摘要

在本文中，我们提出了一种新的用于从乳酸乳球菌染色体中删除片段的反选法。该方法使用非复制型质粒载体，其整合入染色体并使细胞对细菌素敏感。整合载体携带的pUC在大肠杆菌中而不是在乳酸乳球菌中，红霉素抗性基因，用于选择单交换整合ORI功能，和从乳酸乳球菌染色体的两个片段进行同源重组。此外，整合载体装配有编码甘露糖 - 磷酸转移酶系统组分IIC（IIa类细菌素受体）的单核细胞增生利斯特氏菌基因mptC。来自整合载体的mptC的表达使天然抗性乳酸乳球菌对IIa类细菌素敏感。然后使用该灵敏度来选择细菌素琼脂上的双交换克隆。只有通过缺失mptC质粒而恢复了内源性细菌素抗性的细胞才能存活下来。然后可以通过PCR将携带所需缺失的菌落与野生型回复菌区分开来。通过使用IIa类细菌素leucocin A，leucocin C或者pediocin AcH作为反选择剂，我们从野生型乳酸链球菌素产生的乳酸乳球菌N8株中删除了22-和33-kb染色体片段。总之，这里提出的这种反选方法是在乳酸乳球菌染色体中产生连续缺失的方便，有效和廉价的技术。

In this paper, we present a new counterselection method for deleting fragments fromLactococcus lactis chromosome. The method uses a non-replicating plasmid vector, which integrates into the chromosome and makes the cell sensitive to bacteriocins. The integration vector carries pUC ori functional in Escherichia coli but not in L. lactis, an erythromycin resistance gene for selecting single crossover integrants, and two fragments from L. lactischromosome for homologous recombinations. In addition, the integration vector is equipped with the Listeria monocytogenes gene mptC encoding the mannose-phosphotransferase system component IIC, the receptor for class IIa bacteriocins. Expression of mptC from the integration vector renders the naturally resistant L. lactis sensitive to class IIa bacteriocins. This sensitivity is then used to select the double crossover colonies on bacteriocin agar. Only the cells which have regained the endogenous bacteriocin resistance through the loss of the mptC plasmid will survive. The colonies carrying the desired deletion can then be distinguished from the wild-type revertants by PCR. By using the class IIa bacteriocins leucocin A, leucocin C or pediocin AcH as the counterselective agents, we deleted 22- and 33-kb chromosomal fragments from the wild-type nisin producing L. lactis strain N8. In conclusion, this counterselection method presented here is a convenient, efficient and inexpensive technique to generate successive deletions in L. lactis chromosome.

https://link.springer.com/article/10.1007/s00253-016-7828-6