Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, Helsinki, Finland.
Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, Espoo, Finland.
Appl Microbiol Biotechnol. 2016 Nov;100(22):9661-9669. doi: 10.1007/s00253-016-7828-6. Epub 2016 Sep 22.
In this paper, we present a new counterselection method for deleting fragments from Lactococcus 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. lactis chromosome 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.
在本文中，我们提出了一种从乳球菌染色体中删除片段的新的反选择方法。该方法使用一种非复制性的质粒载体，该载体整合到染色体中，使细胞对细菌素敏感。整合载体携带 pUC ori 在大肠杆菌中功能，但不在乳球菌中，一个用于选择单交换整合子的红霉素抗性基因，以及来自乳球菌染色体的两个片段用于同源重组。此外，整合载体配备有李斯特菌mptC 基因，该基因编码甘露糖磷酸转移酶系统成分 IIC，是 IIa 类细菌素的受体。来自整合载体的 mptC 的表达使天然耐药的乳球菌对 IIa 类细菌素敏感。这种敏感性随后用于在细菌素琼脂上选择双交换菌落。只有通过失去 mptC 质粒而恢复内源性细菌素抗性的细胞才能存活。然后可以通过 PCR 将携带所需缺失的菌落与野生型回复突变体区分开来。通过使用 IIa 类细菌素亮白菌素 A、亮白菌素 C 或乳杆菌素 AcH 作为反选择剂，我们从野生型乳链菌肽生产菌株 N8 中删除了 22kb 和 33kb 的染色体片段。总之，这里提出的这种反选择方法是一种方便、高效和廉价的在乳球菌染色体中产生连续缺失的技术。
