Abstract:To study the biological function of the methyl-accepting chemotaxis protein (MCP) gene 05SSU0273 and its products in Streptococcus suis serotype 2, a pair of specific primers was designed for the gene 05SSU0273, and the target DNA fragment was successfully amplified using the genomic template of Chinese strains 05ZYH33. Subsequently, target gene was inserted into pMD18-T vector, and then subcloned into prokaryotic expression vector pET32a, generating a recombinant expression plasmid pET32a:: 05SSU0273. Direct DNA sequencing method was used to confirm its correctness. The recombinant plasmid was then transformed into E. coli BL21. After IPTG induction, this bacteria could produce the target MCP protein with 6×his-tag which could be purified by affinity chromatography system. The results of SDS-PAGE and Western-blot showed that the fusion protein had a relative molecular weight of 43 kD. Moreover, MCP gene had been detected in 20 of 35 S. suis strains with different serotypes. This prevalence indicated that protein MCP might play a role in the survival of S. suis 2 in different hosts. Together, our work laid foundation for functional research on MCP in S. suis 2.
郭静静,杜骁杰,刘文静,郑峰,胡丹,王长军,潘秀珍. 猪链球菌2型甲基受体趋化蛋白编码基因05SSU0273的原核表达及纯化[J]. 中国人兽共患病学报, 2013, 29(3): 220-225.
GUO Jing-jing,DU Xiao-jie,LIU Wen-jing,ZHENG Feng, HU Dan,WANG Chang-jun,PAN Xiu-zhen. Prokaryotic expression and purification of the recombinant methyl-acceptingchemotaxis protein gene 05SSU0273 from Streptococcus suis serotype 2. Chinese Journal of Zoonoses, 2013, 29(3): 220-225.
[1] Staats JJ, Feder I, Okwumabua O, et al. Streptococcus suis: past and present[J] .Vet Res Commun,1997, 21(6): 381-407. [2] Gottschalk M, Segura M. The pathogenesis of the meningitis caused by Streptococcus suis: the unresolved questions[J] . Vet Microbiol, 2000, 76(3): 259-272. DOI: 10.1016/S0378-1135(00)00250-9 [3] Tang J, Wang C, Feng Y, et al. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2[J] . PLoS Med, 2006, 3(5): 668-676. DOI: 10.1371/journal.pmed.0030151 [4] Lun Z, Wang Q, Chen X, et al. Streptococcus suis: an emerging zoonotic pathogen[J] . Lancet Infect Dis, 2007, 7(3): 201-209. DOI: 10.1016/S1473-3099(07)70001-4 [5] Ye C, Zhu X, Jing H, et al. Streptococcus suis sequence type 7 outbreak, Sichuan,China[J] . Emerg Infec Dis, 2006, 12(8): 1203-1208. DOI: 10.3201/eid1208.060232 [6] Fittipaldi N, Segura M, Grenier D, et al. Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis[J] . Future Microbiol, 2012, 7(2): 259-279. DOI: 10.2217/fmb.11.149 [7] Baums CG, Kock C, Beineke A, et al. Streptococcus suis bacterin and subunit vaccine immunogenicities and protective efficacies against serotypes 2[J] . Clin Vaccine Immunol, 2009, 16( 2 ): 200-208. DOI: 10.1128/CVI.00371-08 [8] Shao Z, Pan X, Li X, et al. HtpS, a novel immunogenic cell surface-exposed protein of Streptococcus suis, confers protection in mice[J] . FEMS Microbiol Lett, 2011, 314(2): 174-182. DOI: 10.1111/j.1574-6968.2010.02162.x [9] De Greeff A, Buys H, van Alphen L, et al. Response regulator important in pathogenesis of Streptococcus suis serotype 2[J] . Microb Pathog, 2002, 33(4): 185-192. DOI: 10.1006/mpat.2002.0526 [10] Li M, Wang C, Feng Y, et al. Sal K/ SalR, a two-component signal t ransduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2[J] . PLoS ONE, 2008, 3(5): e2080. DOI:10.1371/journal.pone.0002080 [11] Feng XD,Cheng G, Wang J, et al. Construction and characterization of a two-component signal transduction system ciaR gene knock-out mutant of Streptococcus suis serotype 2[J] . Chin J Zoonoses, 2009, 25(3): 214-219. (in Chinese) 冯晓丹,程功,王晶,等.猪链球菌2型二元信号转导系统ciaR基因敲除突变株的构建及生物学功能研究[J] .中国人兽共患病学报,2009,25(3):214-219. [12] Marilyn RK, Frederick WD. The methyl-accepting chemotaxis proteinof Escherichia coli[J] . Biological Chem, 1982, 257(17): 10378-10386. [13] Sourjik V, Berg HC. Receptor sensitivity in bacterial chemotaxis[J] . PNAS, 2002, 99(1): 123-127. DOI: 10.1073/pnas.011589998 [14] Calperin MY, Nikolskaya AN, Koonin EV. Novel domains of prolulryorkieic two-component signal transduction systems[J] . FEMS Microbiol Lett, 2001, 203(2001): 11-21. DOI: 10.1016/S0378-1097(01)00326-3 [15] Kristich CJ, Ordal GW. Analysis of chimeric chemoreceptors in Bacillus subtilis reveals a role for CheD in the function of the McpC HAMP domain[J] . J Bacteriol, 2004 , 186(17): 5950-5955. DOI: 10.1128/JB.186.17.5950-5955.2004 [16] Amati G, Bisicchia P, Galizzi A. DegU-P represses expression of the motility fla-che operon in Bacillus subtilis[J] .J Bacteriol,2004, 186(18): 6003-6014. DOI: 10.1128/JB.186.18.6003-6014.2004 [17] Mariconda S, Wang Q, Harshey RM. A mechanical role for the chemotaxis system in swarming motility[J] .Mol Microbiol, 2006, 60(6): 1590-1602. DOI:10.1111/j.1365-2958.2006.05208.x [18] Morehouse KA, Goodfellow IG, Sockett RE. A chimeric N-terminal Escherichia coli--C-terminal Rhodobacter sphaeroides FliG rotor protein supports bidirectional E. coli flagellar rotation and chemotaxis[J] .J Bacteriol, 2005, 187(5): 1695-1701. DOI:10.1128/JB.187.5.1695-1701.2005 [19] Smith JG, Latiolais JA, Guanga GP, et al. Investigation of the role of electrostatic charge in activation of the Escherichia coli response regulator CheY[J] .J Bacteriol, 2001, 85(21): 6385-6391. DOI: 10.1128/JB.185.21.6385-6391.2003 [20] Terry K, Williams SM, Connolly L, et al. Chemotaxis plays multiple roles during Helicobacter pylori animal infection[J] .Infect Immun,2005, 73(2): 803-811. DOI:10.1128/IAI.73.2.803-811.2005 [21] Oleksiuk O, Jakovljevic V, Vladimirov N, et al. Thermal robustness of signaling in bacterial chemotaxis[J] . Cell,2011, 145(2): 312-321. DOI:10.1016/j.cell.2011.03.013 [22] Sourjik V. Receptor clustering and signal processing in E. coli chemotaxis[J] . Trends Microbiol, 2004, 12(12): 569-576. DOI:10.1016/j.tim.2004.10.003 [23] Xin X, Othmer HG. A "trimer of dimers"-based model for the chemotactic signal transduction network in bacterial chemotaxis[J] . Bull Math Biol, 2012, 74(10): 2339-2382. DOI: 10.1007/s11538-012-9756-7 [24] Yuan J, Branch RW, Hosu BG, et al. Adaptation at the output of the chemotaxis signalling pathway[J] . Nature, 2012, 484(7393): 233-236. DOI: 10.1038/nature10964 [25] Silva MA, Lucas TG, Salgueiro CA, et al. Protein folding modulates the swapped dimerization mechanism of methyl-accepting chemotaxis heme sensors[J] .PloS One,2012, 7(9): e46328. DOI: 10.1371/journal.pone.0046328 [26] Glekas GD, Mulhern BJ, Kroc A, et al. The Bacillus subtilis chemoreceptor McpC senses multiple ligands using two discrete mechanisms[J] . J Biol Chem, 2012, 287(47): 39412-39418. DOI: 10.1074/jbc.M112.413518 [27] Van Der DC, Duiverman J, Bexkens H, et al. Chemotaxis of a motile Streptococcus toward sugars and amino acids[J] . J Bacteriol, 1975, 124(3): 1142-1147. [28] Pan X, Ge J, Li M, et al. The orphan response regulator covR: a globally negative modulator of virulencein Streptococcus suis serotype 2[J] . J Bacteriol, 2009, 191(8): 2601-2612. DOI: 10.1128/JB.01309-08 [29] Chuiko NV. Chemotaxis of Bacillus subtilis toward organic compounds[J] . Mikrobiol Z, 2011, 73(3): 39-45 [30] Le Moual H, Koshland DE Jr. Molecular evolution of the C-terminal cytoplasmic domain of a superfamily of bacterial receptors involved in Taxis[J] . J Mol Biol, 1996, 261 (4): 568-585. DOI: 10.1006/jmbi.1996.0483