吉兰泰盐湖土壤嗜盐菌的分离及其产四氢嘧啶的研究
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摘要
【目的】对吉兰泰盐湖土壤嗜盐菌进行分离鉴定及生物学特征和系统发育分析,探索利用菌株合成四氢嘧啶的可能性。【方法】从内蒙古吉兰泰盐湖采集土壤样本,通过0,1%,2%,3%,4%,5%,6%,8%,10%,12%,14%,16%,18%,20%(质量分数)NaCl梯度耐盐试验,对分离放线菌进行分类统计。对分离得到的2株边界极端嗜盐菌JG5、JG9进行培养特征观察和生物学特性研究,基于16SrRNA基因序列构建系统发育树。利用四氢嘧啶基因簇中最为保守的ectB基因,合成简并引物,PCR扩增筛选含有四氢嘧啶基因簇的菌株。体积分数80%乙醇法抽提胞内四氢嘧啶,薄层层析(TLC)定性检测菌株胞内四氢嘧啶的存在情况,高效液相色谱(HPLC)法定量检测不同NaCl处理(质量分数为0,5%,10%,15%)JG5和JG9的胞内四氢嘧啶积累量。【结果】从吉兰泰盐湖土壤中分离得到嗜盐放线菌28株,其中主要为中度嗜盐放线菌,占总数的60.7%;弱嗜盐菌次之,占21.4%;非嗜盐菌3株,占10.7%;边界极端嗜盐菌最少,有2株,分别为JG5和JG9,占7.2%。PCR结果显示,28株中有17株含四氢嘧啶基因簇,占60.7%。生理生化特征研究结果显示,2株边界极端嗜盐菌JG5、JG9能利用的碳源种类丰富,也可利用多种氨基酸作为氮源,但JG9不能利用柠檬酸钠作为碳源,JG5和JG9均不能利用谷氨酸作为氮源;JG5和JG9都能使硝酸盐还原、明胶分解、牛奶凝固与胨化,都能产生H_2S,能降解纤维素,两者都不能分解尿素。系统发育树表明,2株极端嗜盐菌皆属于链霉菌属(Streptomyces)的密旋链霉菌(Streptomyces pactum),且亲缘关系较近。薄层层析结果表明,17株中有11株菌株可产四氢嘧啶。高效液相色谱分析结果显示,NaCl质量分数为10%时,JG5胞内四氢嘧啶积累量达最大值,为80.35mg/L;NaCl质量分数为5%时,JG9积累量达到最大值,为97.89mg/L。【结论】分离菌株大多含有四氢嘧啶基因簇,部分能够生产四氢嘧啶。2株边界极端嗜盐菌JG5和JG9在不同盐环境下,胞内均能产生四氢嘧啶。
【Objective】Isolation,identification,biological characteristics and phylogenetic analysis of halophiles from Jilantai Saline Lake soil were conducted to explore the feasibility of producing ectoine using the isolated strain.【Method】Soil samples were collected from Jilantai Salt Lake,and classified by NaCl test with mass fractions of 0,1%,2%,3%,4%,5%,6%,8%,10%,12%,14%,16%,18%,and 20%.Two strains of borderline halophilic bacteria of JG5 and JG9 were isolated and cultured.The phylogenetic tree was constructed based on 16 SrRNA gene sequence.Using the most conserved ectBgene in the ectoine gene cluster,degenerate primers were synthesized to screen the strains containing ectoine gene cluster.The intracellular ectoine was extracted by 80%(volume fraction)ethanol.The existence of intracellular ectoine was qualitatively detected by TLC.The accumulation of intracellular ectoine in JG5 and JG9 at different concentrations of NaCl(0,5%,10%,and 15%,mass fraction)was quantitatively determined by HPLC.【Result】A total of 28 halophilic actinomycetes were isolated,with moderately halophilic actinomycetes of60.7%,weak halophilic bacteria of 21.4%,and non-halophilic bacteria of 10.7%.The borderline extreme halophilic bacteria were the least(7.2%)with two strains of JG5 and JG9.PCR showed that there were 17 strains containing ectoine gene cluster,accounting for 60.7% of the total.Physiological and biochemical studies showed that the two strains could use abundant carbon sources and use many amino acids as nitrogen sources.JG9 cannot use sodium citrate as carbon source,while neither JG5 nor JG9 can use glutamate as nitrogen source.Both JG5 and JG9 can reduce nitrate,decompose gelatin,and coagulate milk and peptone.Both of them can produce H_2S and degrade cellulose,while neither can decompose urea.Phylogenetic analysis of JG5 and JG9 showed that the two strains belonged to Streptomyces pactum,and the relationship between JG5 and JG9 was close.TLC showed that 11 strains can produce intracellular ectoine.HPLC showed that the intracellular accumulation of ectoine of strain JG5 reached the maximum concentration of80.35 mg/L in 10% NaCl,and intracellular accumulation of ectoine of strain JG9 reached the maximum concentration of 97.89 mg/L in 5% NaCl.【Conclusion】Most of the isolated strains contained ectoine gene cluster,and some could produce intracellular ectoine.Two strains of extreme halophilic bacteria,JG5 and JG9,could produce intracellular ectoine in different salt environments.
【Objective】Isolation,identification,biological characteristics and phylogenetic analysis of halophiles from Jilantai Saline Lake soil were conducted to explore the feasibility of producing ectoine using the isolated strain.【Method】Soil samples were collected from Jilantai Salt Lake,and classified by NaCl test with mass fractions of 0,1%,2%,3%,4%,5%,6%,8%,10%,12%,14%,16%,18%,and 20%.Two strains of borderline halophilic bacteria of JG5 and JG9 were isolated and cultured.The phylogenetic tree was constructed based on 16 SrRNA gene sequence.Using the most conserved ectBgene in the ectoine gene cluster,degenerate primers were synthesized to screen the strains containing ectoine gene cluster.The intracellular ectoine was extracted by 80%(volume fraction)ethanol.The existence of intracellular ectoine was qualitatively detected by TLC.The accumulation of intracellular ectoine in JG5 and JG9 at different concentrations of NaCl(0,5%,10%,and 15%,mass fraction)was quantitatively determined by HPLC.【Result】A total of 28 halophilic actinomycetes were isolated,with moderately halophilic actinomycetes of60.7%,weak halophilic bacteria of 21.4%,and non-halophilic bacteria of 10.7%.The borderline extreme halophilic bacteria were the least(7.2%)with two strains of JG5 and JG9.PCR showed that there were 17 strains containing ectoine gene cluster,accounting for 60.7% of the total.Physiological and biochemical studies showed that the two strains could use abundant carbon sources and use many amino acids as nitrogen sources.JG9 cannot use sodium citrate as carbon source,while neither JG5 nor JG9 can use glutamate as nitrogen source.Both JG5 and JG9 can reduce nitrate,decompose gelatin,and coagulate milk and peptone.Both of them can produce H_2S and degrade cellulose,while neither can decompose urea.Phylogenetic analysis of JG5 and JG9 showed that the two strains belonged to Streptomyces pactum,and the relationship between JG5 and JG9 was close.TLC showed that 11 strains can produce intracellular ectoine.HPLC showed that the intracellular accumulation of ectoine of strain JG5 reached the maximum concentration of80.35 mg/L in 10% NaCl,and intracellular accumulation of ectoine of strain JG9 reached the maximum concentration of 97.89 mg/L in 5% NaCl.【Conclusion】Most of the isolated strains contained ectoine gene cluster,and some could produce intracellular ectoine.Two strains of extreme halophilic bacteria,JG5 and JG9,could produce intracellular ectoine in different salt environments.
引文
[1]任培根,周培瑾.中度嗜盐菌的研究进展[J].微生物学报,2003,43(3):427-431.Ren P G,Zhou P J.Research progress of moderately Halophilic eubacteria[J].Acta Microbiologica Sinica,2003,43(3):427-431.
[2]Hanelt I,Muller V.Molecular mechanisms of adaptation of the moderately halophilic bacterium halobacillis halophilus to its environment[J].Life(Basel),2013,3(1):234-243.
[3]赵百锁,杨礼富,宋蕾,等.中度嗜盐菌在生物技术中的应用[J].微生物学通报,2007,34(2):359-362.Zhao B S,Yang L F,Song L,et al.Biotechnology applications in moderately Halophilic eubacteria[J].Microbiology China,2007,34(2):359-362.
[4]Perez-Garcia F,Ziert C,Risse J M,et al.Improved fermentative production of the compatible solute ectoine by Corynebacterium glutamicumfrom glucose and alternative carbon sources[J].JBiotechnol,2017,258:59-68.
[5]郭艳丽,张培玉,于德爽,等.嗜盐菌与高盐度废水生物处理研究进展[J].环境科学与管理,2008,33(9):79-83.Guo Y L,Zhang P Y,Yu D S,et al.Advance of halophile and biological treatment of high-salinity wastewater[J].Environmental Science and Management,2008,33(9):79-83.
[6]Gan L,Zhang H,Tian J,et al.Planococcus salinus sp.nov.,a moderately halophilic bacterium isolated from a saline-alkali soil[J].Int J Syst Evol Microbiol,2018,68(2):589-595.
[7]Bursy J,Kuhlmann A U,Pittelkow M,et al.Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2)in response to salt and heat stresses[J].Appl Environ Microbiol,2008,74(23):7286-7296.
[8]Obruca S,Sedlacek P,Mravec F,et al.Evaluation of 3-hydroxybutyrate as an enzyme-protective agent against heating and oxidative damage and its potential role in stress response of poly(3-hydroxybutyrate)accumulating cells[J].Appl Microbiol Biotechnol,2016,100(3):1365-1376.
[9]Roychoudhury A,Haussinger D,Oesterhelt F.Effect of the compatible solute ectoine on the stability of the membrane proteins[J].Protein and Peptide Letters,2012,19(8):791-794.
[10]Harishchandra R K,Wulff S,Lentzen G,et al.The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes[J].Biophysical Chemistry,2010,150(121):37-46.
[11]Pastor J M,Salvador M,Argandona M,et al.Ectoines in cell stress protection:uses and biotechnological production[J].Biotechnology Advances,2010,28(6):782-801.
[12]Graf R,Anzali S,Buenger J,et al.The multifunctional role of ectoine as a natural cell protectant[J].Clin Dermatol,2008,26(4):326-333.
[13]Ma Y,Wang Q,Xu W,et al.Stationary phase-dependent accumulation of ectoine is an efficient adaptation strategy in Vibrio anguillarum against cold stress[J].Microbiol Res,2017,205:8-18.
[14]朱道展,刘杏荣.相容性溶质四氢嘧啶及其羟基化衍生物的研究进展[J].中国生物工程杂志,2011,31(2):95-101.Zhu D C,Liu X R.Research progress of compatible solute ectoine and its hydroxylation derivatives[J].China Biotechnology,2011,31(2):95-101.
[15]Kanapathipillai M,Lentzen G,Sierks M,et al.Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer’s beta-amyloid[J].FEBS Letters,2005,579(21):4775-4780.
[16]安晓英.缬草内生及根际土壤放线菌的分离鉴定及次级代谢产物生物合成基因的克隆[D].陕西杨凌:西北农林科技大学,2015.An X Y.Isolation,identification and antibiotics biosynthesis genes cloning of endophytic and rhizosphere actinomycetes of valerian[D].Yangling,Shaanxi:Northwest A&F University,2015.
[17]Kieser T,Bibb M J,Buttner M J,et al.Practical streptomyces genetics[M].Norwich,UK:the John Innes Foundation,2000:161-210.
[18]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:364-398.Dong X Z,Cai M Y.Manual for identification of common bacterial systems[M].Beijing:Science Press,2001:364-398.
[19]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870.
[20]Liu L,Xi Z,Wu S,et al.Estimating phylogenetic trees from genome-scale data[J].Annals of the New York Academy of Sciences,2015,1360:36-53.
[21]Willems M,Tahiri N,Makarenkov V.A new efficient algorithm for inferring explicit hybridization networks following the Neighbor-Joining principle[J].J Bioinform Comput Biol,2014,12(5):1450024.
[22]Tamura K,Nei M,Kumar S.Prospects for inferring very large phylogenies by using the neighbor-joining method[J].Proceedings of the National Academy of Sciences(USA),2004,101(30):11030-11035.
[23]Zapf A,Brunner E,Konietschke F.A wild bootstrap approach for the selection of biomarkers in early diagnostic trials[J].BMC Med Res Methodol,2015,15:43.
[24]朱德锐,刘建,刘静静,等.青海湖嗜盐菌的分离与优势菌株QHL5的特性[J].应用与环境生物学报,2012,18(5):797-803.Zhu D R,Liu J,Liu J J,et al.Isolation of halophiles from Qinghai Lake and growth characteristics of dominant strain QHL5[J].Chin J Appl Environ Biol,2012,18(5):797-803.
[25]李耀东,龙启福,李文军,等.高效液相色谱检测青海湖嗜盐菌胞内积聚的相溶物质四氢嘧啶[J].环境化学,2013,32(9):1687-1691.Li Y D,Long Q F,Li W J,et al.Detection of soluble substance intracellular ectoine of halophiles in Qinghai Lake by HPLC[J].Environmental Chemistry,2013,32(9):1687-1691.
[26]于志同,刘兴起,王永,等.13.8ka以来内蒙古吉兰泰盐湖的演化过程[J].湖泊科学,2012,24(4):629-636.Yu Z T,Liu X Q,Wang Y,et al.Evolution of Jilantai Salt Lake,Inner Mongolia in the last 13.8ka[J].Journal of Lake Sciences,2012,24(4):629-636.
[27]Nakayama H,Yoshida K,Ono H,et al.Ectoine,the compatible solute of Halomonas elongata,confers hyperosmotic tolerance in cultured tobacco cells[J].Plant Physiology,2000,122(4):1239-1247.
[28]高波.吉兰泰盐湖土壤中嗜盐碱放线菌的分离鉴定及四氢嘧啶工程菌株的构建[D].陕西杨凌:西北农林科技大学,2016.Gao B.Isolation and identification of basophilic actinomycetes and halophilic actinomycetes in the soil of Jilantai Saline Lake and construction of ectoine engineering strains[D].Yangling,Shaanxi:Northwest A&F University,2016.
[29]吕爱军,胡斌,温洪宇,等.极端嗜盐菌的特性及其应用前景[J].微生物学杂志,2005,25(2):65-68.LüA J,Hu B,Wen H Y,et al.Characteristics of extreme halophile and its application prospects[J].Journal of Microbiology,2005,25(2):65-68.
[2]Hanelt I,Muller V.Molecular mechanisms of adaptation of the moderately halophilic bacterium halobacillis halophilus to its environment[J].Life(Basel),2013,3(1):234-243.
[3]赵百锁,杨礼富,宋蕾,等.中度嗜盐菌在生物技术中的应用[J].微生物学通报,2007,34(2):359-362.Zhao B S,Yang L F,Song L,et al.Biotechnology applications in moderately Halophilic eubacteria[J].Microbiology China,2007,34(2):359-362.
[4]Perez-Garcia F,Ziert C,Risse J M,et al.Improved fermentative production of the compatible solute ectoine by Corynebacterium glutamicumfrom glucose and alternative carbon sources[J].JBiotechnol,2017,258:59-68.
[5]郭艳丽,张培玉,于德爽,等.嗜盐菌与高盐度废水生物处理研究进展[J].环境科学与管理,2008,33(9):79-83.Guo Y L,Zhang P Y,Yu D S,et al.Advance of halophile and biological treatment of high-salinity wastewater[J].Environmental Science and Management,2008,33(9):79-83.
[6]Gan L,Zhang H,Tian J,et al.Planococcus salinus sp.nov.,a moderately halophilic bacterium isolated from a saline-alkali soil[J].Int J Syst Evol Microbiol,2018,68(2):589-595.
[7]Bursy J,Kuhlmann A U,Pittelkow M,et al.Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2)in response to salt and heat stresses[J].Appl Environ Microbiol,2008,74(23):7286-7296.
[8]Obruca S,Sedlacek P,Mravec F,et al.Evaluation of 3-hydroxybutyrate as an enzyme-protective agent against heating and oxidative damage and its potential role in stress response of poly(3-hydroxybutyrate)accumulating cells[J].Appl Microbiol Biotechnol,2016,100(3):1365-1376.
[9]Roychoudhury A,Haussinger D,Oesterhelt F.Effect of the compatible solute ectoine on the stability of the membrane proteins[J].Protein and Peptide Letters,2012,19(8):791-794.
[10]Harishchandra R K,Wulff S,Lentzen G,et al.The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes[J].Biophysical Chemistry,2010,150(121):37-46.
[11]Pastor J M,Salvador M,Argandona M,et al.Ectoines in cell stress protection:uses and biotechnological production[J].Biotechnology Advances,2010,28(6):782-801.
[12]Graf R,Anzali S,Buenger J,et al.The multifunctional role of ectoine as a natural cell protectant[J].Clin Dermatol,2008,26(4):326-333.
[13]Ma Y,Wang Q,Xu W,et al.Stationary phase-dependent accumulation of ectoine is an efficient adaptation strategy in Vibrio anguillarum against cold stress[J].Microbiol Res,2017,205:8-18.
[14]朱道展,刘杏荣.相容性溶质四氢嘧啶及其羟基化衍生物的研究进展[J].中国生物工程杂志,2011,31(2):95-101.Zhu D C,Liu X R.Research progress of compatible solute ectoine and its hydroxylation derivatives[J].China Biotechnology,2011,31(2):95-101.
[15]Kanapathipillai M,Lentzen G,Sierks M,et al.Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer’s beta-amyloid[J].FEBS Letters,2005,579(21):4775-4780.
[16]安晓英.缬草内生及根际土壤放线菌的分离鉴定及次级代谢产物生物合成基因的克隆[D].陕西杨凌:西北农林科技大学,2015.An X Y.Isolation,identification and antibiotics biosynthesis genes cloning of endophytic and rhizosphere actinomycetes of valerian[D].Yangling,Shaanxi:Northwest A&F University,2015.
[17]Kieser T,Bibb M J,Buttner M J,et al.Practical streptomyces genetics[M].Norwich,UK:the John Innes Foundation,2000:161-210.
[18]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:364-398.Dong X Z,Cai M Y.Manual for identification of common bacterial systems[M].Beijing:Science Press,2001:364-398.
[19]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870.
[20]Liu L,Xi Z,Wu S,et al.Estimating phylogenetic trees from genome-scale data[J].Annals of the New York Academy of Sciences,2015,1360:36-53.
[21]Willems M,Tahiri N,Makarenkov V.A new efficient algorithm for inferring explicit hybridization networks following the Neighbor-Joining principle[J].J Bioinform Comput Biol,2014,12(5):1450024.
[22]Tamura K,Nei M,Kumar S.Prospects for inferring very large phylogenies by using the neighbor-joining method[J].Proceedings of the National Academy of Sciences(USA),2004,101(30):11030-11035.
[23]Zapf A,Brunner E,Konietschke F.A wild bootstrap approach for the selection of biomarkers in early diagnostic trials[J].BMC Med Res Methodol,2015,15:43.
[24]朱德锐,刘建,刘静静,等.青海湖嗜盐菌的分离与优势菌株QHL5的特性[J].应用与环境生物学报,2012,18(5):797-803.Zhu D R,Liu J,Liu J J,et al.Isolation of halophiles from Qinghai Lake and growth characteristics of dominant strain QHL5[J].Chin J Appl Environ Biol,2012,18(5):797-803.
[25]李耀东,龙启福,李文军,等.高效液相色谱检测青海湖嗜盐菌胞内积聚的相溶物质四氢嘧啶[J].环境化学,2013,32(9):1687-1691.Li Y D,Long Q F,Li W J,et al.Detection of soluble substance intracellular ectoine of halophiles in Qinghai Lake by HPLC[J].Environmental Chemistry,2013,32(9):1687-1691.
[26]于志同,刘兴起,王永,等.13.8ka以来内蒙古吉兰泰盐湖的演化过程[J].湖泊科学,2012,24(4):629-636.Yu Z T,Liu X Q,Wang Y,et al.Evolution of Jilantai Salt Lake,Inner Mongolia in the last 13.8ka[J].Journal of Lake Sciences,2012,24(4):629-636.
[27]Nakayama H,Yoshida K,Ono H,et al.Ectoine,the compatible solute of Halomonas elongata,confers hyperosmotic tolerance in cultured tobacco cells[J].Plant Physiology,2000,122(4):1239-1247.
[28]高波.吉兰泰盐湖土壤中嗜盐碱放线菌的分离鉴定及四氢嘧啶工程菌株的构建[D].陕西杨凌:西北农林科技大学,2016.Gao B.Isolation and identification of basophilic actinomycetes and halophilic actinomycetes in the soil of Jilantai Saline Lake and construction of ectoine engineering strains[D].Yangling,Shaanxi:Northwest A&F University,2016.
[29]吕爱军,胡斌,温洪宇,等.极端嗜盐菌的特性及其应用前景[J].微生物学杂志,2005,25(2):65-68.LüA J,Hu B,Wen H Y,et al.Characteristics of extreme halophile and its application prospects[J].Journal of Microbiology,2005,25(2):65-68.