Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis
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 diffe...
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| Format: | Article |
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Frontiers Media S.A.
2017-07-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fmicb.2017.01405/full |
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| author | Nuria Castro-Bravo Claudio Hidalgo-Cantabrana Miguel A. Rodriguez-Carvajal Patricia Ruas-Madiedo Abelardo Margolles |
| author_facet | Nuria Castro-Bravo Claudio Hidalgo-Cantabrana Miguel A. Rodriguez-Carvajal Patricia Ruas-Madiedo Abelardo Margolles |
| author_sort | Nuria Castro-Bravo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T20:30:33Z |
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| id | doaj.art-d4e42f91537846a1b78190d3a7f4766e |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-14T20:30:33Z |
| publishDate | 2017-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-d4e42f91537846a1b78190d3a7f4766e2022-12-21T22:48:32ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2017-07-01810.3389/fmicb.2017.01405286215Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactisNuria Castro-Bravo0Claudio Hidalgo-Cantabrana1Miguel A. Rodriguez-Carvajal2Patricia Ruas-Madiedo3Abelardo Margolles4Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones CientíficasVillaviciosa, SpainDepartment of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones CientíficasVillaviciosa, SpainDepartment of Organic Chemistry, Universidad de SevillaSevilla, SpainDepartment of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones CientíficasVillaviciosa, SpainDepartment of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones CientíficasVillaviciosa, SpainAn 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.http://journal.frontiersin.org/article/10.3389/fmicb.2017.01405/fullBifidobacteriumexopolysaccharidegene replacementfluorescent proteinsNMRSEC-MALS |
| spellingShingle | Nuria Castro-Bravo Claudio Hidalgo-Cantabrana Miguel A. Rodriguez-Carvajal Patricia Ruas-Madiedo Abelardo Margolles Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis Frontiers in Microbiology Bifidobacterium exopolysaccharide gene replacement fluorescent proteins NMR SEC-MALS |
| title | Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis |
| title_full | Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis |
| title_fullStr | Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis |
| title_full_unstemmed | Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis |
| title_short | Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis |
| title_sort | gene replacement and fluorescent labeling to study the functional role of exopolysaccharides in bifidobacterium animalis subsp lactis |
| topic | Bifidobacterium exopolysaccharide gene replacement fluorescent proteins NMR SEC-MALS |
| url | http://journal.frontiersin.org/article/10.3389/fmicb.2017.01405/full |
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