Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.
Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.
复制标题
用于斑马鱼模型系统的革兰氏阴性细菌红色荧光蛋白标记的广泛宿主范围质粒。
DOI：
10.1128/aem.01679-09
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发表时间：
2010
影响因子：
4.4
通讯作者：
Kim,CarolH
中科院分区：
文献类型：
--
作者：
Singer,JohnT;Phennicie,RyanT;Sullivan,MatthewJ;Porter,LauraA;Shaffer,ValerieJ;Kim,CarolH
为了观察绿色荧光蛋白标记的斑马鱼免疫细胞与入侵细菌之间的实时相互作用，构建了一系列用于标记多种鱼类和人类病原体的红色荧光蛋白（RFP）质粒。本研究的目的是建立一个质粒的集合，将表达RFP色素组成型和undertacpromoter调节，这将是无毒的，广泛传播到各种革兰氏阴性菌。将编码RFP二聚体（d）、单体（m）和串联二聚体（td）衍生物d-Tomato、td-Tomato、m-Orange和m-Cherry的DNA片段克隆到大肠杆菌中基于IncQ的载体pMMB 66 EH中。通过接合交配将质粒动员到受体菌株中。异丙基-β-d-硫代半乳糖苷（IPTG）可诱导大肠杆菌、铜绿假单胞菌、迟缓爱德华氏菌和鳗弧菌产色素。一个自发突变的接合子。分离出组成型表达td-Tomato的aerodiosaPA 14。互补分析表明，组成型表型可能是由于pMMB 66 EH上携带的lacIq突变所致。DNA序列分析证实了在14 bp的lacI区域内存在5个转换、4个颠换和2 bp的添加。从该组成型突变体中纯化载体DNA，并将RFP色素的结构DNA序列克隆到组成型载体中。P. aeruginosa、铜绿假单胞菌E. tarda和V. anguillarumexpressed所有色素在一个IPTG独立的方式。斑马鱼感染性研究的结果表明，RFP标记的病原体将是有用的先天免疫系统的宿主细胞和感染病原体之间的实时相互作用的研究。
To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and undertacpromoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH inEscherichia coli.Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible inEscherichia coli,Pseudomonas aeruginosa,Edwardsiella tarda, andVibrio(Listonella)anguillarumstrains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant ofP. aeruginosaPA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation inlacIqcarried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region oflacI.Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants ofP. aeruginosa,E. tarda, andV. anguillarumexpressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.