Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI
Abstract
1. Introduction
2. Results
2.1. Genes Encoding Glycosyltransferases Involved in EPS Biosynthesis in R. leguminosarum bv. trifolii Are Part of a Single Transcriptional Unit
2.2. PssG and PssI Proteins—Highly Similar, Yet Different
2.3. PssG and PssI Interact with the Inner Membrane through Amphipathic Helices
2.4. PssG but Not PssI Localization to the Inner Membrane Requires Other Proteins Involved in EPS Synthesis
2.5. PssI Has a More Expanded BTH Interaction Network Than PssG
2.6. PssG and PssI Interact In Vitro
2.7. Single pssG or pssI Deletion Decreases the EPS Amount by Half
2.8. PssG and PssI Serve Complementary Functions in EPS Synthesis
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Culture Conditions
4.2. Bioinformatic Analyses
4.3. Total RNA Isolation and cDNA Synthesis
4.4. DNA Techniques
4.5. β-Galactosidase Activity Measurements of Transcriptional Fusions
4.6. Construction of the ΔpssI and ΔpssG Single Mutants, ΔpssIΔpssG Double Mutant, ΔGT10 Mutant, and Their Complemented Derivatives
4.7. General Analyses of Proteins
4.8. Subcellular Localization Study
4.8.1. Localization of Proteins in the Heterologous System
4.8.2. Localization of Proteins in the Homologous System
4.9. Purification and Solubilization of Inclusion Bodies
4.10. Protein Refolding and Pull-Down Assay
4.11. Topology Analyses
4.12. Bacterial Two-Hybrid Analysis
4.13. Exopolysaccharide Analyses
4.14. Sensitivity Tests
4.15. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Żebracki, K.; Horbowicz, A.; Marczak, M.; Turska-Szewczuk, A.; Koper, P.; Wójcik, K.; Romańczuk, M.; Wójcik, M.; Mazur, A. Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI. Int. J. Mol. Sci. 2023, 24, 4248. https://doi.org/10.3390/ijms24044248
Żebracki K, Horbowicz A, Marczak M, Turska-Szewczuk A, Koper P, Wójcik K, Romańczuk M, Wójcik M, Mazur A. Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI. International Journal of Molecular Sciences. 2023; 24(4):4248. https://doi.org/10.3390/ijms24044248
Chicago/Turabian StyleŻebracki, Kamil, Aleksandra Horbowicz, Małgorzata Marczak, Anna Turska-Szewczuk, Piotr Koper, Klaudia Wójcik, Marceli Romańczuk, Magdalena Wójcik, and Andrzej Mazur. 2023. "Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI" International Journal of Molecular Sciences 24, no. 4: 4248. https://doi.org/10.3390/ijms24044248
APA StyleŻebracki, K., Horbowicz, A., Marczak, M., Turska-Szewczuk, A., Koper, P., Wójcik, K., Romańczuk, M., Wójcik, M., & Mazur, A. (2023). Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI. International Journal of Molecular Sciences, 24(4), 4248. https://doi.org/10.3390/ijms24044248