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Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria.

Romero-Jiménez L, Rodríguez-Carbonell D, Gallegos MT, Sanjuán J, Pérez-Mendoza D - BMC Microbiol. (2015)

Bottom Line: One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria.Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility.

View Article: PubMed Central - PubMed

Affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. lorena.romero@eez.csic.es.

ABSTRACT

Background: The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.

Results: We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises "on demand".

Conclusions: mini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.

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Quantification of calcofluor-derived fluorescence of R. etli CFN42 (Ret), R. leguminosarum bv. viciae UPM791 (Rle), S. meliloti 8530 (Sme), and P. syringae pv.tomato DC3000 (Pto) expressing plasmid-encoded (pJBpleD*) or chromosomally integrated pleD* gene, and their respective control strains. Mean values from three independent cultures ± standard deviation
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Fig3: Quantification of calcofluor-derived fluorescence of R. etli CFN42 (Ret), R. leguminosarum bv. viciae UPM791 (Rle), S. meliloti 8530 (Sme), and P. syringae pv.tomato DC3000 (Pto) expressing plasmid-encoded (pJBpleD*) or chromosomally integrated pleD* gene, and their respective control strains. Mean values from three independent cultures ± standard deviation

Mentions: Similar to plasmid pJBpleD*, all mini-Tn7pleD* generated Congo Red (CR+) and Calcofluor (CF+) phenotypes (Additional file 1: Figure S1), with an enhanced CF-derived fluorescence (Fig. 3). These phenotypes were not observed in non-pleD* strains (Fig. 3 and Additional file 1: Figure S1). CR binds to D-glucopyranosyl units, basic or neutral polysaccharides, as well as to some proteins, whereas CF binds to β(1–4) and β(1–3) glycosidic bonds of polysaccharides [49]. In Pto, Ret and Rle the CR+ and CF+ stainings are likely due to overproduction of cellulose [13], whereas in S. meliloti 8530 this is due to another CR+/CF+ related polymer, a recently described mixed-linkage β-glucan [15]. Stronger CR and CF stainings were observed for the Pto Tn7pleD*Tc than for the Pto Tn7pleD*Km strain, in agreement with the higher c-di-GMP intracellular and pleD* expression levels in that transposant, as described above (Fig. 2 and Additional file 1: Figure S1).Fig. 3


Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria.

Romero-Jiménez L, Rodríguez-Carbonell D, Gallegos MT, Sanjuán J, Pérez-Mendoza D - BMC Microbiol. (2015)

Quantification of calcofluor-derived fluorescence of R. etli CFN42 (Ret), R. leguminosarum bv. viciae UPM791 (Rle), S. meliloti 8530 (Sme), and P. syringae pv.tomato DC3000 (Pto) expressing plasmid-encoded (pJBpleD*) or chromosomally integrated pleD* gene, and their respective control strains. Mean values from three independent cultures ± standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587759&req=5

Fig3: Quantification of calcofluor-derived fluorescence of R. etli CFN42 (Ret), R. leguminosarum bv. viciae UPM791 (Rle), S. meliloti 8530 (Sme), and P. syringae pv.tomato DC3000 (Pto) expressing plasmid-encoded (pJBpleD*) or chromosomally integrated pleD* gene, and their respective control strains. Mean values from three independent cultures ± standard deviation
Mentions: Similar to plasmid pJBpleD*, all mini-Tn7pleD* generated Congo Red (CR+) and Calcofluor (CF+) phenotypes (Additional file 1: Figure S1), with an enhanced CF-derived fluorescence (Fig. 3). These phenotypes were not observed in non-pleD* strains (Fig. 3 and Additional file 1: Figure S1). CR binds to D-glucopyranosyl units, basic or neutral polysaccharides, as well as to some proteins, whereas CF binds to β(1–4) and β(1–3) glycosidic bonds of polysaccharides [49]. In Pto, Ret and Rle the CR+ and CF+ stainings are likely due to overproduction of cellulose [13], whereas in S. meliloti 8530 this is due to another CR+/CF+ related polymer, a recently described mixed-linkage β-glucan [15]. Stronger CR and CF stainings were observed for the Pto Tn7pleD*Tc than for the Pto Tn7pleD*Km strain, in agreement with the higher c-di-GMP intracellular and pleD* expression levels in that transposant, as described above (Fig. 2 and Additional file 1: Figure S1).Fig. 3

Bottom Line: One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria.Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility.

View Article: PubMed Central - PubMed

Affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. lorena.romero@eez.csic.es.

ABSTRACT

Background: The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.

Results: We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises "on demand".

Conclusions: mini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.

Show MeSH
Related in: MedlinePlus