Determination of DNA Transfer Frequency, Related to the Presence of comX Gene, in Natural Streptococcus thermophilus Isolates

Yıl 2018, Cilt: 44 Sayı: 1, 9 - 23, 30.04.2018

Ferit Can Yazdıç , Altuğ Karaman İsmail Akyol , Mehmet Sait Ekinci , Emin Özköse

Öz

Activity of comX, alternative sigma factor, is crucial in the expression of the genes required for conjugation takes place in Streptococci and hence it is related to the frequency of competency. Determination of these genes in natural isolates, therefore, is a pivotal issue to understand the conjugation capacity of those microorganisms. In the current study, 106 Streptococcus thermophilus isolates are investigated for the presence of comX gene. Total 46 of these isolates are found to be comX positive using polymerase chain reaction (PCR) and nucleotide sequencing data of them were obtained. The plasmid pNZ276 and catalase tests were used to investigate the conjugation frequency of competent cells. Conjugation characteristics of the isolates BioSt-7, 16, 19, 23, 24, 25, 29, 30, 36, 37, 42 and 46 were studied and conjugation frequency of the isolates BioSt-19, 24, 25,29 and 42 are found to be over 40% and moreover a notable relationship between conjugation ratio and comX gene is determined. Substitution of isoleucine (I) for asparagine (N) in 72nd amino acid position of comX protein increased the conjugation frequency of the isolates. The findings of current study suggest that, some mutations occurred on comX gene may affect the frequency of competency and conjugated characteristics of streptococcal isolates.

Anahtar Kelimeler

Streptococcus thermophilus, comX gene, conjugation frequency

Doğal Streptococcus thermophilus İzolatlarında comX Gen Varlığına Bağlı DNA Transfer Frekansının Belirlenmesi

Öz

Alternatif sigma faktör olan comX, streptokoklarda dışarıdan DNA alabilme kapasitesini belirleyen genlerin transkripsiyonunu kontrol eden ve dolaysıyla bu suşların yabancı genetik materyali kabul etme frekansı ile ilişkili olan bir gendir. Bu nedenle doğal izolatlardaki varlığının tespit edilmesi, bu izolatların konjugasyon kapasitelerinin belirlenmesi açısından önem arz etmektedir. Bu çalışmada 106 farklı Streptococcus thermophilus izolatında comX geninin varlığı araştırılmıştır. Bu izolatların 46 tanesinde polimeraz zincir tepkimesi (PZT) yöntemi ile comX gen varlığı tespit edilmiş ve nükleotid dizileri belirlenmiştir. Kompetent hücrelerin konjugasyon frekansları için pNZ276 plazmiti ve katalaz testi kullanılmıştır. BioSt-7, 16, 19, 23, 24, 25, 29, 30, 36, 37, 42 ve 46 nolu izolatların konjugasyon özellikleri tespit edilmiştir. BioSt-19, 24, 25,29 ve 42 nolu izolatların da konjugasyon frekansları % 40’tan yüksek bulunmuş ve konjugasyon oranı ile comX gen dizisi arasında ilişki tespit edilmiştir. ComX proteininde 72. amino asit pozisyonundaki asparajinin yerine izolösin değişimi (N→I), konjugasyon frekansını artırmıştır. Bu çalışmanın sonuçları, comX gen dizisi üzerindeki değişim mutasyonlarının izolatların gen transfer sıklığını önemli derecede etkileyebileceğini, suşlara göre farklılık içereceğini ve konjugasyon özelliğini değiştirebileceğini göstermiştir.

Anahtar Kelimeler

Streptococcus thermophilus, comX geni, konjugasyon frekansı

Kaynakça

• Blomqvist, T., Steinmoen, H., & Håvarstein, L. S. (2006). Natural genetic transformation: a novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus. Applied and environmental microbiology, 72(10), 6751-6756.
• Bolotin, A., Quinquis, B., Renault, P., Sorokin, A., Ehrlich, S.D., Kulakauskas, S., Lapidus, A., Goltsman, E., Mazur, M., Pusch, G.D., Fonstein, M., Overbeek, R., Kyprides, N., Purnelle, B., Prozzi, D., Ngui, K., Masuy, D., Hancy, F., Burteau, S., Boutry, M., Delcour, J., Goffeau, A. and Hols, P. (2004). Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus. Nat. Biotechnol. 22, 1554–1558.
• Boutry C, Wahl A, Delplace B, Clippe A, Fontaine L, Hols P. (2012). Adaptor protein MecA is a negative regulator of the expression of late competence genes in Streptococcus thermophilus. J. Bacteriol. 194:1777–1788.
• Boutry, C., Delplace, B., Clippe, A., Fontaine, L., & Hols, P. (2013). SOS response activation and competence development are antagonistic mechanisms in Streptococcus thermophilus. Journal of bacteriology, 195(4), 696-707.
• Claverys, J.P. and Havarstein, L.S. (2002). Extracellular-peptide control of competence for genetic transformation in Streptococcus pneumoniae. Front. Biosci. 7, 1798–1814.
• Claverys, J.P. and Martin, B. (2003). Bacterial competence genes: signatures of active transformation, or only remnants?. Trends Microbiol. 11, 161–165. Cvitkovitch, D.G. (2001). Genetic competence and transformation in oral streptococci. Crit. Rev. Oral Biol. Med. 12, 217–243.
• Dagan, T., (2011). Phylogenomic networks. Trends Microbiol. 19, 483–491.
• Fontaine L, Boutry C, de Frahan MH, Delplace B, Fremaux C, Horvath P, Boyaval P, Hols P. (2010). A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius. J. Bacteriol. 192:1444 –1454.
• Fontaine, L., Wahl, A., Fléchard, M., Mignolet, J., & Hols, P. (2015). Regulation of competence for natural transformation in streptococci. Infection, Genetics and Evolution, 33, 343-360.
• Fox, P.F. (1993) Cheese: Chemistry, Physics and Microbiology. Chapman & Hall, London, UK.
• Gardan R, Besset C, Guillot A, Gitton C, Monnet V. (2009). The oligopeptide transport system is essential for the development of natural competence in Streptococcus thermophilus strain LMD-9. J. Bacteriol. 191: 4647–4655.
• Ho, T. D., & Ellermeier, C. D. (2012). Extra cytoplasmic function σ factor activation. Current opinion in microbiology, 15(2), 182-188.
• Hols P, Hancy F, Fontaine L, Grossiord B, Prozzi D, Leblond-Bourget N, Decaris B, Bolotin A, Delorme C, Dusko Erlich S, Guedon E, Monnet V, Renault P, Kleerebezem M. (2005). New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics. FEMS Microbiol. Rev. 29:435– 463.
• Hsu, H. H., Chung, K. M., Chen, T. C., & Chang, B. Y. (2006). Role of the sigma factor in transcription initiation in the absence of core RNA polymerase. Cell, 127(2), 317-327.
• Johnsborg O, Havarstein LS. (2009). Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae. FEMS Microbiol. Rev. 33:627– 642.
• Luo P, Li HY, Morrison DA (2003). ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae. Mol Microbiol 50: 623–633.
• Luo, P., & Morrison, D. A. (2003). Transient association of an alternative sigma factor, ComX, with RNA polymerase during the period of competence for genetic transformation in Streptococcus pneumoniae. Journal of bacteriology, 185(1), 349-358.
• Madigan, M., and J. Martinko. "Brock Biology of Microorganisms, 11th edn." (2005): 149-152.
• Mitchell, T.J. (2003) The pathogenesis of streptococcal infections: from tooth decay to meningitis. Nat. Rev. Microbiol. 1, 219–230.
• Nei, M., and Kumar, S. (2000). Molecular Evolution and Phylogenetics, Oxford University Press, Oxford.
• Perry, J.A., M.B. Jones, S.N. Peterson, D.G. Cvitkovitch and C.M. Lévesque. (2009). Peptide alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence. Mol. Microbiol. 72: 905–917.
• Peterson, S.N., Sung, C.K., Cline, R., Desai, B.V., Snesrud, E.C., Luo, P., Walling, J., Li, H., Mintz, M., Tsegaye, G., Burr, P.C., Do, Y., Ahn, S., Gilbert, J., Fleischmann, R.D. and Morrison, D.A. (2004) Identification of competence pheromone responsive genes in Streptococcus pneumoniae by use of DNA microarrays. Mol. Microbiol. 51, 1051–1070.
• Prudhomme, M., L. Attaiech, G. Sanchez, B. Martin and J.P. Claverys. (2006). Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae. Science 313: 89–92.
• Sambrook, J., & Russell, D. W. (2001). Molecular cloning: a laboratory manual 3rd edition. Coldspring-Harbour Laboratory Press, UK.
• Tamime, A.Y. and Deeth, H.C. (1980) Yogurt: technology and biochemistry. J. Food Protect. 43, 939–977.
• Tettelin, H. (2004) Streptococcal genomes provide food for thought. Nat. Biotechnol. 22, 1523–1524.
• Tettelin, H., Nelson, K.E., Paulsen, I.T., Eisen, J.A., Read, T.D., Peterson, S., Heidelberg, J., DeBoy, R.T., Haft, D.H., Dodson, R.J., Durkin, A.S., Gwinn, M., Kolonay, J.F., Nelson, W.C., Peterson, J.D., Umayam, L.A., White, O., Salzberg, S.L., Lewis, M.R., Radune, D., Holtzapple, E., Khouri, H., Wolf, A.M., Utterback, T.R., Hansen, C.L., McDonald, L.A., Feldblyum, T.V., Angiuoli, S., Dickinson, T., Hickey, E.K., Holt, I.E., Loftus, B.J., Yang, F., Smith, H.O., Venter, J.C., Dougherty, B.A., Morrison, D.A., Hollingshead, S.K. and Fraser, C.M. (2001) Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498–506.
• Thompson JD, Gibson TJ, Plewniak F, et al (1997): The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic acids research, 25, 4876-4882.