Identification and Analysis of a C-Phycocyanin Beta Subunit Gene from Thermosynechococcus sp. TUBT-T01
Abstract
C-Phycocyanin, a blue-colored pigment, is a type of phycobiliproteins that has an important role in light-dependent photosynthetic reactions of cyanobacteria. It is used as a natural pigment in food and cosmetics and has also been shown to possess antioxidant properties. The aim of this study was to identify and analyze the nucleotide sequences of a gene encoding a C-phycocyanin beta subunit (cpcB) belonging to Thermosynechococcus sp. TUBT-T01, a thermophilic cyanobacterium. Primers were designed to amplify cpcB gene by polymerase chain reaction and DNA fragment was ligated to pGEM-T easy vector for nucleotide sequencing. The sequencing results showed that the cpcB gene was 519 base pairs in length and its nucleotide sequences had 97% identity to those of thermophilic Thermosynechococcus sp. NK55, T. elongatus BP-1 and Synechococcus vulcanus. At the protein level, CpcB protein was made up of 172 amino acids. The presence of glutamine at position 68 and asparagine at position 21 were noticeable and their presence could involve in the stability of C-phycocyanin at high temperature. Keywords : Hot spring cyanobacteria, C-phycocyanin, C-phycocyanin beta subunit geneReferences
Adir, N., Dobrovetsky, Y., & Lerner, N. (2001). Structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus at 2.5 angstrom: structural implications for thermal stability in phycobilisome assembly. Journal of Molecular Biology, 313, 71-81.
Adir, N. & Lerner, N. (2003).The Crystal Structure of a Novel Unmethylated Form of C-phycocyanin, a Possible Connector Between Cores and Rods in Phycobilisomes. Journal of Biological Chemistry, 278,
25926–25932.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., & Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410.
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389-3402.
Chaiklahan, R., Chirasuwan, N., & Bunnag, B. (2012). Stability of phycocyanin extracted from Spirulina sp.: influence of temperature, pH and preservatives. Process Biochemistry, 47, 659-664.
Cherdkiatikul, T. & Suwanwong, Y. (2014). Production of the α and β subunits of Spirulina allophycocyanin
and C-phycocyanin in Escherichia coli: a comparative study of their antioxidant activities. Journal of Biomolecular Screening, 19, 959-965.
Conley, P.B., Lemaux, P.G., & Grossman, A. (1988). Molecular characterization and evolution of sequences encoding light-harvesting components in the chromatically adapting cyanobacterium Fremyella diplosiphon. Journal of Molecular Biology, 199, 447-465.
de Lorimier, R., Guglielmi, G., Bryant, D.A., & Stevens, S.E., Jr. (1990a). Structure and mutation of a gene encoding a Mr 33,000 phycocyanin-associated linker polypeptide. Archives of Microbiology, 153,
541-549.
de Lorimier, R., Bryant, D.A., & Stevens, S.E., Jr. (1990b). Genetic analysis of a 9 kDa phycocyanin-associated linker polypeptide. Biochimica et Biophysica Acta (BBA) – Bioenergetics. 1019, 29-41.
Duerring, M., Schmidt, G.B., & Huber, R. (1991). Isolation, crystallization, crystal structure analysis and refinement of constitutive C-phycocyanin from the chromatically adapting cyanobacterium Fremyella diplosiphon at 1.66 A resolution. Journal of Molecular Biology, 217, 577-92.
Estrada, P., Bescos, B.P., & Villar del Fresno, A.M. (2001). Antioxidant activity of different fractions of spirulina platensis protean extract. Il Farmaco, 56, 497–500.
Fujisawa, T., Narikawa, R., Okamoto, S., Ehira, S., Yoshimura, H., Suzuki, I., Masuda, T., Mochimaru, M., Takaichi, S., Awai, K., Sekine, M., Horikawa, H., Yashiro, I., Omata, S., Takarada, H., Katano, Y., Kosugi, H., Tanikawa, S., Ohmori, K., Sato, N., Ikeuchi, M., Fujita, N., & Ohmori, M. (2010). Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39. DNA research, 17,
85-103.
Garnier, J., Gibrat, J.F., & Robson, B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods in Enzymology, 266, 540-553.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In J.M. Walker. (Ed.), The Proteomics Protocols Handbook. (pp. 571-607). Humana Press.
Govindjee & Shevela, D. (2011). Adventures with cyanobacteria: a personal perspective. Frontiers in Plant Science, 2, 28: 1-17.
Grossman, A.R., Schaefer, M.R., Chiang, G.G., & Collier, J.L. (1993). The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiological Reviews, 57, 725–749.
Guruprasad, K., Reddy, B.V., & Pandit, M.W. (1990). Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Engineering, 4, 155-161.
Guex, N., Peitsch, M.C., & Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis, 30, S162-S173.
Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870-1874.
Kyte, J. & Doolittle, R.F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157, 105-132.
Leu, J.Y., Lin, T.H., Selvamani, M.J.P.,Chen, H.C., Liang, J.Z., & Pan, K.M. (2013). Characterization of a novel thermophillic cyanobacterial strain from Taian hot springs in Taiwan for high CO2 mitigation and C-phycocyanin extraction. Process Biochemistry. 48, 41-48.
MacColl, R. (1998). Cyanobacterial phycobilisomes. Journal of Structural Biology, 124, 311-334.
Minkova, K.M., Tchernov, A.A., Tchorbadjieva, M.I., Fournadjieva, S.T., Antova, R.E., & Busheva, M.C. (2003). Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. Journal of Biotechnology, 102,
55–59.
Muthulakshmi, M., Saranya, A., Sudha, M., & Selvakumar, G. (2012). Extraction, partial purification, and antibacterial activity of phycocyanin from Spirulina isolated from fresh water body against various human pathogens. Journal of Algal Biomass Utilization, 3, 7-11.
Nakamura, Y., Kaneko, T., Sato, S., Ikeuchi, M., Katoh, H., Sasamoto, S., Watanabe, A., Iriguchi, M., Kawashima K., Kimura T., Kishida Y., Kiyokawa C., Kohara M., Matsumoto M., Matsuno A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, C., Yamada, M., & Tabata, S. (2002). Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. DNA Research, 9, 123-130.
Prabha, R., Singh, D.P., Gupta, S.K., de Farias, S.T., & Rai, A. (2013). Comparative analysis to identify determinants of changing life style in Thermosynechococcus elongatus BP-1, a thermophilic cyanobacterium. Bioinformation, 9, 299-308.
Pandey, V.D., Pandey, A., & Sharma, V. (2013). Biotechnological applications of cyanobacterial phycobiliproteins. International Journal of Current Microbiology and Applied Sciences, 2, 89-97.
Richa, Kannaujiya, V.K., Kesheri, M., Sihgh, G., & Sinha, R.P. (2011). Biotechnological potentials of phycobiliproteins. International Journal of Pharma and Bio Sciences, 2, 446-454.
Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., & Stanier, R.Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology, 111, 1-61.
Romay, C., Gonzalez, R., Ledon, N., Remirez, D., & Rimbau, V. (2003). C-Phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Current Protein and Peptide Science, 4,
207-216.
Sambrook, J.F. & Russell, D.W. (2001). Screening bacterial colonies using X-gal and IPTG: α-complementation. Molecular cloning: a laboratory manual, 3rd ed. vol.1. (pp. 1.123-1.125). In J. Argentine, N. Irwin, K.A. Janssen, S. Curtis, M. Zierler, M. Dickerson, I. Sialiano, N. Mclnerny, D. Brown, S. Schaefer, D. deBurin, E. Atkeson, D. Weiss, & M.D. Curtis. (Eds.), New York: Cold Spring Harbor Laboratory Press.
Schrödinger, L.L.C., (2015). The PyMOL Molecular Graphics System version 1.8. New York.
Shen, G., Saunée, N.A., Williams, S.R., Gallo, E.F., Schluchter, W.M., & Bryant, D.A. (2006). Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002. Journal of Biological Chemistry, 281(26), 17768-177678.
Shen, G., Schluchter, W. M., & Bryant, D.A. (2008). Biogenesis of phycobiliproteins I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phycocyanobilin lyase specific for β-phycocyanin and allophycocyanin subunits. Journal of Biological Chemistry, 283(12), 7503–7512.
Shih, C.M., Cheng, S.N., Wong, C.S., Kuo, Y.L., & Chou, T.C. (2009). Antiinflammatory and antihyperalgesic activity of C-phycocyanin. Anesthesia and Analgesia, 108, 1303-1310.
Sievers, F., Wilm, A., Dineen, D.G., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D., & Higgins, D. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology. 7, 539: 1-6.
Stolyar, S., Liu, Z., Thiel, V., Tomsho, L.P., Pinel, N., Nelson, W.C., Lindemann, S.R., Romine, M.F., Haruta, S., Schuster, S.C., Bryant, D.A., & Fredrickson, J.K. (2014). Genome sequence of the thermophilic cyanobacterium Thermosynechococcus sp. strain NK55a. Genome Announcements, 2, e01060-e01013.
Suwanmanee, K., Charoenrat, T., & Chittapun, S. (2015). Isolation and cultivation of thermophilic blue green algae from hot springs in Surat Thani province. In Proceeding the 7th National Conference on Algae and Plankton (NCAP) conference. (pp. 1-12), Thailand: Bangkok.
Szilágyi, A. & Závodszky, P. (2000). Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure, 8, 493-504.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M., & Rozen, S.G. (2012). Primer3--new capabilities and interfaces. Nucleic Acids Research, 40, e115: 1-12.
Wang, H., Liu, Y., Gao, X., Carter, C.L., & Liu, Z.R. (2007). The recombinant beta subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis. Cancer Letters, 247, 150-158.
Yuwadee, P. (2006). Phycology. (2nd edition). Chiang Mai: Chotana Print. (Book In Thai).
Zhou, J., Gasparich, G.E., Stirewalt, V.L., de Lorimier, R., & Bryant, D.A. (1992). The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants.
The Journal of Biological Chemistry, 267, 16138-16145.
Adir, N. & Lerner, N. (2003).The Crystal Structure of a Novel Unmethylated Form of C-phycocyanin, a Possible Connector Between Cores and Rods in Phycobilisomes. Journal of Biological Chemistry, 278,
25926–25932.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., & Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410.
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389-3402.
Chaiklahan, R., Chirasuwan, N., & Bunnag, B. (2012). Stability of phycocyanin extracted from Spirulina sp.: influence of temperature, pH and preservatives. Process Biochemistry, 47, 659-664.
Cherdkiatikul, T. & Suwanwong, Y. (2014). Production of the α and β subunits of Spirulina allophycocyanin
and C-phycocyanin in Escherichia coli: a comparative study of their antioxidant activities. Journal of Biomolecular Screening, 19, 959-965.
Conley, P.B., Lemaux, P.G., & Grossman, A. (1988). Molecular characterization and evolution of sequences encoding light-harvesting components in the chromatically adapting cyanobacterium Fremyella diplosiphon. Journal of Molecular Biology, 199, 447-465.
de Lorimier, R., Guglielmi, G., Bryant, D.A., & Stevens, S.E., Jr. (1990a). Structure and mutation of a gene encoding a Mr 33,000 phycocyanin-associated linker polypeptide. Archives of Microbiology, 153,
541-549.
de Lorimier, R., Bryant, D.A., & Stevens, S.E., Jr. (1990b). Genetic analysis of a 9 kDa phycocyanin-associated linker polypeptide. Biochimica et Biophysica Acta (BBA) – Bioenergetics. 1019, 29-41.
Duerring, M., Schmidt, G.B., & Huber, R. (1991). Isolation, crystallization, crystal structure analysis and refinement of constitutive C-phycocyanin from the chromatically adapting cyanobacterium Fremyella diplosiphon at 1.66 A resolution. Journal of Molecular Biology, 217, 577-92.
Estrada, P., Bescos, B.P., & Villar del Fresno, A.M. (2001). Antioxidant activity of different fractions of spirulina platensis protean extract. Il Farmaco, 56, 497–500.
Fujisawa, T., Narikawa, R., Okamoto, S., Ehira, S., Yoshimura, H., Suzuki, I., Masuda, T., Mochimaru, M., Takaichi, S., Awai, K., Sekine, M., Horikawa, H., Yashiro, I., Omata, S., Takarada, H., Katano, Y., Kosugi, H., Tanikawa, S., Ohmori, K., Sato, N., Ikeuchi, M., Fujita, N., & Ohmori, M. (2010). Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39. DNA research, 17,
85-103.
Garnier, J., Gibrat, J.F., & Robson, B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods in Enzymology, 266, 540-553.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In J.M. Walker. (Ed.), The Proteomics Protocols Handbook. (pp. 571-607). Humana Press.
Govindjee & Shevela, D. (2011). Adventures with cyanobacteria: a personal perspective. Frontiers in Plant Science, 2, 28: 1-17.
Grossman, A.R., Schaefer, M.R., Chiang, G.G., & Collier, J.L. (1993). The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiological Reviews, 57, 725–749.
Guruprasad, K., Reddy, B.V., & Pandit, M.W. (1990). Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Engineering, 4, 155-161.
Guex, N., Peitsch, M.C., & Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis, 30, S162-S173.
Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870-1874.
Kyte, J. & Doolittle, R.F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157, 105-132.
Leu, J.Y., Lin, T.H., Selvamani, M.J.P.,Chen, H.C., Liang, J.Z., & Pan, K.M. (2013). Characterization of a novel thermophillic cyanobacterial strain from Taian hot springs in Taiwan for high CO2 mitigation and C-phycocyanin extraction. Process Biochemistry. 48, 41-48.
MacColl, R. (1998). Cyanobacterial phycobilisomes. Journal of Structural Biology, 124, 311-334.
Minkova, K.M., Tchernov, A.A., Tchorbadjieva, M.I., Fournadjieva, S.T., Antova, R.E., & Busheva, M.C. (2003). Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. Journal of Biotechnology, 102,
55–59.
Muthulakshmi, M., Saranya, A., Sudha, M., & Selvakumar, G. (2012). Extraction, partial purification, and antibacterial activity of phycocyanin from Spirulina isolated from fresh water body against various human pathogens. Journal of Algal Biomass Utilization, 3, 7-11.
Nakamura, Y., Kaneko, T., Sato, S., Ikeuchi, M., Katoh, H., Sasamoto, S., Watanabe, A., Iriguchi, M., Kawashima K., Kimura T., Kishida Y., Kiyokawa C., Kohara M., Matsumoto M., Matsuno A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takeuchi, C., Yamada, M., & Tabata, S. (2002). Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. DNA Research, 9, 123-130.
Prabha, R., Singh, D.P., Gupta, S.K., de Farias, S.T., & Rai, A. (2013). Comparative analysis to identify determinants of changing life style in Thermosynechococcus elongatus BP-1, a thermophilic cyanobacterium. Bioinformation, 9, 299-308.
Pandey, V.D., Pandey, A., & Sharma, V. (2013). Biotechnological applications of cyanobacterial phycobiliproteins. International Journal of Current Microbiology and Applied Sciences, 2, 89-97.
Richa, Kannaujiya, V.K., Kesheri, M., Sihgh, G., & Sinha, R.P. (2011). Biotechnological potentials of phycobiliproteins. International Journal of Pharma and Bio Sciences, 2, 446-454.
Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., & Stanier, R.Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology, 111, 1-61.
Romay, C., Gonzalez, R., Ledon, N., Remirez, D., & Rimbau, V. (2003). C-Phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Current Protein and Peptide Science, 4,
207-216.
Sambrook, J.F. & Russell, D.W. (2001). Screening bacterial colonies using X-gal and IPTG: α-complementation. Molecular cloning: a laboratory manual, 3rd ed. vol.1. (pp. 1.123-1.125). In J. Argentine, N. Irwin, K.A. Janssen, S. Curtis, M. Zierler, M. Dickerson, I. Sialiano, N. Mclnerny, D. Brown, S. Schaefer, D. deBurin, E. Atkeson, D. Weiss, & M.D. Curtis. (Eds.), New York: Cold Spring Harbor Laboratory Press.
Schrödinger, L.L.C., (2015). The PyMOL Molecular Graphics System version 1.8. New York.
Shen, G., Saunée, N.A., Williams, S.R., Gallo, E.F., Schluchter, W.M., & Bryant, D.A. (2006). Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002. Journal of Biological Chemistry, 281(26), 17768-177678.
Shen, G., Schluchter, W. M., & Bryant, D.A. (2008). Biogenesis of phycobiliproteins I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phycocyanobilin lyase specific for β-phycocyanin and allophycocyanin subunits. Journal of Biological Chemistry, 283(12), 7503–7512.
Shih, C.M., Cheng, S.N., Wong, C.S., Kuo, Y.L., & Chou, T.C. (2009). Antiinflammatory and antihyperalgesic activity of C-phycocyanin. Anesthesia and Analgesia, 108, 1303-1310.
Sievers, F., Wilm, A., Dineen, D.G., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D., & Higgins, D. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology. 7, 539: 1-6.
Stolyar, S., Liu, Z., Thiel, V., Tomsho, L.P., Pinel, N., Nelson, W.C., Lindemann, S.R., Romine, M.F., Haruta, S., Schuster, S.C., Bryant, D.A., & Fredrickson, J.K. (2014). Genome sequence of the thermophilic cyanobacterium Thermosynechococcus sp. strain NK55a. Genome Announcements, 2, e01060-e01013.
Suwanmanee, K., Charoenrat, T., & Chittapun, S. (2015). Isolation and cultivation of thermophilic blue green algae from hot springs in Surat Thani province. In Proceeding the 7th National Conference on Algae and Plankton (NCAP) conference. (pp. 1-12), Thailand: Bangkok.
Szilágyi, A. & Závodszky, P. (2000). Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure, 8, 493-504.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M., & Rozen, S.G. (2012). Primer3--new capabilities and interfaces. Nucleic Acids Research, 40, e115: 1-12.
Wang, H., Liu, Y., Gao, X., Carter, C.L., & Liu, Z.R. (2007). The recombinant beta subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis. Cancer Letters, 247, 150-158.
Yuwadee, P. (2006). Phycology. (2nd edition). Chiang Mai: Chotana Print. (Book In Thai).
Zhou, J., Gasparich, G.E., Stirewalt, V.L., de Lorimier, R., & Bryant, D.A. (1992). The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants.
The Journal of Biological Chemistry, 267, 16138-16145.
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