Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Plasmids
2.2. Mutants’ Constructions
2.3. Phenotypic Characterization of the Mutants
2.3.1. Plant Nodulation Assay
2.3.2. Motility, Tolerance to Salt, Detergent, Acid and Oxidative Stresses
2.4. Transcirptional Fusion Assays
3. Results and Discussion
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain/Plasmid Name | Description | Source |
---|---|---|
Bacterial strains | ||
LMR001T | Ensifer aridi LMR001T isolated from Vachellia gummifera grown in Merzouga desert sand dune | [4] |
LMR001∆nesR | nesR deletion mutant of LMR001 | This work |
LMR001∆sahR | sahR deletion mutant of LMR001 | This work |
LMR001∆nesR∆sahR | nesR deletion mutant of LMR001∆sahR | This work |
LMR001∆iolR | iolR deletion mutant of LMR001 | This work |
LMR001∆thuR | thuR deletion mutant of LMR001 | This work |
LMR001∆otsB | otsB deletion mutant of LMR001 | This work |
E. coli DH5α | hsdR17 endA1 thi-1 gyrA96 relA1 recA1 supE44 DlacU169 (f80lacZDM15) | [24] |
E. coli S17-1 | recA [SmR], thi, pro, RP4-2-Tc:Mu: aph::Tn7λpir. | [25] |
E. coli XL2 Blue Ultra-competent | endA1 supE44 thi-1 hsdR17 recA1 gyrA96 relA1 lac [F’ proAB lacIqZΔM15 Tn10 (TetR) Amy CamR] | Stratagene |
Plasmids | ||
pJQ200SK | pACYC184-derived (p15A) suicide vector (GmR) | [26] |
pRK2013 | Helper plasmid containing the ColE1 replicon with RK2 tra genes (KmR) | [27] |
pGEM-T Easy | pUC origin, Multi Cloning Sites (MCS), lacZ gene fusion, β-lactamase coding region, lac operon sequences (ApR) | Promega |
pBluescript KS(+) | ColEI, F1 Origin, MCS, lacZ gene fusion, β-lactamase coding region, lac operon sequences (ApR) | Stratagene |
TOPO vecteur | pUC origin, région Plac promoteur, lacZ α-ccdB gene fuision. Topoisomerase enzyme (KmR, ApR) | Invitrogen |
pPROBE-NT | pVS1-derived (p15a) vector, gfp (GmR) | [28] |
pNT-sahRp | pPROBE NT containing sahR (PEG2239) promoter fused to gfp (KmR) | [15] |
pNT-nesRp | pPROBE NT containing nesR (PEG5735) promoter fused to gfp (KmR) | [15] |
pNT-rsiA1p | pPROBE NT containing rsiA1 (PEG2540) promoter fused to gfp (KmR) | [15] |
pNT-rsiB1p | pPROBE NT containing rsiB1 (PEG2541) promoter fused to gfp (KmR) | [15] |
pNT-thuEp | pPROBE NT containing thuE (PEG6268) promoter fused to gfp (KmR) | [15] |
pNT-otsBp | pPROBE NT containing otsB (PEG4868) promoter fused to gfp (KmR) | [15] |
pNT-thuRp | pPROBE NT containing thuR (PEG6269) promoter fused to gfp (KmR) | [15] |
pNT-iolCp | pPROBE NT containing iolC (PEG373) promoter fused to gfp (KmR) | [15] |
pNT-iolRp | pPROBE NT containing iolR (PEG374) promoter fused to gfp (KmR) | [15] |
Target Gene (bp) | Primer * | Sequence (5′-3′) * | 5′ Region Size (bp)/Distance to Start (nt) | 3′ Region Size (bp)/Distance to Stop (nt) | Deletion (bp) |
---|---|---|---|---|---|
iolR (861) | IOLR-P374-A-XI | CCTCTAGACGAGATTTACGGCTCCAAGG | 457/−10 | 689 | |
IOLR-P374-B-HIII | TCAAAGACACCACAAGCTTCTCCAGCTTGCTCGTGTTC | ||||
IOLR-P374-C-HIII | AAGCTTGTGGTGTCTTTGACCGACTC | 658/−183 | |||
IOLR-P374-D-XI | CCTCTAGAGCAATTGTCGCGATAGAAGA | ||||
otsB (750) | OSTB-P4868-A-XI | GGTCTAGATGCGCGATCTTCATGAACAA | 454/+75 | 503 | |
OSTB-P4868-B-HIII | TTCATCCGTCAGAAAGCTTGTCGGAGCAATGTCGAGAAG | ||||
OSTB-P4868-C-HIII | AAGCTTTCTGACGGATGAAGGAATGTT | 500/−172 | |||
OSTB-P4868-D-XI | GGTCTAGAATAGCCGGACATCTCATGCC | ||||
thuR (1020) | THUR-P6269-A-XI | GGTCTAGAGGTCCTCAGAAGCATTGTCA | 473/+87 | 865 | |
THUR-P6269-B-HIII | GTTCGTGAATCTCAAGCTTCTCGCTGACCTCGGGATAG | ||||
THUR-P6269-C-HIII | AAGCTTGAGATTCACGAACTCTGGCC | 453/−69 | |||
THUR-P6269-D-XI | GGTCTAGATAGGTGCGATGAACATGACG | ||||
nesR (783) | NESR-P5735-A-XI | CGCTCTAGAGGCTCGATATCACGCCAC | 186/+39 | 749 | |
NESR-P5735-B | TTAAGCCGCGGCGGTGAACTGGTTCCTGATC | ||||
NESR-P5735-C | GCCGCGGCTTAAAACACC | 160/+6 | |||
NESR-P5735-D-XI | GCGTCTAGAACGACAAGCGGCAGCTTGC | ||||
sahR (1023) | SAHR-P2239-A-AI | GGGCCCGACAACATCTCGAAGG | 1062/+36 | 973 | |
SAHR-P2239-B-PI | CTGCAGAGCATCCAAACCAAGCG | ||||
SAHR-P2239-C-PI | CTGCAGGGGGAGTTTGAGAATGA | 1132/−14 | |||
SAHR-P2239-D-XI | TCTAGACCGAATTAGGGACTATAATTCCG |
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Belfquih, M.; Filali-Maltouf, A.; Le Quéré, A. Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development. Microorganisms 2022, 10, 298. https://doi.org/10.3390/microorganisms10020298
Belfquih M, Filali-Maltouf A, Le Quéré A. Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development. Microorganisms. 2022; 10(2):298. https://doi.org/10.3390/microorganisms10020298
Chicago/Turabian StyleBelfquih, Meryem, Abdelkarim Filali-Maltouf, and Antoine Le Quéré. 2022. "Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development" Microorganisms 10, no. 2: 298. https://doi.org/10.3390/microorganisms10020298
APA StyleBelfquih, M., Filali-Maltouf, A., & Le Quéré, A. (2022). Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development. Microorganisms, 10(2), 298. https://doi.org/10.3390/microorganisms10020298