Modulation of rosR Expression and Exopolysaccharide Production in Rhizobium leguminosarum bv. trifolii by Phosphate and Clover Root Exudates
Abstract
:1. Introduction
2. Results and Discussion
2.1. Identification of Putative Regulatory Motifs in the rosR Upstream Region
2.2. Functional Analysis of the Putative Regulatory Motif IR4 in the rosR Upstream Region
2.3. Functional Analysis of Other Putative Regulatory Motifs in the rosR Upstream Region
2.4. Functional Analysis of a Putative LysR Motif
2.5. Effect of Phosphate and Clover Root Exudates on EPS Production
2.6. Biofilm Formation by R. leguminosarum bv. trifolii on Abiotic Surfaces in the Presence of Phosphate and Clover Root Exudates
2.7. Effect of Phosphate and Flavonoids on Symbiosis of Rt24.2 and Its Derivatives with Clover
3. Experimental Section
3.1. Bacterial Strains, Plasmids, and Growth Conditions
3.2. DNA Methods and Sequence Analysis
3.3. Construction of Transcriptional rosR-lacZ Fusions with Mutated IR4 Sites
3.4. β-Galactosidase Assay
3.5. EPS Isolation
3.6. Biofilm Formation Assay
3.7. Preparation of Exudates from Sprouted Seeds of Clover
3.8. Plant Tests
4. Conclusions
Acknowledgments
References
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Plasmid fusion | LysR motif | ANU843 (wt) | ANU851 (nodD−) | ||
---|---|---|---|---|---|
Clover root exudates (μM) | |||||
0 | 10 | 0 | 10 | ||
pEP1 | + | 2630 ± 288 | 3613 ± 352 | 2682 ± 253 | 2789 ± 245 |
pEP9 | − | 1778 ± 185 | 1866 ± 191 | 1709 ± 167 | 1811 ± 177 |
Strain or plasmid | Relevant characteristics | Reference |
---|---|---|
Rhizobium leguminosarum bv. trifolii | ||
Rt24.2 | Wild type, Rifr, Nxr | [36] |
Rt2472 | Rt24.2 derivative carrying mini-Tn5 between 151–152 bp position of rosR, Rifr, Kmr | [41] |
ANU843 | Wild type, Rifr | [57] |
ANU851 | ANU843 derivative carrying Tn5::nodD, Kmr | [57] |
E. coli | ||
VH1000 | MG1655 derivative, lacZ, lacI, pyrE+ | [58] |
LG01 | MG1655 derivative, phoB519::Tn5, Kmr | [59] |
Plasmids | ||
pUC19 | Cloning and sequencing vector, Apr | [60] |
pMP220 | IncP, mob, promoterless lacZ, Tcr | [61] |
pMJ221 | pUC19 containing 126-bp EcoRI-XbaI fragment of the rosR upstream region (based on primers: pROS2/pREW2 and pRBAM1/pREW3) | This work |
pMJ222 | pUC19 containing 126-bp EcoRI-XbaI fragment of the rosR upstream region (based on primers: pROS2/pREW4 and pRBAM1/pREW3) | This work |
pMJ223 | pUC19 containing 126-bp EcoRI-XbaI fragment of the rosR upstream region (based on primers: pROS2/pREW1) | This work |
pBR1 | pBBR1MCS-2 containing 1100-bp EcoRI-BamHI fragment with rosR of Rt24.2 | [41] |
pEP1 | pMP220 carrying the −403 to +243 bp fragment of the rosR coding region | [36] |
pEP9 | pMP220 carrying the −403 to −185 bp fragment of the rosR upstream region | [36] |
pEP10 | pMP220 carrying the −403 to −232 bp fragment of the rosR upstream region | [36] |
pEP13 | pMP220 carrying the −357 to −232 bp fragment of the rosR upstream region | [42] |
pEP14 | pMP220 carrying the −357 to −268 bp fragment of the rosR upstream region | [42] |
pEP15 | pMP220 carrying the −339 to −268 bp fragment of the rosR upstream region | [42] |
pEP21 | pMP220 containing 126-bp EcoRI-XbaI fragment from pMJ221 | This work |
pEP22 | pMP220 containing 126-bp EcoRI-XbaI fragment from pMJ222 | This work |
pEP23 | pMP220 containing 126-bp EcoRI-XbaI fragment from pMJ223 | This work |
Oligonucleotide primers | Sequence (5′→3′) * | |
pROS2 | GAGCCCCTGAATTCTTCATCTGTCA | This work |
pREW1 | AGGGATCTAGAAGCAGTACGCTAGACATTCAC | This work |
pREW2 | GTAATTCGGATCCAGAACTCTACTTGCA | This work |
pREW3 | GAAATCAAAACTGAGGGATCTAGAAGCA | This work |
pREW4 | GTAATTCGGATCCCGAACTCTACTTGCA | This work |
pRBAM1 | AAATGCAAGTAGAGTTCTGGATCCGAATTA | This work |
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Janczarek, M.; Skorupska, A. Modulation of rosR Expression and Exopolysaccharide Production in Rhizobium leguminosarum bv. trifolii by Phosphate and Clover Root Exudates. Int. J. Mol. Sci. 2011, 12, 4132-4155. https://doi.org/10.3390/ijms12064132
Janczarek M, Skorupska A. Modulation of rosR Expression and Exopolysaccharide Production in Rhizobium leguminosarum bv. trifolii by Phosphate and Clover Root Exudates. International Journal of Molecular Sciences. 2011; 12(6):4132-4155. https://doi.org/10.3390/ijms12064132
Chicago/Turabian StyleJanczarek, Monika, and Anna Skorupska. 2011. "Modulation of rosR Expression and Exopolysaccharide Production in Rhizobium leguminosarum bv. trifolii by Phosphate and Clover Root Exudates" International Journal of Molecular Sciences 12, no. 6: 4132-4155. https://doi.org/10.3390/ijms12064132