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Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria

  • Applied Genetics and Molecular Biotechnology
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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.

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Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We would like to thank undergraduate student Clara Sebastian for her assistance with fluorescence microscopy. pRCR12 plasmid harboring mrfp gene was kindly provided by Paloma López (CIB, Madrid, Spain).

Funding

This work was supported by RTA2017-00002–00-00 project from the Ministry of Economy and Competitiveness (Spain).

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Authors and Affiliations

  1. 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

    Susana Langa, Ángela Peirotén, Juan Luis Arqués & José María Landete

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  1. Susana Langa
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  2. Ángela Peirotén
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  3. Juan Luis Arqués
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  4. José María Landete
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Contributions

JML conceived and designed research. JML, AP, and SL conducted experiments. JML, AP and SL analyzed the data. JML, SL, AP, and JLA interpreted the data and wrote the manuscript. All authors read and approved the submitted manuscript.

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Correspondence to Susana Langa.

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Langa, S., Peirotén, Á., Arqués, J.L. et al. Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria. Appl Microbiol Biotechnol 105, 7367–7378 (2021). https://doi.org/10.1007/s00253-021-11537-y

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  • DOI: https://doi.org/10.1007/s00253-021-11537-y

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