一株产L-天冬氨酸酶大肠埃希氏菌的噬菌体分离和生物学特性
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摘要
【目的】从大肠埃希氏菌CICC 11021S发酵液中分离一株噬菌体,对其生物学特性进行研究。【方法】采用双层平板法分离噬菌体CICC 80003;利用透射电镜观察噬菌体形态;提取噬菌体基因组,核酸内切酶处理并进行凝胶电泳;分析噬菌体最佳感染复数、一步生长曲线、p H和温度稳定性、宿主谱。考察CICC 80003对CICC 11021S生长和L-天冬氨酸酶活力的影响。【结果】CICC 80003噬菌斑圆形透明,有明显晕环;头部规则,直径约50-60 nm,尾部长约120-130 nm;基因组能被核酸内切酶Bam H I和Mlu I切开;最佳感染复数0.1,潜伏期5 min,裂解期25 min,平均裂解量约86个;最适p H值8.0;90°C温育15 min,噬菌体全部失活;能裂解大肠埃希氏菌和沙门氏菌的部分菌株。发生噬菌体污染时,CICC 11021S无法正常生长,基本检测不到L-天冬氨酸酶活力。【结论】CICC 80003属于长尾噬菌体科ds DNA噬菌体,液体环境中能够彻底裂解大肠埃希氏菌CICC 11021S。
[Objective] A phage was isolated from Escherichia coli CICC 11021 S fermentation media, and its biological characterization was studied. [Methods] A phage designated CICC 80003 was isolated using the double-layer agar culture method. Phage morphology was observed by transmission electron microscopy. Gel electrophoresis of phage genome was carried out after genome extraction and treatment by endonucleases. We investigated the optimal multiplicity of infection, one-step growth curve, p H and temperature stability, and host range. Additionally, we analyzed the effects of CICC 80003 on cell growth and L-aspartase activity of CICC 11021 S. [Results] The phage plaque was round and transparent, and showed clear aureole. The phage had a regular head of 50-60 nm in diameter and a long tail with 120-130 nm in length. The phage genome could be cut by endonucleases Bam H I and Mlu I. The optimal multiplicity of infection was 0.1. The one-step growth curve showed a latent period of 5 min and a rise period of 25 min. The average burst size was about 86 phage particles per infected cell. The optimal p H was 8.0. The phage was completely inactivated when incubated at 90 °C for 15 min. Some of Escherichia coli and Salmonella spp. strains could be split by CICC 80003. No cell growth and L-aspartase activity was detected when phage contamination occurred. [Conclusion] CICC 80003 was a ds DNA phage and belonged to the family Siphoviridae, which could completely split E. coli CICC 11021 S in broth.
[Objective] A phage was isolated from Escherichia coli CICC 11021 S fermentation media, and its biological characterization was studied. [Methods] A phage designated CICC 80003 was isolated using the double-layer agar culture method. Phage morphology was observed by transmission electron microscopy. Gel electrophoresis of phage genome was carried out after genome extraction and treatment by endonucleases. We investigated the optimal multiplicity of infection, one-step growth curve, p H and temperature stability, and host range. Additionally, we analyzed the effects of CICC 80003 on cell growth and L-aspartase activity of CICC 11021 S. [Results] The phage plaque was round and transparent, and showed clear aureole. The phage had a regular head of 50-60 nm in diameter and a long tail with 120-130 nm in length. The phage genome could be cut by endonucleases Bam H I and Mlu I. The optimal multiplicity of infection was 0.1. The one-step growth curve showed a latent period of 5 min and a rise period of 25 min. The average burst size was about 86 phage particles per infected cell. The optimal p H was 8.0. The phage was completely inactivated when incubated at 90 °C for 15 min. Some of Escherichia coli and Salmonella spp. strains could be split by CICC 80003. No cell growth and L-aspartase activity was detected when phage contamination occurred. [Conclusion] CICC 80003 was a ds DNA phage and belonged to the family Siphoviridae, which could completely split E. coli CICC 11021 S in broth.
引文
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[17]Zhou HY,Liu L,Du GC,et al.Screening of bacteriophage-resistant strain for L-phenylalanine production[J].Food and Fermentation Industries,2013,39(7):88-93(in Chinese)周海岩,刘龙,堵国成,等.L-苯丙氨酸抗噬菌体生产菌的选育和应用[J].食品与发酵工业,2013,39(7):88-93
[18]Breitbart M,Rohwer F.Here a virus,there a virus,everywhere the same virus?[J].Trends in Microbiology,2005,13(6):278-284
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[22]Anton BP,Raleigh EA.Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli Mcr A endonuclease[J].Journal of Bacteriology,2004,186(17):5699-5707
[23]Meineke B,Shuman S.Determinants of the cytotoxicity of Prr C anticodon nuclease and its amelioration by t RNA repair[J].RNA,2012,18(1):145-154
[24]Hazan R,Engelberg-Kulka H.Escherichia coli maz EF-mediated cell death as a defense mechanism that inhibits the spread of phage P1[J].Molecular Genetics and Genomics,2004,272(2):227-234
[2]Sheng XY,Fu YQ,Li S,et al.Study on a new strategy for the cultivation of L-ASP-producing bacteria utilizing the wasted fumaric acid fermentation broth[J].Food Science and Technology,2010,35(1):23-26(in Chinese)盛晓燕,付永前,李霜,等.利用富马酸发酵废液培养L-天冬氨酸转化菌策略的研究[J].食品科技,2010,35(1):23-26
[3]Huffer S,Roche CM,Blanch HW,et al.Escherichia coli for biofuel production:bridging the gap from promise to practice[J].Trends in Biotechnology,2012,30(10):538-545
[4]Sun WJ,Mo QY,Liu CF,et al.Bacteriophage in fermentation industry:sources,detection and control actions[J].Food Science and Technology,2013,38(8):323-327(in Chinese)孙文敬,莫秋云,刘长峰,等.发酵工业噬菌体污染的来源、检测与防治[J].食品科技,2013,38(8):323-327
[5]Liu Y,Ma YY,Xu YQ,et al.Isolation and physiological characteristics study of a bacteriophage against an Escherichia coli with L-aspartase production[J].Journal of Microbiology,2015,35(5):43-46(in Chinese)刘勇,马玉岳,徐友强,等.一株产L-天冬氨酸酶大肠杆菌噬菌体分离和生理特性初步研究[J].微生物学杂志,2015,35(5):43-46
[6]Ma YY,Jiang GZ,Jiang ZY,et al.An Escherichia coli strain producing high yield of L-aspartase and its application:China,201110382584[P].2012-07-11(in Chinese)马玉岳,姜国政,姜增妍,等.高产L-天冬氨酸酶的大肠杆菌及其应用:中国,201110382584[P].2012-07-11
[7]Nan N,Cao F,Shen JT,et al.Characterization of a bacteriophage of Klebsiella pneumoniae producing1,3-propanediol[J].Acta Microbiologica Sinica,2013,53(9):943-949(in Chinese)南南,曹放,沈俊涛,等.一株产1,3-丙二醇的克雷伯氏菌的噬菌体生物学特性[J].微生物学报,2013,53(9):943-949
[8]Jiang YH,Li FL,Wang LZ,et al.Isolation,identification and biological properties of a lytic phage against Salmonella[J].Microbiology China,2015,42(3):534-542(in Chinese)江艳华,李风铃,王联珠,等.一株沙门氏菌裂解性噬菌体的分离鉴定及生物学特性[J].微生物学通报,2015,42(3):534-542
[9]Bertani G.Lysogeny at mid-twentieth century:P1,P2,and other experimental systems[J].Journal of Bacteriology,2004,186(3):595-600
[10]Yu ZC.Study of the adaptation mechanism of marine Pseudomonas to sea ice environment and construction of their genetic manipulation system[D].Ji’nan:Doctoral Dissertation of Shandong University,2014(in Chinese)于子超.海洋假交替单胞菌适应海冰环境的机制及其遗传操作体系的建立[D].济南:山东大学博士学位论文,2014
[11]Lu Z,Breidt F Jr,Fleming HP,et al.Isolation and characterization of a Lactobacillus plantarum bacteriophage,?JL-1,from a cucumber fermentation[J].International Journal of Food Microbiology,2003,84(2):225-235
[12]Leuschner RGK,Arendt EK,Hammes WP.Characterization of a virulent Lactobacillus sake phage PWH2[J].Applied Microbiology and Biotechnology,1993,39(4/5):617-621
[13]Pajunen M,Kiljunen S,Skurnik M.BacteriophageφYe O3-12,specific for Yersinia enterocolitica serotype O:3,is related to coliphages T3 and T7[J].Journal of Bacteriology,2000,182(18):5114-5120
[14]Sun WJ,Liu CF,Yu L,et al.A novel bacteriophage KSL-1 of2-Keto-gluconic acid producer Pseudomonas fluorescens K1005:isolation,characterization and its remedial action[J].BMC Microbiology,2012,12:127
[15]Feng SZ,Liu J,Sun Y.An introduction to the bacterial viruses in8th report of ICTV[J].Chinese Journal of Veterinary Science,2007,27(4):604-608(in Chinese)冯书章,刘军,孙洋.细菌的病毒——噬菌体最新分类与命名[J].中国兽医学报,2007,27(4):604-608
[16]Hu YF,Li TM,Yang ZY,et al.Phage resistance of Corynebacterium crenatum conferred by the restriction and modification system cgl I[J].Chinese Journal of Biotechnology,2008,24(5):760-765(in Chinese)胡永飞,李铁民,杨智勇,等.限制修饰系统cgl I赋予钝齿棒杆菌抗噬菌体功能活性[J].生物工程学报,2008,24(5):760-765
[17]Zhou HY,Liu L,Du GC,et al.Screening of bacteriophage-resistant strain for L-phenylalanine production[J].Food and Fermentation Industries,2013,39(7):88-93(in Chinese)周海岩,刘龙,堵国成,等.L-苯丙氨酸抗噬菌体生产菌的选育和应用[J].食品与发酵工业,2013,39(7):88-93
[18]Breitbart M,Rohwer F.Here a virus,there a virus,everywhere the same virus?[J].Trends in Microbiology,2005,13(6):278-284
[19]Guglielmotti DM,Binetti AG,Reinheimer JA,et al.Streptococcus thermophilus phage monitoring in a cheese factory:phage characteristics and starter sensitivity[J].International Dairy Journal,2009,19(8):476-480
[20]Stummeyer K,Schwarzer D,Claus H,et al.Evolution of bacteriophages infecting encapsulated bacteria:lessons from Escherichia coli K1-specific phages[J].Molecular Microbiology,2006,60(5):1123-1135
[21]Lu MJ,Stierhof YD,Henning U.Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4[J].Journal of Virology,1993,67(8):4905-4913
[22]Anton BP,Raleigh EA.Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli Mcr A endonuclease[J].Journal of Bacteriology,2004,186(17):5699-5707
[23]Meineke B,Shuman S.Determinants of the cytotoxicity of Prr C anticodon nuclease and its amelioration by t RNA repair[J].RNA,2012,18(1):145-154
[24]Hazan R,Engelberg-Kulka H.Escherichia coli maz EF-mediated cell death as a defense mechanism that inhibits the spread of phage P1[J].Molecular Genetics and Genomics,2004,272(2):227-234