单核细胞增生李斯特菌新型转录调控因子Lmo2672分子特征及其系统进化分析
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摘要
利用PCR方法扩增单核细胞增生李斯特菌(Listeria monocytogenes,LM)野毒株SB5新型转录调控因子lmo2672基因,进行克隆、测序,对其编码蛋白的二三级结构、理化特性、蛋白结构域等分子特征进行预测分析,并进行系统进化分析。结果显示:LM-SB5 lmo2672基因全长807 bp,共编码268 aa;二级结构预测发现,该蛋白含有1个DNA结合域(185～259 aa),2个由螺旋-转角-螺旋(helix-turn-helix)构成的HTH-18结构域(184～198 aa,226～249 aa),位于整个蛋白的C端,无信号肽序列及跨膜结构;序列比较发现,Lmo2672属于AraC家族转录调节蛋白,其核苷酸序列与NTSN株(4b型)同源性为99.63%;与国际标准株EGD-e株(1/2a型)的同源性为97.77%;系统进化树显示,LM-SB5 lmo2672核苷酸序列与谱系Ⅱ型NTSN等菌株亲缘关系较近,而与谱系Ⅰ型菌株标准株EGD-e等亲缘关系较远。该研究结果为下一步探讨lmo2672基因在LM毒力和环境应激中的调控作用奠定了理论基础。
In the present, a novel transcription regulatory factor Lmo2672 gene of Listeria monocytogenes was amplified from the SB5 strain by PCR method and cloned for sequencing in order to predict and analyze the molecular characteristics of the secondary and tertiary structure and domains. The results showed that the lmo2672 gene had a length of 807 bp, encoding 268 amino acids. Prediction of the protein structure showed that Lmo2672 protein included a DNA-binding domain and two HTH-18 domains of helix-turn-helix that were located at the C-terminal but had no signal peptide and transmembrane domains. The alignment of nucleotide sequences of the lmo2672 gene revealed the similarity at 99.63% with NTSN strain(serotype 4 b) and 97.77% with the international standard strain EGD-e(serotype1/2 a). The phylogenetic analysis showed that the lmo2672 gene of the SB5 strain was closely related with lineage Ⅱ such as NTSN strain but genetically far to lineage Ⅰ strains such as standard strain EGD-e. The results could provide a theoretical foundation for the further study of the regulatory lmo2672 gene in LM toxicity and environmental stresses.
In the present, a novel transcription regulatory factor Lmo2672 gene of Listeria monocytogenes was amplified from the SB5 strain by PCR method and cloned for sequencing in order to predict and analyze the molecular characteristics of the secondary and tertiary structure and domains. The results showed that the lmo2672 gene had a length of 807 bp, encoding 268 amino acids. Prediction of the protein structure showed that Lmo2672 protein included a DNA-binding domain and two HTH-18 domains of helix-turn-helix that were located at the C-terminal but had no signal peptide and transmembrane domains. The alignment of nucleotide sequences of the lmo2672 gene revealed the similarity at 99.63% with NTSN strain(serotype 4 b) and 97.77% with the international standard strain EGD-e(serotype1/2 a). The phylogenetic analysis showed that the lmo2672 gene of the SB5 strain was closely related with lineage Ⅱ such as NTSN strain but genetically far to lineage Ⅰ strains such as standard strain EGD-e. The results could provide a theoretical foundation for the further study of the regulatory lmo2672 gene in LM toxicity and environmental stresses.
引文
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[2]Gandhi M,Chikindas M L.Listeria:a foodborne pathogen that knows how to survive[J].Int J Food Microbiol,2007,113(1):1-15.
[3]王少辉,刘萍萍,魏建超,等.单增李斯特菌inl K基因缺失株的构建及其生物学特性分析[J].中国动物传染病学报,2017,25(4):19-23.
[4]江玲丽,陈健舜,方维焕.单核细胞增多性李斯特菌主要毒力因子研究进展[J].中国人兽共患病学报,2007,23(7):733-735,封3.
[5]Bradley A J,Leach K A,Green M J,et al.The impact of dairy cows'bedding material and its microbial content on the quality and safety of milk-A cross sectional study of UK farms[J].Int J Food Microbiol,2018,269:36-45.
[6]de las Heras A,Cain R J,Bielecka M K,et al.Regulation of Listeria virulence:Prf A master and commander[J].Curr Opin Microbiol,2011,14(2):118-127.
[7]陆承平.兽医微生物学[M].3版.北京:中国农业出版社,2001.
[8]Swaminathan B,Gerner-Smidt P.The epidemiology of human listeriosis[J].Microbes Infect,2007,9(10):1236-1243.
[9]V e l e b a M,H i g g i n s P G,G o n z a l e z G,e t a l.Characterization of Rar A,a novel Ara C family multidrug resistance regulator in Klebsiella pneumoniae[J].Antimicrob Agents Chemother,2012,56(8):4450-4458.
[10]Pietack N,Becher D,Schmidl S R,et al.In vitro phosphorylation of key metabolic enzymes from Bacillus subtilis:Prk C phosphorylates enzymes from different branches of basic metabolism[J].J Mol Microbiol Biotechnol,2010,18(3):129-140.
[11]Lowden M J,Skorupski K,Pellegrini M,et al.Structure of Vibrio cholerae Tox T reveals a mechanism for fatty acid regulation of virulence genes[J].Proc Natl Acad Sci USA,2010,107(7):2860-2865.
[12]Coburn P S,Baghdayan A S,Dolan G T,et al.An Aractype transcriptional regulator encoded on the Enterococcus faecalis pathogenicity island contributes to pathogenesis and intracellular macrophage survival[J].Infect Immum,2008,76(12):5668-5676.
[13]Ni L,Tonthat N K,Chinnam N,et al.Structures of the Escherichia coli transcription activator and regulator of diauxie,Xyl R:an Ara C DNA-binding family member with a Lac I/Gal R ligand-binding domain[J].Nucleic Acids Res,2013,41(3):1998-2008.
[14]Chatterjee S S,Hossain H,Otten S,et al.Intracellular gene expression profile of Listeria monocytogenes[J].Infect Immun,2006,74(2):1323-1338.
[15]Toledo-Arana A,Dussurget O,Nikitas G,et al.The Listeria transcriptional landscape from saprophytism to virulence[J].Nature,2009,459(7249):950-956.
[16]Yang J,Tauschek M,Robins-Browne R M.Control of bacterial virulence by Ara C-like regulators that respond to chemical signals[J].Trends Microbiol,2011,19(3):128-135.
[17]Stadtl?nder K H.Handbook of Listeria monocytogenes[J].Microbe,2009,4(5):250-251.
[18]Hadjilouka A,Molfeta C,Panagiotopoulou O,et al.Expression of Listeria monocytogenes key virulence genes during growth in liquid medium,on rocket and melon at 4,10 and 30℃[J].Food Microbiol,2016,55:7-15.
[19]Martin R G,Rosner J L.The Ara C transcriptional activators[J].Curr Opin Microbiol,2001,4(2):132-137.