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Published on: March 4, 2017
Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria
Susana Langa1, Ángela Peirotén2, Juan Luis Arqués2
1Departamento 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. langa.susana@inia.es.
Abstract:
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.
Insights
Researchers developed new plasmids for labeling lactobacilli strains, enabling both red and green fluorescence. The pNZ:TuR.aFP.STOP.mCherry plasmid allows stable dual-color fluorescent protein expression for strain differentiation, even in anaerobic environments.
Area of Science:
- Molecular Biology
- Microbiology
- Biotechnology
Background:
- Fluorescent proteins are essential for tracking cells and proteins, but many require oxygen for fluorescence.
- mCherry offers bright signals and rapid maturation, while Evoglow-Pp1 provides fluorescence under anaerobic conditions.
Purpose of the Study:
- To engineer plasmids for expressing red fluorescent protein mCherry and cyan-green fluorescent protein Evoglow-Pp1 in lactobacilli.
- To evaluate the expression and stability of these fluorescent proteins in various lactobacilli strains.
- To assess the utility of these engineered plasmids for differentiating lactobacilli in complex environments.
Main Methods:
- Modification of the pNZ:TuR.aFP plasmid to create constructs for mCherry (pNZ:TuR.mCherry), a fusion protein (pNZ:TuR.aFP.mCherry), and separate expression (pNZ:TuR.aFP.STOP.mCherry).
- Transformation of six different lactobacilli strains (Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri) with the engineered plasmids.
- Analysis of red and green fluorescence emission stability and differentiation capacity in a fecal microbiota environment.
Main Results:
- All transformed lactobacilli strains exhibited red, green, or combined red and green fluorescence.
- The pNZ:TuR.aFP.STOP.mCherry plasmid demonstrated stable co-expression of both Evoglow-Pp1 and mCherry.
- The engineered plasmids enabled successful differentiation of lactobacilli strains within a complex fecal microbiota environment.
Conclusions:
- The pNZ:TuR.aFP.STOP.mCherry plasmid is a valuable tool for labeling lactobacilli strains.
- This system provides stable, dual-color fluorescence functional under both aerobic and anaerobic conditions.
- The developed fluorescent labeling system aids in distinguishing bacterial strains in complex microbial communities.
