Tyrosine Kinase Self-Phosphorylation Controls Exopolysaccharide Biosynthesis in Gluconacetobacter diazotrophicus Strain Pal5
Abstract
:1. Introduction
2. Materials and Methods
2.1. Annotation of the Putative Wzc Protein
2.2. Bacterial Strains and Growth Conditions
2.3. Construction of Wzc Mutant of G. Diazotrophicus
2.4. EPS Purification and Quantification
2.5. Tyrosine Kinase Assay with Radiolabeled ATP
2.6. In Vitro IP Kinase Assay
2.7. Cloning and Expression of the Wzc Gene in E. coli Strain BL21-AI
2.8. RT-qPCR Analysis
2.9. Statistical Analysis
3. Results
3.1. Homology of the Strain Pal5 Wzc Protein to Other Bacterial Proteins
3.2. Tn5 Insertional Inactivation of the PAL5 Wzc Gene
3.3. EPS Production Is Impaired in a Wzc Mutant
3.4. Glycosyltransferase Phosphorylation in G. diazotrophicus
3.5. Immunoprecipitation Assay for Detection of Protein Tyrosine Kinase Self-Phosphorylation Dynamics with Radiolabeled ATP
3.6. Wzc Expression in E. coli BL21-AI
3.7. Wzc Gene Expression in Bacterial Strains Grown in Different Carbon Sources
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strains and Primers | Characteristics or Sequences | Reference |
---|---|---|
Pal5 | Wild-type, AmpS, KmS, TcS, EPS+ | [1] |
MGD | gumD::Tn5, KmR, gumD−, EPS− | [16] |
MWzc | wzc::Tn5, KmR, wzc−, EPS− | This study |
ΔW_Pal5 | His6-tagged Wzc recombinant protein of Pal5 | This study |
ΔW_MGD | His6-tagged Wzc recombinant protein of MGD | This study |
ΔW_MWzc | His6-tagged Wzc recombinant protein of MWzc | This study |
wzc-sense wzc-antisense | 5′-GACCTGGCCAATATGTTCGT-3′ 5′-ATCAGCAGCTTCTTGCGATT-3′ | This study |
ExpWzcN1-sense ExpWzcN1-antisense | 5′-GACCTGGCCAAAATATGTTCGT-3′ 5′-ATCAGCAGCTATATGTTCGT-3′− | This study |
M13GW-sense M13GW-antisense | 5′-GTAAAACGACGGCCAG-3′ 5′-AGGAAACAGCTATGAC-3′ | This study |
attB1 | 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′ | Invitrogen |
attB2 | 5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′ | |
23S-sense 23S-antisense | 5′AAAGCCGGATCAATCCGTTA3′ 5′AAGCCGTAGTCGATGGAAAC3′ | [20] |
RTwzc-sense RTwzc-antisense | 5′GGGGAAATCGAACAGTTGCG3′ 5′CGGGCGCGCGGTCCGC3′ | This study |
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Wanderley, K.; Sousa, D.; Silva, G.; Maia, J.; Silva, M.; Vidal, M.; Baldani, J.; Meneses, C. Tyrosine Kinase Self-Phosphorylation Controls Exopolysaccharide Biosynthesis in Gluconacetobacter diazotrophicus Strain Pal5. Life 2021, 11, 1231. https://doi.org/10.3390/life11111231
Wanderley K, Sousa D, Silva G, Maia J, Silva M, Vidal M, Baldani J, Meneses C. Tyrosine Kinase Self-Phosphorylation Controls Exopolysaccharide Biosynthesis in Gluconacetobacter diazotrophicus Strain Pal5. Life. 2021; 11(11):1231. https://doi.org/10.3390/life11111231
Chicago/Turabian StyleWanderley, Katyanne, Dayse Sousa, Gabriel Silva, Josemir Maia, Maria Silva, Marcia Vidal, José Baldani, and Carlos Meneses. 2021. "Tyrosine Kinase Self-Phosphorylation Controls Exopolysaccharide Biosynthesis in Gluconacetobacter diazotrophicus Strain Pal5" Life 11, no. 11: 1231. https://doi.org/10.3390/life11111231