The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa
Abstract
:1. Introduction
2. Results and Discussion
2.1. PA14_27940 Deletion Renders Hypersusceptibility to Several Antibiotics in P. aeruginosa
2.2. PA14_27940 Deletion Alters the Motility of P. aeruginosa
2.3. Effect of PA14_27940 in the Production of P. aeruginosa Virulence Determinants
2.4. The Loss of PA14_27940 Impedes the Induction of armZ, Hence Reducing the Induction of mexXY by Tobramycin
2.5. Fosfomycin Hypersusceptibility of the ΔPA14_27940 Mutant Is Due to the Reduced Expression of the Genes Encoding the Alternative Peptidoglycan Recycling Pathway
3. Materials and Methods
3.1. Bacterial Strains, Plasmids and Oligonucleotides
3.2. Bacterial Growth Conditions
3.3. Construction of ΔPA14_27940 P. aeruginosa Mutant
3.4. Whole-Genome Sequencing and Bioinformatics Analysis
3.5. Analysis of Susceptibility to Antibiotics
3.6. Motility Assays
3.7. Elastase Activity and Pyocyanin Production
3.8. Biofilm Formation
3.9. Quantification of Intracellular Fosfomycin
3.10. RNA Extraction and qRT-PCR
3.11. Statistical Analyses
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Antibiotic | MIC (µg/mL) | |
---|---|---|
PA14 | ΔPA14_27940 | |
TOB | 0.75 | 0.19 * |
STR | 6 | 3 * |
AMK | 1.5 | 0.25 * |
KAN | 32 | 6 * |
TGC | 3 | 1 * |
TET | 24 | 12 * |
CIP | 0.032 | 0.032 |
CAZ | 0.5 | 0.5 |
IPM | 0.75 | 0.75 |
ATM | 3 | 2 |
FOF | 16 | 4 * |
ERY | 96 | 32 * |
CHL | 32 | 24 |
CS | 1 | 1 |
PB | 1 | 1 |
Bacterial Strains | Description | Reference/Origin |
---|---|---|
Escherichia coli DH5α | Strain used for cloning | Laboratory collection |
E. coli S17-1 | Donor strain for conjugation | [41] |
Pseudomonas aeruginosa PA14 | Laboratory model strain of P. aeruginosa | Laboratory collection |
ΔPA14_27940 | Mutant of P. aeruginosa PA14 with PA14_27940 deletion | This study |
Plasmids | Description | Origin |
---|---|---|
pGEM®-T Easy | Commercial plasmid used for cloning optimization of PCR products | Promega (Madison, WI, USA) |
pEX18Ap | Cloning vector | [42] |
RGD001 | pEX18Ap containing 500 bp upstream and 500 bp downstream of the gene PA14_27940 | This study |
Oligonucleotides | Sequence 5′3′ | Utilization |
PA14_27940.ups_fw | AAGCTGGGCTTCCAGTTCCAGGC | Mutant construction |
PA14_27940.ups_rv | TTACCGGTACTCATGCAAGGCTTTATGCATGGGTCATCCA | Mutant construction |
PA14_27940.dns_fw | TGGATGACCCATGCATAAAGCCTTGCATGAGTACCGGTAA | Mutant construction |
PA14_27940.dns_rv | AAGCTAAGGGATCGACCGGTTAA | Mutant construction |
PA14_27940.comp_fw | GGCCTGCAGATCTTCGAAAG | Mutant construction |
PA14_27940.comp_rv | GTCACCGGAAGCATGTTCAT | Mutant construction |
rplU_fw | CGCAGTGATTGTTACCGGTG | qRT-PCR |
rplU_rv | AGGCCTGAATGCCGGTGATC | qRT-PCR |
mexX_fw | GTACGAGGAAGGCCAGGAC | qRT-PCR |
mexX_rv | CTTGATCAGGTCGGCGTAG | qRT-PCR |
M13_fw | CGCCAGGGTTTTCCCAGTCACGAC | Sequencing |
M13_rv | CAGGAAACAGCTATGAC | Sequencing |
armZ_RT_F | ATCCTGCAAGAGCATGTCA | qRT-PCR |
armZ_RT_R | GACGTCGAGCAGTTCCAG | qRT-PCR |
agmK_RT_F | AGCTGAATCGCTGGTTGGAC | qRT-PCR |
agmK_RT_R | AACGGTCGGCAGTCTTCCTG | qRT-PCR |
anmK_RT_F | CAACGTGCTGATGGACGCCT | qRT-PCR |
anmK_RT_R | AGCCAGGACAGGTTGAAGCG | qRT-PCR |
nagZ_RT_F | AGGTGGGCGGGCTGATCATCTT | qRT-PCR |
nagZ_RT_R | ATTGGGGTTGTCGGCGATCG | qRT-PCR |
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Genova, R.; Gil-Gil, T.; Cuesta, T.; Martínez, J.L.; Sanz-García, F. The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa. Int. J. Mol. Sci. 2023, 24, 17355. https://doi.org/10.3390/ijms242417355
Genova R, Gil-Gil T, Cuesta T, Martínez JL, Sanz-García F. The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa. International Journal of Molecular Sciences. 2023; 24(24):17355. https://doi.org/10.3390/ijms242417355
Chicago/Turabian StyleGenova, Roberta, Teresa Gil-Gil, Trinidad Cuesta, José Luis Martínez, and Fernando Sanz-García. 2023. "The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa" International Journal of Molecular Sciences 24, no. 24: 17355. https://doi.org/10.3390/ijms242417355