ANALYSIS OF PSEUDOMONAS GENE-PRODUCTS USING LACIQ PTRP-LAC PLASMIDS AND TRANSPOSONS THAT CONFER CONDITIONAL PHENOTYPES
ANALYSIS OF PSEUDOMONAS GENE-PRODUCTS USING LACIQ PTRP-LAC PLASMIDS AND TRANSPOSONS THAT CONFER CONDITIONAL PHENOTYPES
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DOI：
10.1016/0378-1119(93)90533-9
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发表时间：
1993-01-15
期刊：
影响因子：
3.5
通讯作者：
TIMMIS, KN
中科院分区：
文献类型：
--
作者：
DELORENZO, V;ELTIS, L;TIMMIS, KN
新的基于转座子和质粒的广泛宿主表达系统已经被开发出来，以促进假单胞菌和相关革兰氏菌基因产物的遗传分析。利用lacI(q)/Ptrp-lac的特性构建mini-Tn5表达载体转座子和rsf1010衍生质粒，用于控制表达和条件表型的产生。利用这些质粒在不同菌株中而不是在大肠杆菌中超表达假单胞菌LB400多氯联苯降解途径的bphB和bphC基因，表达了p.p putida TOL质粒间操纵子XylS调控因子。与大肠杆菌相比，2,3二羟基联苯双加氧酶（bphC基因产物）在其原生宿主体内大量产生时比活性提高了10倍，但在相同的体内条件下，XylS调节因子形成蛋白聚集体。本文提出的另一种基于lacI(q)/ ptrp - lacp的表达载体是转座子mini-Tn5 lacI(q)/Ptrc，它可以在lac诱导剂的控制下，将含有外源基因的遗传盒插入目标细菌的染色体中，通过监测在mini-Tn5 lacI(q)/Ptrc中克隆并插入到p.p putida染色体中的lacZ报告基因的表达可以证明这一点。
Novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of Pseudomonas and related Gram- bacteria. The properties of lacI(q)/Ptrp-lac were used to construct mini-Tn5 expression vector transposons and RSF1010-derived plasmids for controlled expression and generation of conditional phenotypes. These plasmids were used to hyper-express the XylS regulator of the meta operon of the TOL plasmid of P. putida or the bphB and bphC genes of the polychlorobiphenyl-degrading pathway of Pseudomonas sp. LB400 in different strains of Pseudomonas instead of in Escherichia coli. Specific activity of 2,3 dihydroxybiphenyl dioxygenase (bphC gene product) was increased tenfold when hyperproduced in its native host as compared to E. coli, but under the same in vivo conditions, the XylS regulator formed protein aggregates. The other lacI(q)/Ptrp-lac-based expression vector presented here, transposon mini-Tn5 lacI(q)/Ptrc, facilitates the insertion of genetic cassettes containing heterologous genes under the control of lac inducers in the chromosome of target bacteria, as shown by monitoring expression of a lacZ reporter cloned in mini-Tn5 lacI(q)/Ptrc and inserted in the chromosome of P. putida.