Anaerobic green fluorescent protein as a marker of Bifidobacterium strains

Landete, José María; Peirotén, Ángela; Rodríguez, Eva; Margollés, Abelardo; Médina, Margarita; Arqués, Juan Luís

doi:10.1016/j.ijfoodmicro.2014.01.008

Cited by 43 publications

(37 citation statements)

References 39 publications

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“…Lactococcus lactis MG1363 was used as a host for heterologous gene expression in the pNZ:Tu plasmid (26). Escherichia coli DH10B was used for DNA manipulations.…”

Section: Methodsmentioning

confidence: 99%

“…The F-1092 forward primer introduced an NcoI site at about the initiation codon of the est_1092 gene, and the R-1092 reverse primer introduced an XbaI site downstream of the stop codon. The PCR product was digested with the two restriction enzymes and ligated into the corresponding restriction sites of vector pNZ:Tu (26). The ligation mixture was transformed into L. lactis MG1363 by electroporation (29), and the transformants containing the recombinant pNZ:Tu-1092 plasmid were checked by restriction mapping and sequencing of the inserted fragment.…”

Section: Methodsmentioning

confidence: 99%

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A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

Esteban-Torres

Landete

Reverón

et al. 2015

Appl Environ Microbiol

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bLactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. Lactobacillus plantarum is a highly versatile lactic acid bacterial species found in many different ecological niches, such as vegetables, meat, fish, and dairy products, as well as in the gastrointestinal tract (1). The genome of L. plantarum strain WCFS1 was the first to be fully sequenced, and it was, in fact, the first of any of the Lactobacillus genomes to be published (2). When the genome diversity of L. plantarum on a full genome scale was analyzed, it was revealed that L. plantarum strains are predicted to lack 9 to 20% of the genes of the L. plantarum WCFS1 reference genome, and about 50 genes appeared to be specific to strain WCFS1, as they were not found in any other strain (1). This variability confirms the flexibility of L. plantarum in its ability to adapt to different environments and growth substrates.Phenolic compounds are important constituents of food products of plant origin, as they are related to the sensory characteristics of the food and are beneficial to consumer health (3). Therefore, it is interesting to know the metabolic pathways of biosynthesis or degradation of these compounds in bacteria. L. plantarum is the lactic acid bacterium that is the most frequently found in the fermentation of food products of plant origin, being the bacterial model for the study of phenolic compound metabolism (4). Among these compounds, the metabolism of phenolic esters is greatly relevant, as they are widely spread throughout the plant kingdom (3). Esters of phenolic acids mainly belong to two distingui...

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“…Lactococcus lactis MG1363 was used as a host for heterologous gene expression in the pNZ:Tu plasmid (26). Escherichia coli DH10B was used for DNA manipulations.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

Esteban-Torres

Landete

Reverón

et al. 2015

Appl Environ Microbiol

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“…The flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) used in this paper overcome oxygen restrictions (Drepper et al 2007;Ernst and Tielker 2009). Recently, our group described the development of a noninvasive green fluorescence-based reporter system for real-time tracking of bifidobacteria under both aerobic and anaerobic conditions through the use of this tool (Landete et al 2014). Moreover, LAB are known to sustain growth at low pH because they can quickly adapt their internal pH to the external pH.…”

Section: Discussionmentioning

confidence: 99%

“…Subsequently, plasmids pNZ:AlaR-aFP and pNZ:TuR-aFP ( Fig. S1) were used to transform Lactobacillus and Enterococcus strains shown in Table 1 with the method proposed by Landete et al (2014).…”

Section: Cloning Of Anaerobic Fluorescence Protein Gene In Pnz8048mentioning

confidence: 99%

“…However, many interesting LAB in relation to industrial and probiotic applications exert their beneficial effects in environments without oxygen, which would restrict the use of GFP or bioluminescent reporters. This problem can be solved by using the anaerobic cyan-green fluorescent protein evoglow-Pp1 (Landete et al 2014). This is of special interest in the case of the anaerobic LAB.…”

Section: Introductionmentioning

confidence: 99%

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Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria

Landete

Langa

Revilla

et al. 2015

Appl Microbiol Biotechnol

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Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production.

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Highly Fluorescent Flavins: Rational Molecular Design for Quenching Protection Based on Repulsive and Attractive Control of Molecular Alignment

Suzuki

Inoue

Kawamorita

et al. 2015

Chemistry A European J

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Unprecedented intense fluorescent emission was observed for a variety of flavin compounds bearing a perpendicular cyclic imide moiety at the C7 position of an isoalloxazine platform. A series of alloxan-substituted flavins was prepared selectively by reduction of the corresponding N-aryl-2-nitro-5-alkoxyanilines with zinc dust and subsequent reaction with alloxan monohydrate in the presence of boric acid. Analogues bearing oxazolidine-2,4-dione functionality were obtained on methylation of the alloxan-substituted flavins with methyl iodide and subsequent rearrangement in the presence of an inorganic base. The flavin compounds exhibit intense white-green fluorescent emission in the solution state under UV excitation at 298 K, with emission efficiencies Φ298 K greater than 0.55 in CH3 CN, which are higher than the values for all reported flavin compounds under similar conditions. The highest Φ298 K value of 0.70 was obtained in CH3 CN for isoalloxazine bearing C7-alloxan and N10-2,6-diisopropylphenyl groups. The temperature dependence of the emission intensities indicates that the pronounced emission properties at 298 K are attributable to the highly heat resistant properties towards emission decay with increasing temperature. Mechanistic studies, including X-ray diffraction analysis, revealed that the good emission properties and high heat resistance of the alloxan-substituted flavins are due to a synergetic effect of the associative nature of the C7-alloxan unit and the repulsive nature of the perpendicular bulky substituents at the C7 and N10 positions.

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Anaerobic green fluorescent protein as a marker of Bifidobacterium strains

Cited by 43 publications

References 39 publications

A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria

Highly Fluorescent Flavins: Rational Molecular Design for Quenching Protection Based on Repulsive and Attractive Control of Molecular Alignment

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