Analysis of gene expression of bifidobacteria using as the reporter an anaerobic fluorescent protein

Montenegro-Rodríguez, Cynthia; Peirotén, Ángela; Sánchez‐Jiménez, Abel; Arqués, Juan Luís; Landete, José María

doi:10.1007/s10529-015-1802-8

Cited by 12 publications

(4 citation statements)

References 19 publications

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“…Additionally, a GFP fluorescent protein containing a flavin-mono-nucleotide-based cofactor (evoglow-Pp1), which emits fluorescence in the presence/absence of oxygen, was included in a vector under control of the elongation factor Tu (P tuf from B. longum ) that replicates in B. longum and B. breve (Landete et al, 2014). This system, which supposes an advantage to study bifidobacteria in strict anaerobic conditions, was latterly validated under the control of other promoters (Montenegro-Rodríguez et al, 2015). …”

Section: Introductionmentioning

confidence: 99%

Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

et al. 2017

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An extracellular layer of exopolysaccharides (EPS) covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW)-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter). Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the interaction with the host.

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Section: Introductionmentioning

confidence: 99%

Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

et al. 2017

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“…Here, the behavior of some inducible promoters of genes involved in the transport and the catabolism of carbohydrates and the influence of the presence or absence of cre sites in these promoters were studied by means of a plasmid encoding the evoglow-Pp1 protein as reporter. The use of a flavin mononucleotide-based fluorescent protein presents interesting advantages in some environments and has been successfully used as a reporter (Martínez-Fernández et al 2019;Montenegro-Rodríguez et al 2015). When L. casei BL23 strains transformed with pNZ:gutF.aFP and pNZ:gutF Δ cre .aFP were grown in the presence of sorbitol and ribose, we observed fluorescence induction, being higher in L. casei pNZ:gutF Δcre.…”

Section: Discussionmentioning

confidence: 83%

Catabolite responsive elements as a strategy for the control of heterologous gene expression in lactobacilli

Langa

Peirotén

Arqués

et al. 2020

Appl Microbiol Biotechnol

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Genes involved in the transport and catabolism of carbohydrates are usually controlled through the binding of the catabolite control protein A (CcpA) to the catabolite-responsive elements (cre) of target genes in Gram-positive bacteria. In this work, we show how the elimination of the cre sites in Lactobacillus casei BL23 promoters induced by sorbitol (PgutF), maltose (PmalL), and myoinositol (PiolT) allowed the induction of gene expression in media supplemented with sorbitol, maltose, and myo-inositol, respectively, even in the presence of glucose. This was studied using plasmids encoding the anaerobic fluorescent protein evoglow-Pp1 as a reporter. In addition, gutF cre site was introduced into a bile inducible promoter (P16090) and into the constitutive promoter of the elongation factor P (PEf-P) of L. casei BL23. The existence of the cre site blocked gene expression in the P16090 inducible promoter in the presence of glucose, while it had no influence on the expression of the PEf-P constitutive one. These results demonstrated that the introduction or elimination of cre sites in inducible promoters allows the control and modification of their heterologous genetic expression, showing how the cre site, the transcriptional regulator, and CcpA interact to control gene expression in inducible genes. Key points• Cre sequences regulate gene expression in inducible promoters in L. casei BL23.• Cre sites do not affect gene expression in constitutive promoters in L. casei BL23.• Cre sequences could control heterologous genic expression in lactobacilli.Keywords catabolite response element . inducible promoter . constitutive promoter . genic expression . Lactobacillus

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“…The use of fluorescent proteins as a visible marker and as a transcriptional reporter to monitor bacterial gene expression in real-time in LAB and Bifidobacterium spp. in living cells has been addressed through different strategies [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ].…”

Section: Reporter Genes Used In Lactic Acid Bacteria and Bifidobacmentioning

confidence: 99%

Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors

Landete

Arqués

2017

IJMS

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Control and quantification of effector molecules such as heavy metals, toxins or other target molecules is of great biotechnological, social and economic interest. Microorganisms have regulatory proteins that recognize and modify the gene expression in the presence or absence of these compounds (effector molecules) by means of binding to gene sequences. The association of these recognizing gene sequences to reporter genes will allow the detection of effector molecules of interest with high sensitivity. Once investigators have these two elements—recognizing gene sequences and reporter genes that emit signals—we need a suitable vehicle to introduce both elements. Here, we suggest lactic acid bacteria (LAB) and bifidobacteria as promising carrier microorganisms for these molecular biosensors. The use of fluorescent proteins as well as food-grade vectors and clustered regularly interspaced short palindromic repeats (CRISPR) are indispensable tools for introducing biosensors into these microorganisms. The use of these LAB and bifidobacteria would be of special interest for studying the intestinal environment or other complex ecosystems. The great variety of species adapted to many environments, as well as the possibility of applying several protocols for their transformation with recognizing gene sequences and reporter genes are considerable advantages. Finally, an effort must be made to find recognizable gene sequences.

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Analysis of gene expression of bifidobacteria using as the reporter an anaerobic fluorescent protein

Cited by 12 publications

References 19 publications

Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

Catabolite responsive elements as a strategy for the control of heterologous gene expression in lactobacilli

Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors

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