BINDING OF ppGpp AND cAMP TO TRANSCRIPTIONAL REGULATOR PROTEIN BolA

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Keywords:

Guanosine pentaphosphate (ppGpp), cAMP, glutathione (GSH), protein-ligand interaction, morphogene bolA

Abstract

E. coli BolA is a DNA-binding transcription factor that responds to various stresses during the stationary phase by regulating the transcription of stringent response-related genes. It also protects bacteria from stressful environments by changing the cell wall morphology and inducing biofilm formation. The present study examined the physical interaction between ppGpp and cAMP to BolA by fluorescence spectroscopy. Recombinant BolA from E. coli was cloned, overexpressed, and purified to purity. BolA shows an intrinsic fluorescence light at 339 nm when excited at 280 nm, and the maximum intensity decreases in the presence of cAMP and ppGpp. The dissociation constant for ppGpp and cAMP was determined to be 164 ± 20 and 165 ± 36 µM, respectively. The experimental evaluation suggests ppGpp competes with cAMP for the ligand-binding pocket of BolA. Under stringent conditions, ppGpp bind to BolA with a strong affinity to control gene regulation and biofilm formation. However, when the cell goes back to normal environmental conditions, cAMP occupies the binding site to repress the activity of BolA. This study showed for the first time that ppGpp and cAMP bind to BolA with strong affinity.

References

Costerton, J. W., Stewart, P. S., Greenberg, E. P. (1999): Bacterial biofilms: a common cause of persistent infections. Science (New York, N.Y.)284(5418), 1318–1322.

Mah, T. F., O'Toole, G. A. (2001): Mechanisms of biofilm resistance to antimicrobial agents. Trends in Microbiology9(1), 34–39.

Aldea, M., Hernández-Chico, C., de la Campa, A. G., Kushner, S. R., Vicente, M. (1988): Identification, cloning, and expression of bolA, an ftsZ-dependent morphogene of Escherichia coli. Journal of Bacteriology170(11), 5169–5176.

Adnan, M., Pereira, M. O., Machado, I., Morton, G., Hadi, S. (2010): Morphogene BolA: Its role in biofilm formation and respiration of E. coli K-12 MG1655. Clin Microbiol Infect. 16 S571.

Santos, J. M., Freire, P., Vicente, M., Arraiano, C. M. (1999): The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Molecular Microbiology 32(4), 789–798.

Guinote, I. B., Moreira, R. N., Barahona, S., Freire, P., Vicente, M., Arraiano, C. M. (2014): Breaking through the stress barrier: the role of BolA in Gram-negative survival. World Journal of Microbiology & Biotechnology 30(10), 2559–2566.

Khona, D. K., Dongre, S. S., Arraiano, C. M., D'Souza, J. S. (2013): A BolA-like morphogene from the alga Chlamydomonas reinhardtii changes morphology and induces biofilm formation in Escherichia coli. FEMS Microbiology Letters 339(1), 39–47.

Lange, R., Hengge-Aronis, R. (1991): Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S. Journal of Bacteriology 173(14), 4474–4481.

Freire, P., Amaral, J. D., Santos, J. M., Arraiano, C. M. (2006): Adaptation to carbon starvation: RNase III ensures normal expression levels of bolA1p mRNA and sigma(S). Biochimie 88(3-4), 341–346.

Couturier, J., Jacquot, J. P., Rouhier, N. (2009): Evolution and diversity of glutaredoxins in photosynthetic organisms. Cellular and Molecular Life Sciences: CMLS66(15), 2539–2557.

Kumánovics, A., Chen, O. S., Li, L., Bagley, D., Adkins, E. M., Lin, H., Dingra, N. N., Outten, C. E., Keller, G., Winge, D., Ward, D. M., Kaplan, J. (2008): Identification of FRA1 and FRA2 as genes involved in regulating the yeast iron regulon in response to decreased mitochondrial iron-sulfur cluster synthesis. The Journal of biological chemistry 283(16), 10276–10286.

Li, H., Mapolelo, D. T., Dingra, N. N., Naik, S. G., Lees, N. S., Hoffman, B. M., Riggs-Gelasco, P. J., Huynh, B. H., Johnson, M. K., Outten, C. E. (2009): The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation. Biochemistry 48(40), 9569–9581.

Krohn, M., Wagner, R. (1995): A procedure for the rapid preparation of guanosine tetraphosphate (ppGpp) from Escherichia coli ribosomes. Analytical Biochemistry225(1), 188–190.

Clark, A. R. (1996): Analysis of ligand binding by enzymes. In Enzymology Labfax; Engel, P.C., Ed.; Academic Press: San Diego, CA, USA, pp. 199–221.

Lakowicz, J. R. (1999): Principles of Fluorescence Spectroscopy; Plenum Press: New York, NY, USA, pp. 53–55.

Habig, W. H., Jakoby, W. B. (1981): Assays for differentiation of glutathione S-transferases. Methods in Enzymology 77, 398–405.

Shin, W. H., Heo, L., Lee, J., Ko, J., Seok, C., Lee, J. (2011): LigDockCSA: protein-ligand docking using conformational space annealing. Journal of Computational Chemistry 32(15), 3226–3232.

Shin, W. H., Kim, J. K., Kim, D. S., Seok, C. (2013). GalaxyDock2: protein-ligand docking using beta-complex and global optimization. Journal of Computational Chemistry34(30), 2647–2656.

The PyMOL Molecular Graphics System, version 2.0; Schrödinger, Inc.: New York, NY, USA, 2019.

Galego, L., Barahona, S., Romão, C. V., Arraiano, C. M. (2021): Phosphorylation status of BolA affects its role in transcription and biofilm development. The FEBS Journal 288(3), 961–979.

Aldea, M., Garrido, T., Hernández-Chico, C., Vicente, M., Kushner, S. R. (1989): Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene. The EMBO Journal 8(12), 3923–3931.

Kasai, T., Kigawa, T., Koshiba, S., Yokoyama, S. Solution structure of the BolA protein from Escherichia coli (To be published.)

Reeh, S., Pedersen, S., Friesen, J. D. (1976): Biosynthetic regulation of individual proteins in relA+ and relA strains of Escherichia coli during amino acid starvation. Molecular & General Genetics: MGG 149(3), 279–289.

Cashel, M., Gallant, J. (1969): Two compounds implicated in the function of the RC gene of Escherichia coli. Nature 221(5183), 838–841.

Haseltine, W. A., Block, R. (1973): Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proceedings of the National Academy of Sciences of the United States of America 70(5), 1564–1568.

Riggs, D. L., Mueller, R. D., Kwan, H. S., Artz, S. W. (1986): Promoter domain mediates guanosine tetraphosphate activation of the histidine operon. Proceedings of the National Academy of Sciences of the United States of America 83(24), 9333–9337.

Mocz, G., Ross, J. A. (2013): Fluorescence techniques in analysis of protein-ligand interactions. Methods in molecular biology (Clifton, N.J.), 1008 169–210.

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Published

2022-05-29

How to Cite

Duysak, T. (2022). BINDING OF ppGpp AND cAMP TO TRANSCRIPTIONAL REGULATOR PROTEIN BolA. Journal of Applied Biological Sciences, 16(2), 274–282. Retrieved from https://jabsonline.org/index.php/jabs/article/view/996

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