Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922
Abstract
:1. Introduction
2. Materials and Methods
2.1. Culture of E. coli and Electrocompetent Cells
2.2. Transposon Mutagenesis
2.3. Biofilm Analysis
2.4. Phenotypic Characterization of Mutants
2.4.1. Growth Curves
2.4.2. Swimming Assay
2.4.3. Congo Red Assay
2.4.4. Hemagglutination Assays
2.5. Genotypic Characterization of Mutants
2.5.1. DNA Sequencing
2.5.2. purL Gene Disruption
2.5.3. purL Gene Complementation
2.5.4. Effect of Inosine Addition to the Biofilm Formation Ability of the purL Mutant.
2.6. Proteomic Characterization of Mutants
2.6.1. Protein Isolation
2.6.2. Two-Dimensional SDS-PAGE Analysis
2.6.3. Gel Staining and Protein Detection
2.6.4. Mass-Spectrometry Analysis for Orbitrap
2.6.5. RNA Isolation and cDNA Synthesis
2.6.6. Gene Expression by qRT-PCR
2.7. Data Plotting and Statistical Analysis
3. Results
3.1. Mutant Selection by Biofilm Assays
3.2. Phenotypic Characterization of Mutants
3.2.1. Growth Curves in LB Broth
3.2.2. Swimming, Congo Red and Hemagglutination Assays
3.3. Genotypic Characterization of Mutants
3.3.1. DNA Sequencing
3.3.2. Confirmation of the Role of the purL Gene in Biofilm Formation Using Its Knockout Strain
3.4. Proteomic Characterization of Mutant Tn 263
3.4.1. Comparative Proteomic Analyses of the wt and Tn263 by Two-Dimensional SDS-PAGE
3.4.2. Verification of Biofilm-Related Genes by q-RT-PCR
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample | Swimming | Curli Production | Hemagglutination (Log2) |
---|---|---|---|
Tn5 | + | + | 10 |
Tn29 | + | + | 10 |
Tn42 | + | + | 10 |
Tn82 | + | + | 10 |
Tn90 | + | + | 10 |
Tn119 | + | + | 10 |
Tn143 | + | + | 10 |
Tn 249 | + | + | 10 |
Tn 251 | + | + | 10 |
Tn 262 | + | + | 10 |
Tn263 | + | - | 10 |
Tn 337 | + | + | 10 |
Tn 373 | + | + | 10 |
Tn 406 | + | + | 10 |
Tn 420 | + | + | 10 |
Tn 425 | + | + | 10 |
Tn 457 | + | + | 10 |
Tn 463 | + | - | 10 |
Tn 467 | + | + | 10 |
Tn 474 | + | + | 10 |
Log10 CFU/mL ± SD | Live/Dead | ||||
---|---|---|---|---|---|
Biofilm | Planktonic | Integrated Density | Live (% ± SD) | Dead (% ± SD) | |
wild type | 9.09 ± 0.24 | 8.99 ± 0.05 | 2.03 × 107 ± 4.86 × 106 | 85.40 ± 6.30 | 14.60 ± 6.30 |
Tn263 | 6.40 ± 0.51 | 6.96 ± 0.01 | 9.69 × 106 ± 2.79 × 106 | 86.32 ± 4.01 | 13.68 ± 4.01 |
ΔpurL::cat | 6.22 ± 0.44 | 7.02 ± 0.02 | 1.11 × 107 ± 2.34 × 106 | 80.38 ± 6.88 | 19.62 ± 6.88 |
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Cepas, V.; Ballén, V.; Gabasa, Y.; Ramírez, M.; López, Y.; Soto, S.M. Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922. Pathogens 2020, 9, 774. https://doi.org/10.3390/pathogens9090774
Cepas V, Ballén V, Gabasa Y, Ramírez M, López Y, Soto SM. Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922. Pathogens. 2020; 9(9):774. https://doi.org/10.3390/pathogens9090774
Chicago/Turabian StyleCepas, Virginio, Victoria Ballén, Yaiza Gabasa, Miriam Ramírez, Yuly López, and Sara Mª Soto. 2020. "Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922" Pathogens 9, no. 9: 774. https://doi.org/10.3390/pathogens9090774