Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa
Abstract
1. Introduction
2. Materials and Methods
2.1. DNA, Plasmids, and Bacterial Cultures
2.2. Plasmid Construction
2.3. Plasmid-Based ts-Mutant Strain Construction
2.4. Spot-Plating Assay
2.5. Fluorescence Microscopic Analysis
2.6. Fluorescence Activated Cell Sorting (FACS) Analysis
2.7. ATP Content Measurement
2.8. RNA Extraction and RNA-Seq Analysis
2.9. Statistics
2.10. Data Availability
3. Results
3.1. pa0665 Gene Is Essential for Growth on LB-Agar Plate
3.2. Putative Ortholog erpA in E. coli Functionally Complements the Defect of pa0665 in P. aeruginosa
3.3. The Δpa0665/pTS-pa0665 Mutant Exhibits Petite Cell Morphology under Restrictive Temperature
3.4. ATP Content Was Decreased in Δpa0665/pTS-pa0665 Mutant under Restrictive Temperature
3.5. The Δpa0665/pTS-pa0665 Mutant Is Hypersensitive to Oxidative Stress Mediated by H2O2
3.6. Transcriptomic Analysis Reveals Impaired Oxidative Phosphorylation in pa0665-Deficient P. aeruginosa
3.7. Impairment of pa4067/oprG Possibly Linked to the Altered Morphology of Δpa0665/pTS-pa0665 Mutant at 42 °C
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Oligonucleotides | ||
---|---|---|
Name | Sequence (5′-3′) | Usage |
F1 | CTGGAACTGCCTGCCAGCGT | Assay pa0665 alleles in chr and TS plasmid |
R1 | CGGCAACTGCCCTGATGTGA | Ditto |
F2 | CTCCGGCATTTCCAGTCGAT | Assay pa0665 alleles in chr but not TS plasmid |
R2 | AGGTGAACCACGCACTGCTG | Ditto |
Plasmids | Relevant genotype | Reference |
pDEL | pUC-Gmr-sacB | [10,11] |
pRES or pTS | pUC-Tcr-orits | [10,11] |
pOE | pBBRMCS-5-araC-PBAD-Gmr | [10,11] |
pDEL-pa0665 | pa0665 deletion cassette in pDEL | This study |
pRES-pa0665 | pa0665 rescue cassette in pTS | This study |
pOE-pa0665 | araC-PBAD- pa0665 in pOE | This study |
pOE-ec.erpA | araC-PBAD-ec.erpA in pOE | This study |
Strains | Rel genotype/Usage | Reference |
PAO1 | Wild type | [10,11] |
Δpa0665/pTS-pa0665 | pa0665 ts-allele | This study |
Δpa0665/pTS-pa0665/pOE-pa0665 | pa0665-OE in ts | This study |
Δpa0665/pTS-pa0665/pOE-ec.erpA | ec.erpA-OE in ts | This study |
Δpa0665/pTS-pa0665/pOE | pOE in ts | This study |
Δpa4067/oprG | pa4067-deletion | This study |
Δpa3337/rfaD | pa3337-deletion | This study |
wt/pOE-pa0665 | pa0665-OE in wt | This study |
wt/pOE-ec.erpA | ec.erpA-OE in wt | This study |
wt/pOE | pOE in wt | This study |
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Zhu, J.; Zhao, H.; Yang, Z. Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa. Genes 2024, 15, 590. https://doi.org/10.3390/genes15050590
Zhu J, Zhao H, Yang Z. Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa. Genes. 2024; 15(5):590. https://doi.org/10.3390/genes15050590
Chicago/Turabian StyleZhu, Jiayin, Hulin Zhao, and Zhili Yang. 2024. "Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa" Genes 15, no. 5: 590. https://doi.org/10.3390/genes15050590
APA StyleZhu, J., Zhao, H., & Yang, Z. (2024). Genetic Analysis of the ts-Lethal Mutant Δpa0665/pTS-pa0665 Reveals Its Role in Cell Morphology and Oxidative Phosphorylation in Pseudomonas aeruginosa. Genes, 15(5), 590. https://doi.org/10.3390/genes15050590