State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Appl Environ Microbiol. 2023 Feb 28;89(2):e0189122. doi: 10.1128/aem.01891-22. Epub 2023 Feb 6.
The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter- fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding -di-GMP etabolism-elated (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter- fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter- fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter- transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.
机会性病原体铜绿假单胞菌是一种环境微生物，也是生物膜研究的模式生物。环二鸟苷酸（c-di-GMP）是一种细菌第二信使，在生物膜形成中起着关键作用。铜绿假单胞菌含有约 40 个基因，这些基因编码参与 c-di-GMP（生物合成或降解）代谢的酶，但它缺乏有助于研究这些基因系统表达模式的工具。在本研究中，我们构建了一个启动子融合报告基因文库，该文库由 41 个报告质粒组成。每个质粒都包含铜绿假单胞菌参考菌株 PAO1 中相应 c-di-GMP 代谢相关（CMR）基因的启动子；因此，每个启动子融合报告基因都可用于检测相应基因的启动子活性和转录。在铜绿假单胞菌和大肠杆菌中测试了启动子活性。在 41 个基因中，有 26 个基因的启动子在铜绿假单胞菌和大肠杆菌中均具有活性。该文库用于确定不同温度、生长培养基和亚抑菌浓度抗生素对铜绿假单胞菌 41 个 CMR 基因转录谱的影响。结果表明，不同的生长条件确实会影响不同基因的转录，而少数基因的启动子活性在几种不同的生长条件下保持相同水平。总之，我们提供了一个启动子融合报告基因文库，用于系统监测或研究铜绿假单胞菌 CMR 基因的调控。此外，功能性启动子也可用作合成生物学研究的生物积木。机会性病原体铜绿假单胞菌可引起人类急性和慢性感染，是医院感染的主要病原体之一。生物膜形成是铜绿假单胞菌在宿主中持续存在和逃避免疫和抗生素攻击的最重要原因之一。c-di-GMP 是控制生物膜形成的关键第二信使。在铜绿假单胞菌参考菌株 PAO1 中，有 41 个基因被预测参与这种二核苷酸的合成和分解。该领域缺失的主要信息是这些基因在应对环境变化时的系统表达谱。为了实现这一目标，我们构建了一个启动子-转录融合报告基因文库，该文库由 41 个报告质粒组成，每个质粒都包含铜绿假单胞菌中相应 c-di-GMP 代谢相关基因的启动子。该文库提供了一种有用的工具，可以帮助理解与 c-di-GMP 相关的复杂调控网络，并发现潜在的治疗靶点。
