Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (05): 27-35    DOI:
    
Preliminary Screening of ComE Binding Sequences in Streptococcus mutans
PENG Lei,WU Dan,HUANG Bo,LIU Xiao-di,GUO Li-hong
Department of Oral Biology, School and Hospital of Stomatology, Peking University, Beijing 100081, China
Download: HTML   PDF(1123KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective: To screen the binding sequences of ComE from the genome of Streptococcus mutans. Methods: ⑴The genome of Streptococcus mutans UA159 was extracted and sheared by sonication, and then incubated with two different random primers successively. Finally, the extention product was purified from gel extraction and amplified by PCR, and the genomic library of Streptococcus mutans was constructed.⑵Genomic SELEX for eight times to obtain the binding sequences of ComE. Selected DNA fragments were cloned into the pGEM-T Easy Vector and sent to sequence analysis. The sequences were further analyzed by bioinformatics programs. ⑶Two obtained sequences were cloned into pFW5-luc,and then the corresponding recombinant plasmids were transformed into Streptococcus mutans UA159 and Streptococcus mutans comE mutant respectively. Finally, the expression of luc were tested by RT-PCR. Results: ⑴The genomic library of Streptococcus mutans UA159 was obtained with the sequences ranging from 100 bp to 300 bp. ⑵54 clones were selected randomly and sent to sequencing. Two candidates were identified through bioinformatics analysis. Through primary validation, one possible binding sequence was obtained. Conclusion: According to genomic SELEX, eight selection cycles were finished and one possible binding sequence was obtained, which laid the basis for the explanation of the regulation mechanism of ComE in Streptococcus mutans.



Key wordsStreptococcus mutans      ComE      genomic SELEX     
Received: 21 December 2009      Published: 25 May 2010
Cite this article:

BANG Lei, TUN Dan, HUANG Bo, LIU Xiao-Di, GUO Li-Hong. Preliminary Screening of ComE Binding Sequences in Streptococcus mutans. China Biotechnology, 2010, 30(05): 27-35.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2010/V30/I05/27

[1] Li Y H, Lau P C Y, Lee J H, et al. Natural genetic transformation of Streptococcus mutans growing in biofilms. J Bacteriol, 2001, 183(3):897908. 
[2] Li Y H, Hanna M N, Svensater G, et al. Cell density modulates acid adaptation in Streptococcus mutans: Implications for survival in biofilms. J Bacteriol, 2001, 183(23):68756884. 
[3] Li Y H, Tang N, Aspiras M B, et al. A quorumsensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J Bacteriol, 2002, 184(10):26992708. 
[4] van der Ploeg J R. Regulation of bacteriocin production in Streptococcus mutans by the quorumsensing system required for development of genetic competence. J Bacteriol, 2005, 187(12):39803989. 
[5] Podbielski A, Kreikemeyer B. Cell densitydependent regulation: basic principles and effects on the virulence of Grampositive cocci. Int J Infect Dis, 2004, 8(2):8195. 
[6] Spoering A L, Gilmore M S. Quorum sensing and DNA release in bacterial biofilms. Curr Opin Microbiol, 2006, 9(2):133137. 
[7] Kreth J, Merritt J, Shi W, et al. Coordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species. Mol Microbiol, 2005, 57(2):392404. 
[8] Kreth J, Merritt J, Zhu L, et al. Cell density and ComEdependent expression of a group of mutacin and mutacinlike genes in Streptococcus mutans. FEMS Microbiol Lett, 2006, 265(1):1117. 
[9] Kreth J, Hung D C I, Merritt J, et al. The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis. MicrobiologySgm, 2007, 153:17991807. 
[10] Raffa R B, Iannuzzo J R, Levine D R, et al. Bacterial communication (“Quorum sensing”) via ligands and receptors: A novel pharmacologic target for the design of antibiotic drugs. J Pharmacol Exp Ther, 2005, 312(2):417423. 
[11]Liu X D, Du N, Guo L H. Construction of Streptococcus mutans comE Mutant Strain Using Inframe Deletion System. China Biotechnology, 2009, 29 (7):8086. 
[12] Peng L, Liu X D, Guo L H. Highlevel expression and purification of ComE of Streptococcus mutans in Escherichia coli. J Pract Stomatol, 2009, 25(2):4449. 
[13] Singer B S, Shtatland T, Brown D, et al. Libraries for genomic SELEX. Nucleic Acids Res, 1997, 25(4):781786. 
[14] Ferrieres L, FrancezCharlot A, Gouzy J, et al. FIXregulated genes evolved through promoter duplication in Sinorhizobium meliloti. MicrobiologySgm, 2004, 150:23352345. 
[15] Itou H, Okada U, Suzuki H, et al. The CGL2612 protein from Corynebacterium glutamicum is a drug resistancerelated transcriptional repressor  Structural and functional analysis of a newly identified transcription factor from genomic DNA analysis. J Biol Chem, 2005, 280(46):3871138719. 
[16] Rasmussen T B, Givskov M. Quorumsensing inhibitors as antipathogenic drugs. Int J Med Microbiol, 2006, 296(23):149161. 
[17] Abraham W R. Controlling biofilms of grampositive pathogenic bacteria. Curr Med Chem, 2006, 13(13):15091524. 
[18] West A H, Stock A M. Histidine kinases and response regulator proteins in twocomponent signaling systems. Trends Biochem Sci, 2001, 26(6):369376. 
[19] Nikolskaya A N, Galperin M Y. A novel type of conserved DNAbinding domain in the transcriptional regulators of the AlgR/AgrA/LytR family. Nucleic Acids Res, 2002, 30(11):24532459. 
[20] Russell R R, AduseOpoku J, Sutcliffe I C, et al. A binding proteindependent transport system in Streptococcus mutans responsible for multiple sugar metabolism. J Biol Chem, 1992, 267(7):46314637. 
[21] Ajdic D, McShan W M, McLaughlin R E, et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A, 2002, 99(22):1443414439.

[1] DAI Han-ying,XU Ke-qian. Research Progress on DNA Double-Strand Break Assay[J]. China Biotechnology, 2020, 40(8): 55-62.
[2] LI Chuan-bao, HUA Tian, DU Hong-wu. Different Influences Between Long-term Treated IGF-Ⅰand MGF About Glycometabolism in Mice[J]. China Biotechnology, 2012, 32(04): 7-11.
[3] LIU Xiao-Di, DU Ning, GUO Li-Hong. Construction of streptococcus mutans comE mutant strain using In-frame deletion system[J]. China Biotechnology, 2009, 29(07): 80-86.