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O serotype-independent susceptibility of Pseudomonas aeruginosa to lectin-like pyocins.

Ghequire MG, Dingemans J, Pirnay JP, De Vos D, Cornelis P, De Mot R - Microbiologyopen (2014)

Bottom Line: The recombinant proteins exhibit species-specific antagonistic activities down to nanomolar concentrations against clinical and environmental P. aeruginosa strains, including several multidrug-resistant isolates.No correlation was found between L pyocin susceptibility and phylogenetic relatedness of P. aeruginosa isolates.Sensitive strains were retrieved in 13 out of 15 O serotypes tested, excluding the possibility that the highly variable and immunogenic O serotype antigen of the LPS coating would represent a dominant susceptibility-discriminating factor.

View Article: PubMed Central - PubMed

Affiliation: Centre of Microbial and Plant Genetics, University of Leuven, 3001, Heverlee, Belgium.

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Purification and activity of L pyocins. (A) SDS-PAGE electrophoresis of purified pyocins. Lane 1, size marker (kDa); lane 2, PyoL1; lane 3, PyoL2; lane 4, PyoL3. (B–D) Bacteriocin activity of a 5-fold dilution series (starting concentration 2 mg/mL, left halo) of PyoL1 (B), PyoL2 (C) and PyoL3 (D) against indicator strains Pseudomonas aeruginosa Bu002 (B), A19 (C) and Pr335 (D).
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fig03: Purification and activity of L pyocins. (A) SDS-PAGE electrophoresis of purified pyocins. Lane 1, size marker (kDa); lane 2, PyoL1; lane 3, PyoL2; lane 4, PyoL3. (B–D) Bacteriocin activity of a 5-fold dilution series (starting concentration 2 mg/mL, left halo) of PyoL1 (B), PyoL2 (C) and PyoL3 (D) against indicator strains Pseudomonas aeruginosa Bu002 (B), A19 (C) and Pr335 (D).

Mentions: In addition to PyoL1, its most diverged group member, protein P997_04049 (PyoL2 from strain 62) and protein Q020_01808 (PyoL3 from strain BWHSPA007) as a representative for the second cluster (Fig. 1A), were cloned in pET28(a) and expressed in E. coli BL21(DE3). For convenient purification, a sequence encoding a His6-tag was incorporated at the 5′-end of the respective pyocin coding regions since amino-terminal hexa-His fusions have no detectable effect on activity or specificity of recombinant LlpA proteins, contrary to carboxy-terminal fusions in some cases (Parret et al. 2004; Ghequire et al. 2012b). For Q020_01808, the tag was immediately fused to the predicted mature protein without signal peptide. Recombinant proteins were purified by affinity chromatography on Ni-NTA and polished by gel filtration. The calculated molecular weights of PyoL1 (30,445 Da), PyoL2 (30,445 Da), and PyoL3 (31,945 Da) match well with the apparent sizes of the respective recombinant proteins as estimated by SDS-PAGE: 29.0, 28.1 and 30.9 kDa, respectively (Fig. 3A).


O serotype-independent susceptibility of Pseudomonas aeruginosa to lectin-like pyocins.

Ghequire MG, Dingemans J, Pirnay JP, De Vos D, Cornelis P, De Mot R - Microbiologyopen (2014)

Purification and activity of L pyocins. (A) SDS-PAGE electrophoresis of purified pyocins. Lane 1, size marker (kDa); lane 2, PyoL1; lane 3, PyoL2; lane 4, PyoL3. (B–D) Bacteriocin activity of a 5-fold dilution series (starting concentration 2 mg/mL, left halo) of PyoL1 (B), PyoL2 (C) and PyoL3 (D) against indicator strains Pseudomonas aeruginosa Bu002 (B), A19 (C) and Pr335 (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263511&req=5

fig03: Purification and activity of L pyocins. (A) SDS-PAGE electrophoresis of purified pyocins. Lane 1, size marker (kDa); lane 2, PyoL1; lane 3, PyoL2; lane 4, PyoL3. (B–D) Bacteriocin activity of a 5-fold dilution series (starting concentration 2 mg/mL, left halo) of PyoL1 (B), PyoL2 (C) and PyoL3 (D) against indicator strains Pseudomonas aeruginosa Bu002 (B), A19 (C) and Pr335 (D).
Mentions: In addition to PyoL1, its most diverged group member, protein P997_04049 (PyoL2 from strain 62) and protein Q020_01808 (PyoL3 from strain BWHSPA007) as a representative for the second cluster (Fig. 1A), were cloned in pET28(a) and expressed in E. coli BL21(DE3). For convenient purification, a sequence encoding a His6-tag was incorporated at the 5′-end of the respective pyocin coding regions since amino-terminal hexa-His fusions have no detectable effect on activity or specificity of recombinant LlpA proteins, contrary to carboxy-terminal fusions in some cases (Parret et al. 2004; Ghequire et al. 2012b). For Q020_01808, the tag was immediately fused to the predicted mature protein without signal peptide. Recombinant proteins were purified by affinity chromatography on Ni-NTA and polished by gel filtration. The calculated molecular weights of PyoL1 (30,445 Da), PyoL2 (30,445 Da), and PyoL3 (31,945 Da) match well with the apparent sizes of the respective recombinant proteins as estimated by SDS-PAGE: 29.0, 28.1 and 30.9 kDa, respectively (Fig. 3A).

Bottom Line: The recombinant proteins exhibit species-specific antagonistic activities down to nanomolar concentrations against clinical and environmental P. aeruginosa strains, including several multidrug-resistant isolates.No correlation was found between L pyocin susceptibility and phylogenetic relatedness of P. aeruginosa isolates.Sensitive strains were retrieved in 13 out of 15 O serotypes tested, excluding the possibility that the highly variable and immunogenic O serotype antigen of the LPS coating would represent a dominant susceptibility-discriminating factor.

View Article: PubMed Central - PubMed

Affiliation: Centre of Microbial and Plant Genetics, University of Leuven, 3001, Heverlee, Belgium.

Show MeSH
Related in: MedlinePlus