A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.
A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.
复制标题
鼠伤寒沙门氏菌单拷贝 lac 融合体的构建方法及其在 hemA-prfA 操纵子中的应用。
DOI：
10.1128/jb.174.1.245-253.1992
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发表时间：
1992
影响因子：
3.2
通讯作者：
Elliott,T
中科院分区：
文献类型：
--
作者：
Elliott,T
本报告描述了一组大肠杆菌和鼠伤寒沙门氏菌菌株，它们允许在质粒和细菌染色体之间进行lac融合的可逆转移。该系统依靠大肠杆菌recD宿主的同源重组从质粒转移到染色体。该大肠杆菌菌株携带鼠伤寒沙门氏菌put操纵子插入到trp中，产生的融合体形式为trp::put::[Kanr-X-lac]，其中X为所研究的启动子或基因片段。put同源性位于lac融合片段的两侧，因此融合可以转导到鼠伤寒沙门氏菌中，取代常驻的put操纵子。随后转导到具有大的染色体缺失覆盖的鼠伤寒沙门氏菌菌株中，允许选择在质粒上继承融合的重组体。构建了put操纵子的转座版本，并用于将lac融合到新的位置，包括F质粒和ara位点。在鼠伤寒沙门氏菌中，携带不同hemA-prfA操纵子片段的菌株间进行转导杂交，以确定hemA启动子区域对prfA基因和其他hemA下游基因表达的贡献。
This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.