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Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Tucker AT, Nowicki EM, Boll JM, Knauf GA, Burdis NC, Trent MS, Davies BW - MBio (2014)

Bottom Line: Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques.Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products.This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.

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Biofilm formation assay of A. baumannii wild type, ΔpilUT::Kanr strain, ΔpilH::Kanr strain, and ΔpilG::Kanr strain, (A) Crystal violet staining of each strain in a PVC microtiter plate. (B) Biofilm formation measured by crystal violet staining for optical density at 540 nm (OD540). Asterisks denote significant differences in biofilm formation (t test; *, P < 0.0001; n = 8).
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fig4: Biofilm formation assay of A. baumannii wild type, ΔpilUT::Kanr strain, ΔpilH::Kanr strain, and ΔpilG::Kanr strain, (A) Crystal violet staining of each strain in a PVC microtiter plate. (B) Biofilm formation measured by crystal violet staining for optical density at 540 nm (OD540). Asterisks denote significant differences in biofilm formation (t test; *, P < 0.0001; n = 8).

Mentions: To explore the potential role of type IV pili in A. baumannii biofilm formation, we began by deleting the putative A. baumannii 17978 operon encoding homologs of the type IV pilus proteins PilU (A1S_0896) and PilT (A1S_0897) (see Fig. S2 in the supplemental material). PilU and PilT have been shown to be important for pilus function in A. baumannii and P. aeruginosa and have been implicated in pilus depolymerization and retraction (25). Using a crystal violet staining assay, we found that the pilUT mutant showed a marked decrease in biofilm formation compared to the parental strain (Fig. 4). Transmission electron microscopy showed type IV pili present on the surface of the parental A. baumannii 17978 strain but absent from our pilUT mutant (Fig. 5A and B). This supports the conclusion that fully functional type IV pili are important for biofilm production in this strain. Interestingly, a pilT mutant in A. nosocomialis strain M2 was shown to be defective for type IV-dependent natural transformation and twitching motility, indicating impaired pilus function, but it still produced visible pili on the cell surface (23). This suggests either strain variation in pilus formation between A. baumannii 17978 and M2 or that deleting both pilU and pilT is required to lose pilus formation on the cell surface.


Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Tucker AT, Nowicki EM, Boll JM, Knauf GA, Burdis NC, Trent MS, Davies BW - MBio (2014)

Biofilm formation assay of A. baumannii wild type, ΔpilUT::Kanr strain, ΔpilH::Kanr strain, and ΔpilG::Kanr strain, (A) Crystal violet staining of each strain in a PVC microtiter plate. (B) Biofilm formation measured by crystal violet staining for optical density at 540 nm (OD540). Asterisks denote significant differences in biofilm formation (t test; *, P < 0.0001; n = 8).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128354&req=5

fig4: Biofilm formation assay of A. baumannii wild type, ΔpilUT::Kanr strain, ΔpilH::Kanr strain, and ΔpilG::Kanr strain, (A) Crystal violet staining of each strain in a PVC microtiter plate. (B) Biofilm formation measured by crystal violet staining for optical density at 540 nm (OD540). Asterisks denote significant differences in biofilm formation (t test; *, P < 0.0001; n = 8).
Mentions: To explore the potential role of type IV pili in A. baumannii biofilm formation, we began by deleting the putative A. baumannii 17978 operon encoding homologs of the type IV pilus proteins PilU (A1S_0896) and PilT (A1S_0897) (see Fig. S2 in the supplemental material). PilU and PilT have been shown to be important for pilus function in A. baumannii and P. aeruginosa and have been implicated in pilus depolymerization and retraction (25). Using a crystal violet staining assay, we found that the pilUT mutant showed a marked decrease in biofilm formation compared to the parental strain (Fig. 4). Transmission electron microscopy showed type IV pili present on the surface of the parental A. baumannii 17978 strain but absent from our pilUT mutant (Fig. 5A and B). This supports the conclusion that fully functional type IV pili are important for biofilm production in this strain. Interestingly, a pilT mutant in A. nosocomialis strain M2 was shown to be defective for type IV-dependent natural transformation and twitching motility, indicating impaired pilus function, but it still produced visible pili on the cell surface (23). This suggests either strain variation in pilus formation between A. baumannii 17978 and M2 or that deleting both pilU and pilT is required to lose pilus formation on the cell surface.

Bottom Line: Analysis of A. baumannii cellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques.Here we have pioneered a novel recombineering system that facilitates efficient genome editing in A. baumannii by single PCR products.This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.

Show MeSH
Related in: MedlinePlus