Description
Autofluorescent proteins do not require a substrate for their presence to be detected. They are therefore ideal for the detection of bacteria expressing these proteins in vivo and in real time.There are many plasmid vectors that encode fluorescent proteins that function in Escherichia coli.
However, there are few genetic tools of this type that function efficiently in Gram-positive bacteria (Bacillus subtilis excepted).Therefore, our research group, drawing on our experience in the analysis of plasmid replication and the regulation of gene expression, has developed and continues to optimize various plasmid vectors that permit i) the expression of genes, ii) the detection and quantification of gene expression in Gram-positive bacteria, and iii) the use of labelled bacteria in in vitro models (interactions between bacteria and eukaryotic cells) and also in in vivo models (bacterial colonization of the digestive tract of Dario renio (zebrafish) and mice).
These vectors function in (among others) Lactococcus lactis, Streptococcus pneumoniae, Staphylococcus aureus, Listeria monocytogenes, Pediococcus parvulus, as well as in species belonging to the genera Lactobacillus, Leuconostoc and Enterococcus, and are being commercialized by the company Solmeglas (https://solmeglas.com/categoria-producto/biologia-molecular). The vectors encode versions of the fluorescence proteins: green (GFP) from Aqueora Victoria and red (monomeric version, mCherry) from Dicosoma sp., whose coding genes have been optimized for expression in bacteria. Furthermore, the spectra of excitation and emission allow the simultaneous detection of both proteins.
Among other vectors, we have developed, in collaboration with the groups of Dr. Teresa Requena, of the Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC, Madrid), and Prof. Jerry Wells, of the University of Wageningen (Holand), two families of expression vectors of mCherry, that function both in Gram-positive bacteria, including lactic acid bacteria (LAB) and in Escherichia coli due to the presence of two replicons or to the presence of a promiscuous replicon of the pMV158 family. These vectors allow the evaluation uni- and bi-directional promotor sequences and are currently being used to evaluate the expression of genes from different LAB in the natural of heterospecific hosts. Additionally, we have constructed a vector based on the pMV158 replicon for cloning and maltose induced gene expression. The above mentioned vectors have permitted the development of plasmids for fluorescent labelling of bacteria either by cloning a functional promotor that controls the expression of the mrfp gene, encoding the mCherry, or by cloning the gfp gene under the control of the promotor regulated by maltose. Thus, funcionality of promoters of Lactobacillus, Leuconostoc and Pediococcus have been validated. Furthermore, the vectors have been used for expresion of the dextrasucrase of Leuconostoc lactis in Leuconostoc mesenteroides, increasing the spectrum of LAB for analysis of gene expression and production of enzymes of biotechnological interest.
References:
Ruiz-Masó, J. A., López-Aguilar, C., Nieto, C., Sanz, M., Burón, P., Espinosa, M. and del Solar, G. (2012). Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae. Plasmid 67: 53–59.
García-Cayuela, T., Gómez de Cadiñanos, L.P., Mohedano, M.L., Fernández de Palencia, P., Boden, D., Wells, J., Peláez, C., López, P. and Requena, T. (2012). Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli. Appl. Microbiol. Biotechnol. 96:171-181.
Domingues, S., Cunha Aires, A., Mohedano, M.L., López, P. and Arraiano, C.M. (2013) A new tool for cloning and gene expression in Streptococcus pneumoniae. Plasmid 70: 247–253.
Campelo, A.B., Roces, C., Mohedano, M.L., López, P., Rodríguez, A. and Martínez, B. (2014).A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis. Microb. Cell Fact. 13:77.
Mohedano, M.L., García-Cayuela, T., Pérez-RamosA., Gaiser, A.R., Requena, T., and López, P. (2015). Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus. J. Ind. Microbiol. Biotechnol. 42:247-253.
Russo P., Iturria I., Mohedano M.L., Caggianiello G., Rainieri S., Fiocco D., Pardo M. A., López P. and Spano G. (2015). Zebrafish gut colonization by mCherry-labelled lactic acid bacteria. Appl. Microbiol. Biotechnol. 99:3479-3490.
Nácher-Vázquez, M., Ruiz-Masó, J. A., Mohedano, M. L., del Solar, G., Aznar, R. and López, P. (2017). Dextransucrase expression is concomitant with that of replication and maintenance functions of the pMN1 plasmid in Lactobacillus sakei MN1. Front. Microbiol. 8:2281. In “The Good, the Bad and the Ugly: Multiple roles of bacteria in human life” (Venkova, T., Yeo, C.C. y Espinosa, M., editors. Series Title: Frontiers Research Topics (Lausanne: Frontiers Media Ed) pp. 159-174, (2018) ISBN 978-2-88945-574-4.
Pérez-Ramos, A., Mohedano, M.L., Pardo, M.A. and López, P. (2018). β-glucan-producing Pediococcus parvulus 2.6: test of probiotic and immunomodulatory properties in zebrafish models. Front. Microbiol. 9:1684.
Mohedano, M.L., Hernández-Recio, S., Yépez, A., Requena, T., Martínez-Cuesta, M.C., Peláez, C. Peláez MC, Russo. P, LeBlanc, J.G., Spano, G., Aznar, R. and López, P. (2019). Real-time detection of riboflavin production by Lactobacillus plantarum strains and tracking of their gastrointestinal survival and functionality in vitro and in vivo using mCherry labeling. Front. Microbiol. 10:1748.
Besrour-Aouam N., Mohedano, M.L., Fhoula, I., Zarour, K., Najjari, A., Aznar, R., Prieto, A., Ouzari, H.-I. López, P. 2019. Different modes of regulation of the expression of dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1. Front. Microbiol. 10:959.
Garay-Novillo, J.N., García-Morena, D., Ruiz-Masó, J.A., Barra, J.L. and del Solar, G. (2019). Combining modules for versatile and optimal labeling of lactic acid bacteria: two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins. Front. Microbiol. 10:1431. doi: 10.3389/fmicb.2019.01431.
Jara, J., Pérez-Ramos, A., del Solar, G., Rodríguez, J.M., Fernández, L. and Orgaz, B. (2020). Role of Lactobacillus biofilms in Listeria monocytogenes adhesion to glass surfaces. Int. J. Food Microbiol. 334. doi: 10.1016/j.ijfoodmicro.2020.108804.
Hernández-Alcántara, A., Pardo, S., Mohedano, M.L., Vignolo, G., de Moreno de LeBlanc, A., LeBlanc, J.G., Aznar, R. and López, P. (2020). The ability of riboflavin-overproducing Lactiplantibacillus plantarum strains to survive under gastrointestinal conditions. Front. Microbiol. 11:591945.
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