开发出一套来自米拉贝氏变形杆菌的尿素诱导启动子。

IF 3.9 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Applied and Environmental Microbiology Pub Date : 2024-11-20 Epub Date: 2024-10-30 DOI:10.1128/aem.01273-24
Madison J Fitzgerald, Abigail Scavone, Chaitra Moolaveesala, Melanie M Pearson, Harry L T Mobley
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引用次数: 0

摘要

导尿管相关性尿路感染(CAUTI)是医疗系统的一大负担,占全球医院获得性感染的 40%。普遍存在的 CAUTI 病原体 mirabilis 变形杆菌是一种未被充分研究的革兰氏阴性细菌。奇异变形杆菌 CAUTI 的后遗症之一是产生尿路结石,这使得治疗和清除感染变得更加复杂。结石的形成是由尿素酶的活性诱发的,尿素酶是一种镍金属酶,受 UreR 以尿素依赖的方式调节。由于尿素在尿路中含量丰富,尿素酶基因在实验性UTI期间高度表达。我们试图利用尿素酶启动子创建一个表达系统,在体外实验和实验性UTI期间实现尿素诱导的基因表达。在初步研究中,我们观察到尿素酶启动子的基础表达水平出乎意料地高。这在一定程度上取决于调节因子 UreR 的存在。为了进一步开发这一表达系统,我们生成了一系列报告构建体,以评估特定启动子元件在尿素存在和不存在的情况下对启动子活性的影响。感兴趣的元件包括已知的调控结合位点、替代翻译起始位点以及通过比较基因组学确定的单核苷酸多态性。这项工作描述了在这项研究中构建的一套尿素诱导启动子,这些启动子表现出多种表达动态,为基因表达提供了一个可定制的平台。尿素诱导型启动子还能被尿素天然存在的环境(如尿道)激活。因此,开发尿素诱导的选择性基因表达系统对尿路发病机制领域具有重大意义,因为它能在实验性尿路感染过程中进行选择性基因诱导。这种表达系统在生物技术和制造业的重组蛋白生产中也有重要应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of a suite of Proteus mirabilis-derived urea-inducible promoters.

Catheter-associated urinary tract infections (CAUTIs) are a significant burden on healthcare systems, accounting for up to 40% of hospital-acquired infections globally. A prevalent CAUTI pathogen, Proteus mirabilis, is an understudied Gram-negative bacterium. One sequela of P. mirabilis CAUTI is the production of urinary stones, which complicates treatment and clearing of the infection. Stone formation is induced by the activity of urease, a nickel-metalloenzyme that is regulated by UreR in a urea-dependent manner. As urea is abundant in the urinary tract, urease genes are highly expressed during experimental UTI. We sought to leverage the urease promoter to create an expression system that would enable urea-inducible expression of genes during in vitro experiments as well as during experimental UTI. During preliminary studies, we observed unexpectedly high levels of basal expression of the urease promoter. This was somewhat dependent on the presence of regulator UreR. To further develop this expression system, we generated a series of reporter constructs to assess the impact of specific promoter elements on promoter activity in the presence and absence of urea. Elements of interest included known regulatory binding sites, alternative translational start sites, and single-nucleotide polymorphisms identified through comparative genomics. This work describes a suite of urea-inducible promoters, constructed during this study, that exhibit a variety of expression dynamics, providing a customizable platform for gene expression.IMPORTANCEUrea is an inexpensive molecule that can easily be supplied during in vitro experiments. A urea-inducible promoter would also be activated by environments where urea naturally occurs, such as in the urinary tract. Thus, the development of a urea-inducible system for selective gene expression is of great interest to the field of uropathogenesis as it would enable selective gene induction during experimental urinary tract infection. This expression system would also have important applications for recombinant protein production in biotech and manufacturing.

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来源期刊
Applied and Environmental Microbiology
Applied and Environmental Microbiology 生物-生物工程与应用微生物
CiteScore
7.70
自引率
2.30%
发文量
730
审稿时长
1.9 months
期刊介绍: Applied and Environmental Microbiology (AEM) publishes papers that make significant contributions to (a) applied microbiology, including biotechnology, protein engineering, bioremediation, and food microbiology, (b) microbial ecology, including environmental, organismic, and genomic microbiology, and (c) interdisciplinary microbiology, including invertebrate microbiology, plant microbiology, aquatic microbiology, and geomicrobiology.
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