A SYSTEM TO GENERATE CHROMOSOMAL MUTATIONS IN LACTOCOCCUS-LACTIS WHICH ALLOWS FAST ANALYSIS OF TARGETED GENES
A SYSTEM TO GENERATE CHROMOSOMAL MUTATIONS IN LACTOCOCCUS-LACTIS WHICH ALLOWS FAST ANALYSIS OF TARGETED GENES
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DOI：
10.1128/jb.177.24.7011-7018.1995
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发表时间：
1995-12-01
影响因子：
3.2
通讯作者：
LEENHOUTS, K
中科院分区：
文献类型：
--
作者：
LAW, J;BUIST, G;LEENHOUTS, K
描述了一种用于在乳球菌中产生染色体插入的系统。它基于乳球菌pWV 01衍生的Ori（+）RepA（-）载体pORI 19的条件复制，包含lacZ alpha和pUC 19的多克隆位点。将乳酸乳球菌（Lactococcus lactis）染色体的Campbell Ⅰ片段克隆到RepA（+）辅助菌株大肠杆菌（Escherichia coli）EC 101的pORT 19中。通过将该系统与温度敏感性pWV 01衍生物pVE 6007组合，增加了乳酸菌（RepA（-））的表达。改造L.乳酸菌MG 1363（pVE 6007）与乳球菌染色体片段的pORI 19库在允许温度下的融合，由于提供了来自pVE 6007的RepA-Ts的反式，允许来自库的重组质粒的几个拷贝在细胞内复制。温度变化至37 ℃导致pVE 6007的损失和pORI 19衍生物在高频下的整合。一个银行的乳球菌突变体，以这种方式，并成功地筛选存在两个突变：一个在单顺反子的1.3-kb的肽聚糖水解酶基因（acmA）和一个在迄今为止未表征的麦芽糖发酵途径。在30 ℃下将pVE 6007重新引入Mal（-）突变体中，导致整合质粒的切除和麦芽糖发酵能力的恢复。通过使用恢复的Mal（+）菌落的分离质粒内容物转化E. coli EC101。对pMAL中564 bp的染色体片段进行核苷酸序列测定，发现其翻译产物与大肠杆菌的ATP结合蛋白MalK有明显的同源性。大肠杆菌、鼠伤寒沙门氏菌和产气肠杆菌以及变形链球菌的MsmK。两种类型的条件复制型pWV 01衍生载体的组合使用代表了用于染色体基因失活、靶向、克隆和标记基因测序的新的有力工具。
A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori(+) RepA(-) vector pORI19, containing lacZ alpha and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORT19 in RepA(+) helper strain Escherichia coli EC101, The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA(-)), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. transformation of L. lactis MG1363(pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal(-) mutant at 30 degrees C resulted in excision of the integrated plasmid and restoration of the ability to ferment maltose, The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal(+) colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.