Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence
Abstract
:1. Introduction
2. Results
2.1. In Silico Identification of Putative ADH Genes in A. baumannii
2.2. Biochemical Properties of ADH4
2.3. Biological Function of ADH3, ADH4, and ADH6
2.4. Gene Expression Patterns of Wild-Type and Mutant Strains in the Presence of Ethanol
2.5. Adh3 and Adh6 Are Involved in Inorganic and Organic Oxidative Stress Responses
2.6. The Adh3 Stress Resistance Response Does Not Disrupt Homeostasis of Cytosolic NADH/NAD+
2.7. Adh3 and Adh6 Are Associated with Virulence against G. mellonella
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids, and Primers
4.2. Marker-Less Mutation
4.3. Recombinant Alcohol Dehydrogenase Purification and Enzyme Activity Assay
4.4. Stress Resistance Assays
4.5. RNA Extraction and qRT-PCR
4.6. Fluorescence Analysis
4.7. Virulence Assay with G. mellonella
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Carbonate-Bicarbonate (CB) Buffer, pH = 10.1 | |||
---|---|---|---|
Ethanol | 1-Propanol | 1-Butanol | |
Vmax (mmole min−1) | 0.0064 ± 0.0002 | 0.027 ± 0.0028 | 0.005 ± 0.001 |
KM (mM) | 5.11 ± 0.71 | 206.8 ± 79.61 | 196.5 ± 91.21 |
kcat (s−1) | 0.2118 | 0.9003 | 0.1683 |
kcat/KM (s−1M−1) | 41.4481 | 4.3535 | 0.8565 |
Name | Sequences (5′–3′) | Function |
---|---|---|
pK18_Adh3upF | CGAGCTCGGTACCCGGGACGCCCTTTAACATGACCAG | Construction of Adh3 mutant |
pK18_Adh3downR | AACGACGGCCAGTGCCACAGATGCGCTAAGGGAAAAC | Construction of Adh3 mutant |
pK18_Adh4upF | CGAGCTCGGTACCCGGGTGCCCTTCATTATCAATTTCG | Construction of Adh4 mutant |
pK18_Adh4downR | AACGACGGCCAGTGCCAGACATCGCTTTGAGTTGCAT | Construction of Adh4 mutant |
pK18_Adh6upF | CGAGCTCGGTACCCGGGACGCACATTGGTCAGTTTTG | Construction of Adh6 mutant |
pK18_Adh6downR | AACGACGGCCAGTGCCATTGCTGCAACCATAACAGGT | Construction of Adh6 mutant |
Adh1_rF | TGTGATTGCCTGTGGTGAAT | qRT-PCR |
Adh1_rR | ACACCGCCGTAAAGATGACT | qRT-PCR |
Adh2_rF | GGTCGATTCATGCCGTACTT | qRT-PCR |
Adh2_rR | TGTGGTAATACCCGCACAAA | qRT-PCR |
Adh3_rF | TCAGTTACACCTGCCTATTCTTCA | qRT-PCR |
Adh3_rR | CCCAAAGCCGACAATAACAT | qRT-PCR |
Adh4_rF | TGCAAGATGAAGGGCTATTT | qRT-PCR |
Adh4_rR | CACCGCCTAACGACACAATA | qRT-PCR |
Adh5_rF | GCCAGCAGATAAAGCGGATT | qRT-PCR |
Adh5_rR | TGTTGCCCCATATACATTACCA | qRT-PCR |
Adh6_rF | TCTGGTGCACACAACCTACC | qRT-PCR |
Adh6_rR | TCTAAAATCGCAGCATGTGG | qRT-PCR |
Adh7_rF | GGCGAAAATATCGCAACAAT | qRT-PCR |
Adh7_rR | ACCCAAACCACCAATACCAA | qRT-PCR |
pRsetB-His7-Peredox-mCherry_F | GCCCTTTCGTCTTCAAGTAATACGACTCACTATAGGG | Construction of peredox plasmid |
pRsetB-His7-Peredox-mCherry_R | AGCTGTCAAACATGAGTCACTTGTACAGTTCGTCCA | Construction of peredox plasmid |
Plasmid | Description | Antibiotic Resistance (µg/mL) | Reference/Source |
---|---|---|---|
pK18mobsacB | Suicide vector for homologous recombination | Kan50 | [31] |
pK18ΔAdh1 | pK18mobsacB contains the upstream and downstream regions of Adh1 | Kan50 | This study |
pK18ΔAdh2 | pK18mobsacB contains the upstream and downstream regions of Adh2 | Kan50 | This study |
pK18ΔAdh3 | pK18mobsacB contains the upstream and downstream regions of Adh3 | Kan50 | This study |
pK18ΔAdh4 | pK18mobsacB contains the upstream and downstream regions of Adh4 | Kan50 | This study |
pK18ΔAdh5 | pK18mobsacB contains the upstream and downstream regions of Adh5 | Kan50 | This study |
pK18ΔAdh6 | pK18mobsacB contains the upstream and downstream regions of Adh6 | Kan50 | This study |
pK18ΔAdh7 | pK18mobsacB contains the upstream and downstream regions of Adh7 | Kan50 | This study |
pQE80L | Expression vector with colE1 origin for His-tag fusion protein purification | Amp50 | Qiagen |
pAdh3 | Apr; Adh3 cloned into BamHI-HindIII sites of pQE80L | Amp50 | This study |
pAdh4 | Apr; Adh4 cloned into BamHI-HindIII sites of pQE80L | Amp50 | This study |
pAdh6 | Apr; Adh6 cloned into BamHI-SmaI sites of pQE80L | Amp50 | This study |
pWH1266 | Apr; Tcr; shuttle vector for E. coli and A. baumannii | Amp50, Tc12.5 | [32] |
pRsetB-His7tag- Peredox-mCherry | Apr; fluorescent biosensor of the cytosolic NADH/NAD+ redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex, and the red fluorescence of a tandemly attached mCherry. | Amp50 | [33,34] |
pWH1266_peredox-mCherry | Apr; Tcr; permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex, and the red fluorescence of a tandemly attached mCherry from pRsetB-His7tag- Peredox-mCherry was cloned into EcoRI sites of pWH1266 | Amp50, Tc12.5 | This study |
Strain | Description | Reference/Source | |
E. coli DH5α | F−, supE44, hsdR17, recA1, gyrA96, endA1, thi-1, relA1, deoR, λ− | ATCC53868 | |
Acinetobacter baumannii ATCC 19606 | Primary strain used in this study | [31] | |
∆Adh3(∆3) | Marker-less Adh3 deletion mutant | This study | |
∆Adh4(∆4) | Marker-less Adh4 deletion mutant | This study | |
∆Adh6(∆6) | Marker-less Adh6 deletion mutant | This study | |
∆Adh34(∆34) | Marker-less Adh34 double deletion mutant | This study | |
∆Adh36(∆36) | Marker-less Adh36 double deletion mutant | This study | |
∆Adh46(∆46) | Marker-less Adh46 double deletion mutant | This study | |
∆Adh346(∆346) | Marker-less Adh346 triple deletion mutant | This study |
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Lin, G.-H.; Hsieh, M.-C.; Shu, H.-Y. Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence. Int. J. Mol. Sci. 2021, 22, 9921. https://doi.org/10.3390/ijms22189921
Lin G-H, Hsieh M-C, Shu H-Y. Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence. International Journal of Molecular Sciences. 2021; 22(18):9921. https://doi.org/10.3390/ijms22189921
Chicago/Turabian StyleLin, Guang-Huey, Ming-Chuan Hsieh, and Hung-Yu Shu. 2021. "Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence" International Journal of Molecular Sciences 22, no. 18: 9921. https://doi.org/10.3390/ijms22189921