer process. In addition, the transformation ferquency was found to be correlative with the living cell density of the donor and dependent greatly on the physical contact of the DNA donor and recipient cells. The latter phenomenon made the process looks like conjucation rather than transformation. Therefore, the obersed process is actually different from all the three typical bacterial genetic exchange pathways, a new bacterial hozizatonal gene transfer mechanism could be expected upon further researches.

自然转化、接合和转导是目前已知的三种细菌基因水平转移途径，可以通过测试其是否依赖于细胞间接触以及对DNase处理是否敏感对它们进行区分。我们对枯草芽孢杆菌的研究发现，该菌可在不添加外源DNA的情况进行细胞间遗传重组，这个过程对DNase敏感，因此曾被认为是自然转化的一种形式，转化DNA则来自缺陷性原噬菌体PBSX的活动。但进一步的研究证实，PBSX相关基因缺失突变株和野生株相比在重组频率上并无明显变化，细胞间遗传重组的频率也远高于相同条件下游离DNA和受体细胞间的转化频率,说明PBSX活动产生的胞外DNA并非该遗传重组过程的决定因素。此外，这种发生在细胞间的遗传重组过程不仅和给体活细胞的密度关系密切，还严重依赖于给体和受体间的接触，使其看起来又类似于接合。因此，这个过程与目前已知的三种传统的细菌水平基因转移途径都有差异，对其开展深入研究将有可能导致一种新的细菌间遗传交换机制的发现。

在项目的资助下，我们通过系列实验确证枯草芽胞杆菌在固态基质表面发生的高效率细胞间水平基因转移过程总体而言属于自然转化，而非接合或转导。但是，不仅缺陷性原噬菌体PBSX活动产生的胞外DNA与该转化过程关系不大，而且在相同条件下细胞-细胞的转化效率明显高于等量游离DNA-细胞，且进行细胞间转化时的共转化片段至少可达1.2M，远高于以原生质体或人工提取的游离DNA作为转化DNA来源时所能达到的742kb和63KB。此外，该过程只能在发生在加有卡那霉素等抗生素或者基本培养基的选择性条件下，且细胞混合液涂布到固态基质上大约10 h后才能检测到重组子的产生。这种DNA给体细胞特性不仅普遍适用于不同枯草芽孢杆菌菌株，而且也适用于地衣芽孢杆菌。上述结果说明在环境中进行的这类基因水平转移过程中DNA给体细胞确实可能具有我们尚不太了解的重要生物学功能。对枯草菌单基因突变库进行了筛查，尚未发现能产生转化效率明显改变的DNA给体突变株，说明该过程可能受到多基因的复杂调控，有必要进一步通过系统的转录组分析来寻找关键的调控因子。此外，我们也证实对PBSX等相关自溶基因进行缺失突变确实可显著提升发酵培养物的生物量，这对于用芽胞杆菌生产益生菌或者表达基因产品的工业企业也有重要意义。