Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Carretera de La Coruña Km 7.5, 28040, Madrid, Spain.
Appl Microbiol Biotechnol. 2021 Oct;105(19):7367-7378. doi: 10.1007/s00253-021-11537-y. Epub 2021 Sep 18.
Fluorescent proteins are widely used for cell and protein tracking. Most of these proteins show a high signal and need the presence of oxygen to emit fluorescence. Among them, the fluorescent protein mCherry stands up because of its bright signal and fast maturation. Furthermore, the anaerobic cyan-green fluorescent protein Evoglow-Pp1 allows fluorescent detection under anaerobic conditions. In this work, we modified the pNZ:TuR.aFP plasmid, which harbors the gene encoding Evoglow-Pp1 and the promoter of elongation factor Tu from Limosilactobacillus reuteri CECT925, to obtain a plasmid containing the mrfp gene encoding the monomeric mCherry (pNZ:TuR.mCherry). Moreover, both genes were cloned together (pNZ:TuR.aFP.mCherry) developing a chimeric protein; and with a stop codon between them (pNZ:TuR.aFP.STOP.mCherry) resulting in the expression of both Evoglow-Pp1 and mCherry proteins separately under the influence of the same promoter. Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri strains were transformed with the previously mentioned plasmids, showing an excellent red (pNZ:TuR.mCherry), green (pNZ:TuR.aFP), and red combined with green (pNZ:TuR.aFP.mCherry and pNZ:TuR.aFP.STOP.mCherry) fluorescence signal. Both fluorescence emissions were stable in strains transformed with pNZ:TuR.aFP.STOP.mCherry, while differences in the red or green fluorescence emission were observed in some of the strains harboring pNZ:TuR.aFP.mCherry. Moreover, these plasmids allowed strains differentiation in a complex environment, such as fecal microbiota. Hence, we present the plasmid pNZ:TuR.aFP.STOP.mCherry as a useful tool for the labeling of lactobacilli strains, which would be functional under anoxic conditions, thanks to Evoglow-Pp1, while having the high brightness and good photostability of mCherry. KEY POINTS: • LAB transformed with pNZ:TuR.mCherry expressed the red fluorescent protein mCherry. • LAB transformed with pNZ:TuR.aFP.mCherry developed a fusion of both proteins Evoglow-Pp1 and mCherry. • LAB with pNZ:TuR.aFP.STOP.mCherry expressed both fluorescent proteins separately.
荧光蛋白被广泛用于细胞和蛋白质追踪。其中大多数蛋白具有较高的信号强度，需要氧气的存在才能发出荧光。在这些荧光蛋白中，mCherry 因其明亮的信号和快速成熟而脱颖而出。此外，厌氧型的 cyan-green 荧光蛋白 Evoglow-Pp1 允许在厌氧条件下进行荧光检测。在这项工作中，我们对 pNZ:TuR.aFP 质粒进行了修饰，该质粒包含编码 Evoglow-Pp1 的基因和来自雷氏乳杆菌 CECT925 的延伸因子 Tu 的启动子，以获得一个包含编码单体 mCherry 的 mrfp 基因的质粒（pNZ:TuR.mCherry）。此外，我们将这两个基因克隆在一起（pNZ:TuR.aFP.mCherry），构建了一个嵌合蛋白；并在它们之间插入一个终止密码子（pNZ:TuR.aFP.STOP.mCherry），导致在相同启动子的影响下，Evoglow-Pp1 和 mCherry 蛋白分别表达。将上述质粒转化入乳球菌、干酪乳杆菌、植物乳杆菌、发酵乳杆菌、鼠李糖乳杆菌和雷氏乳杆菌，显示出极好的红色（pNZ:TuR.mCherry）、绿色（pNZ:TuR.aFP）和红色与绿色组合（pNZ:TuR.aFP.mCherry 和 pNZ:TuR.aFP.STOP.mCherry）荧光信号。在转化了 pNZ:TuR.aFP.STOP.mCherry 的菌株中，两种荧光发射都很稳定，而在一些携带 pNZ:TuR.aFP.mCherry 的菌株中则观察到红色或绿色荧光发射的差异。此外，这些质粒允许在复杂环境（如粪便微生物群）中对菌株进行区分。因此，我们提出了质粒 pNZ:TuR.aFP.STOP.mCherry，作为一种有用的工具，可以对乳酸杆菌菌株进行标记，该质粒得益于 Evoglow-Pp1，可以在缺氧条件下发挥功能，同时具有 mCherry 的高亮度和良好的光稳定性。关键点： • 转化了 pNZ:TuR.mCherry 的 LAB 表达了红色荧光蛋白 mCherry。 • 转化了 pNZ:TuR.aFP.mCherry 的 LAB 表达了 Evoglow-Pp1 和 mCherry 的融合蛋白。 • 转化了 pNZ:TuR.aFP.STOP.mCherry 的 LAB 分别表达了两种荧光蛋白。
