Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes
Abstract
:1. Introduction
2. Results and Discussion
2.1. Whole-Cell Proteomic Response to GraT
2.2. Proteome Changes Indicating the Cellular Response to the Ribosome Biogenesis Defect of the ΔgraA Strain
2.3. Proteome Changes Reveal Altered Carbon Metabolism of the ΔgraA Strain
2.4. Metabolomic Analysis Indicates Accumulation of Oxaloacetate in the ΔgraA Strain
3. Conclusions
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
4.2. Construction of Plasmids and Strains
4.3. Proteomics
4.4. Metabolomics
4.5. Temperature Tolerance Plate Assay
4.6. Sucrose Gradient Profiles
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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ΔgraA vs. WT, 30 °C | ||||
---|---|---|---|---|
Locus | Gene | Protein | Fold Change * | p-Value |
PP_2645 | mgtA | Magnesium transporter, ATP-dependent | −2.19 | 0.00016 |
PP_2487 | aldA | Putative aldehyde dehydrogenase | −2.01 | 0.00028 |
PP_3760 | cheR3 | Putative methyltransferase CheR3 | 2.23 | 0.00024 |
PP_1868 | deaD | ATP-dependent RNA helicase DeaD | 2.52 | 0.00017 |
PP_1433 | rnc | Ribonuclease 3 (RNase III) | 2.55 | 0.00043 |
PP_2909 | csiD | Protein CsiD | 4.06 | 0.00022 |
ΔgraA vs. WT, 25 °C | ||||
PP_2486 | NADH-dependent flavin oxidoreductase, Oye family | −3.16 | 0.00001 | |
PP_0988 | gcvP1 | Glycine dehydrogenase (decarboxylating) 1 | −3.16 | 0.00013 |
PP_4012 | idh | Isocitrate dehydrogenase | −2.87 | 0.00002 |
PP_4185 | sucD | Succinate--CoA ligase [ADP-forming] subunit alpha | −2.52 | 0.00005 |
PP_4189 | sucA | 2-oxoglutarate decarboxylase, thiamine-requiring E1 subunit | −2.12 | 0.00017 |
PP_1245 | Uncharacterized protein | −2.01 | 0.00018 | |
PP_1800 | rmd | Oxidoreductase Rmd | 2.01 | 0.00001 |
PP_0637 PP_4025 PP_4745 | tnpA | Transposase | 2.17 | 0.00003 |
PP_1868 | deaD | ATP-dependent RNA helicase DeaD | 3.08 | 2.57E-06 |
PP_1236 | Putative Glycine cleavage system transcriptional repressor | 3.68 | 0.00003 | |
PP_5270 | dadA2 | D-amino acid dehydrogenase 2 | 5.01 | 0.0003 |
PP_1835 | Uncharacterized protein | 8.79 | 0.00014 | |
PP_2909 | csiD | Protein CsiD | 11.08 | 0.00007 |
Strain or Plasmid | Genotype or Characteristics | Source or Reference |
---|---|---|
E. coli strains | ||
DH5α λpir | λpir lysogen of DH5α | [90] |
P. putida strains | ||
PaW85 | Wild-type, isogenic to KT2440 | [87] |
ΔgraA | graA is deleted from PaW85 | [46] |
ΔdeaD | deaD (PP_1868) is deleted from PaW85 | This study |
ΔgraA ΔdeaD | deaD is deleted from ΔgraA strain | This study |
ΔcsiD | csiD (PP_2909) is deleted from PaW85 | This study |
ΔgraA ΔcsiD | csiD is deleted from ΔgraA strain | This study |
ΔlhgO | lhgO (PP_2910) is deleted from PaW85 | This study |
ΔgraA ΔlhgO | lhgO is deleted from ΔgraA strain | This study |
Plasmids | ||
pEMG | Suicide plasmid containing lacZα with two flanking I-SceI sites (Kmr) | [90] |
pSW(I-SceI) | For I-SceI endonuclease expression (Apr) | [91] |
pEMG-ΔdeaD | pEMG with a PCR-designed 1180 bp KpnI-SacI insert for deleting the deaD gene (Kmr) | This study |
pEMG-ΔcsiD | pEMG with a PCR-designed 1200 bp EcoRI-BamHI insert for deleting the csiD gene (Kmr) | This study |
pEMG-ΔlhgO | pEMG with a PCR-designed 1158 bp EcoRI-BamHI insert for deleting the lhgO gene (Kmr) | This study |
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Ainelo, A.; Porosk, R.; Kilk, K.; Rosendahl, S.; Remme, J.; Hõrak, R. Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes. Toxins 2019, 11, 103. https://doi.org/10.3390/toxins11020103
Ainelo A, Porosk R, Kilk K, Rosendahl S, Remme J, Hõrak R. Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes. Toxins. 2019; 11(2):103. https://doi.org/10.3390/toxins11020103
Chicago/Turabian StyleAinelo, Andres, Rando Porosk, Kalle Kilk, Sirli Rosendahl, Jaanus Remme, and Rita Hõrak. 2019. "Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes" Toxins 11, no. 2: 103. https://doi.org/10.3390/toxins11020103
APA StyleAinelo, A., Porosk, R., Kilk, K., Rosendahl, S., Remme, J., & Hõrak, R. (2019). Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes. Toxins, 11(2), 103. https://doi.org/10.3390/toxins11020103