喜温嗜酸硫杆菌SM-1在不同温度下基因组的可塑性
|
摘要
【目的】驯化得到喜温嗜酸硫杆菌(Acidithiobacillus caldus)SM-1在低于最适生长温度下具有较高生长活力的突变菌株,并认知喜温嗜酸硫杆菌在不同温度下的基因组可塑性。【方法】利用实验室长期进化实验对菌株进行3个温度的驯化:37、40、45°C。运用454测序技术对驯化获得的菌株进行基因组重测序,通过比较基因组分析驯化株基因组单核苷酸位点变化(SNPs),对包含位点变化的基因从功能上进行分类,在此基础上,分析可能与温度适应性相关的基因。【结果】通过不同温度下的长期驯化,得到了在低于最适生长温度下具有较高活力的菌株;重测序结果发现,SM-1基因组具有较好的可塑性,不同温度(37、40、45°C)生长的菌株中,基因组中分别有418、384和347个核苷酸位点发生累计变化,其中3个温度下有20个相同的非同义突变位点,分别分布于编码重金属和毒性抗性系统、DNA甲基化和蛋白乙酰化酶、核酸代谢相关酶类等相关基因上;相比而言,在低于最适生长温度(37、40°C)下生长菌株特有的位点变化涉及能量代谢、信号转导以及DNA/RNA稳定性相关基因;其中,2个低温菌株共同发生位点变化的基因有3个,其中两个编码转座相关的蛋白Atc_1031与Atc_1623,另一个编码假想蛋白Atc_1130,该蛋白分别与外膜蛋白组装因子B和二硫键形成蛋白具有23%和35%的相似性。另外,不同生长温度下相关蛋白中氨基酸的组成也发生变化。【结论】喜温嗜酸硫杆菌SM-1基因组具有较好的可塑性,对于其基因组变化的研究结果为认识微生物温度适应性提供了组学数据。本研究揭示喜温嗜酸硫杆菌(At.caldus)SM-1可能通过多种途径适应向低温过渡生长,既包括微生物通用的环境适应机制,也存在菌株特有的温度适应途径。
[Objective] The aim of this study is to gain the acclimated strain of Acidithiobacillus caldus SM-1 with high activity when growing at the temperatures below the optimum, and elaborate its genomic plasticity and adaptation at different temperatures. [Methods] Strains were incubated at 37 °C, 40 °C and 45 °C. We used 454 genome resequencing technology to find the single nucleotide mutant sites in genome. The genes including mutant sites were studied to understand their relationship with the temperature adaptation. [Results] By long-term breeding, we obtained strains with higher viability than unacclimated strain at temperatures(37 °C), which is lower than initial optimum growth temperature(45 °C). Resequencing results showed the genome of SM-1 with high plasticity. In the genome of different strains(incubated at 37 °C, 40 °C and 45 °C), 418, 384 and 347 single nucleotide mutation sites were accumulated, respectively. Among them, 20 mutant sites were commonly occurred in the three strains. The genes they affected are involved in heavy metals and toxic resistance system, DNA methylation and protein acylation, and nucleic acid metabolism. In comparison, the specific mutations of the strains, grown at the 37 °C and 40 °C, were related to energy metabolism, signal transduction and DNA/RNA stability. Three genes contained mutant sites are common in L37 and M40, of which, Atc_1031 and Atc_1623 encode proteins related to transposon insertion, Atc_1130 encodes a hypothetical protein that is similar to the out membrane protein assembly factor B or the disulfide bond formation protein with 23% and 35% similarity. In addition, some single nucleotide mutations cause the amino acid changes of the related proteins during the adaption. [Conclusion] The genome of At. caldus SM-1 showed highly plasticity at different temperatures. The study provided a set of genomic data for understanding the temperature adaptability molecular mechanism of microorganisms. The study revealed that At. caldus SM-1 evolved to fit lower temperature through multiple pathways, not only by the general environment adaptation mechanism of microorganism, but also by the specific pathway of the strain.
[Objective] The aim of this study is to gain the acclimated strain of Acidithiobacillus caldus SM-1 with high activity when growing at the temperatures below the optimum, and elaborate its genomic plasticity and adaptation at different temperatures. [Methods] Strains were incubated at 37 °C, 40 °C and 45 °C. We used 454 genome resequencing technology to find the single nucleotide mutant sites in genome. The genes including mutant sites were studied to understand their relationship with the temperature adaptation. [Results] By long-term breeding, we obtained strains with higher viability than unacclimated strain at temperatures(37 °C), which is lower than initial optimum growth temperature(45 °C). Resequencing results showed the genome of SM-1 with high plasticity. In the genome of different strains(incubated at 37 °C, 40 °C and 45 °C), 418, 384 and 347 single nucleotide mutation sites were accumulated, respectively. Among them, 20 mutant sites were commonly occurred in the three strains. The genes they affected are involved in heavy metals and toxic resistance system, DNA methylation and protein acylation, and nucleic acid metabolism. In comparison, the specific mutations of the strains, grown at the 37 °C and 40 °C, were related to energy metabolism, signal transduction and DNA/RNA stability. Three genes contained mutant sites are common in L37 and M40, of which, Atc_1031 and Atc_1623 encode proteins related to transposon insertion, Atc_1130 encodes a hypothetical protein that is similar to the out membrane protein assembly factor B or the disulfide bond formation protein with 23% and 35% similarity. In addition, some single nucleotide mutations cause the amino acid changes of the related proteins during the adaption. [Conclusion] The genome of At. caldus SM-1 showed highly plasticity at different temperatures. The study provided a set of genomic data for understanding the temperature adaptability molecular mechanism of microorganisms. The study revealed that At. caldus SM-1 evolved to fit lower temperature through multiple pathways, not only by the general environment adaptation mechanism of microorganism, but also by the specific pathway of the strain.
引文
[1]Watling HR.The bioleaching of sulphide minerals with emphasis on copper sulphides-A review.Hydrometallurgy,2006,84(1/2):81-108.
[2]Meng CY,Jing QK,Ma J,Liu WY,Liu XY,Wen JK.Overview of microbiological technology for recovery of rare metal resources.Chinese Journal of Rare Metals,2015,39(4):371-378.(in Chinese)孟春瑜,荆乾坤,马骏,刘文彦,刘兴宇,温建康.微生物技术在稀有金属资源利用中的研究概况.稀有金属,2015,39(4):371-378.
[3]Dopson M,Halinen AK,Rahunen N,?zkaya B,Sahinkaya E,Kaksonen AH,Lindstr?m EB,Puhakka JA.Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms.Biotechnology and Bioengineering,2007,97(5):1205-1215.
[4]Rawlings DE.Characteristics and adaptability of iron-and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates.Microbial Cell Factories,2005,4:13.
[5]Lenski RE.Bacterial evolution and the cost of antibiotic resistance.International Microbiology,1998,1(4):265-270.
[6]Sonderegger M,Sauer U.Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose.Appllied and Environmental Microbiology,2003,69(4):1990-1998.
[7]Kuyper M,Toirkens MJ,Diderich JA,Winkler AA,van Dijken JP,Pronk JT.Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain.FEMS Yeast Research,2005,5(10):925-934.
[8]Wang QL,Hu EM,Yu RL,Zhang XW,Qiu GZ.Low-temperature bacteria domestication and leaching of sandstone-type refractory uranium ore.Mining and Metallurgical Engineering,2011,31(6):6-8,12.(in Chinese)王清良,胡鄂明,余润兰,张晓文,邱冠周.细菌低温驯化及其浸出砂岩型铀矿试验研究.冶矿工程,2011,31(6):6-8,12.
[9]Chen ZW,Liu YY,Wu JF,She Q,Jiang CY,Liu SJ.Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates.Appllied Microbiology and Biotechnology,2007,74(3):688-698.
[10]You XY,Guo X,Zheng HJ,Zhang MJ,Liu LJ,Zhu YQ,Zhu BL,Wang SY,Zhao GP,Poetsch A,Jiang CY,Liu SJ.Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation.Journal of Genetics and Genomics,2011,38(6):243-252.
[11]Russell RJM,Gerike U,Danson MJ,Hough DW,Taylor GL.Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium.Structure,1998,6(3):351-361.
[12]Kube M,Chernikova TN,Al-Ramahi Y,Beloqui A,Lopez-Cortez N,Guazzaroni ME,Heipieper HJ,Klages S,Kotsyurbenko OR,Langer I,Nechitaylo TY,Lünsdorf H,Fernández M,Juárez S,Ciordia S,Singer A,Kagan O,Egorova O,Petit PA,Stogios P,Kim Y,Tchigvintsev A,Flick R,Denaro R,Genovese M,Albar JP,Reva ON,Martínez-Gomariz M,Tran H,Ferrer M,Savchenko A,Yakunin AF,Yakimov MM,Golyshina OV,Reinhardt R,Golyshin PN.Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.Nature Communications,2013,4:2156.
[13]Ferrer M,Chernikova TN,Yakimov MM,Golyshin PN,Timmis KN.Chaperonins govern growth of Escherichia coli at low temperatures.Nature Biotechnology,2003,21(11):1266-1267.
[14]Hunger K,Beckering CL,Wiegeshoff F,Graumann PL,Marahiel MA.Cold-induced putative DEAD box RNAhelicases Csh A and Csh B are essential for cold adaptation and interact with cold shock protein B in Bacillus subtilis.Journal of Bacteriology,2006,188(1):240-248.
[15]Chintalapati S,Kiran MD,Shivaji S.Role of membrane lipid fatty acids in cold adaptation.Cellular and Molecular Biology,2004,50(5):631-642.
[16]Yancey PH.Organic osmolytes as compatible,metabolic and counteracting cytoprotectants in high osmolarity and other stresses.Journal of Experimental Biology,2005,208(15):2819-2830.
[17]Kandror O,De Leon A,Goldberg AL.Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures.Proceedings of the National Academy of Science of the United States of America,2002,99(15):9727-9732.
[18]Zhang MJ,Jiang CY,You XY,Liu SJ.Construction and application of an expression vector from the new plasmid p LAtc1 of Acidithiobacillus caldus.Appllied Microbiology and Biotechnology,2014,98(9):4083-4094.
[19]Marmur J.A procedure for the isolation of deoxyribonucleic acid from micro-organisms.Journal of Molecular Biology,1961,3(2):208-218.
[20]Jones PG,Krah R,Tafuri SR,Wolffe AP.DNA gyrase,Cs7.4,and the cold shock response in Escherichia coli.Journal of Bacteriology,1992,174(18):5798-5802.
[21]Mizushima T,Kataoka K,Ogata Y,Inoue R,Sekimizu K.Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock.Molecular Microbiology,1997,23(2):381-386.
[22]Esparza M,Cárdenas JP,Bowien B,Jedlicki E,Holmes DS.Genes and pathways for CO2 fixation in the obligate,chemolithoautotrophic acidophile,Acidithiobacillus ferrooxidans,carbon fixation in A.ferrooxidans.BMC Microbiology,2010,10:229.
[23]Vashisht AA,Tuteja N.Stress responsive DEAD-box helicases:a new pathway to engineer plant stress tolerance.Journal of Photochemistry and Photobiology B:Biology,2006,84(2):150-160.
[24]Watanabe T,Kojima H,Fukui M.Draft Genome sequence of a psychrotolerant sulfur-oxidizing bacterium,Sulfuricella denitrificans sk B26,and proteomic insights into cold adaptation.Appllied and Environmental Microbiology,2012,78(18):6545-6549.
[25]Mazzon RR,Lang EAS,Braz VS,Marques MV.Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance.FEMS Microbiology Letters,2008,288(2):178-185.
[26]Metpally RPR,Reddy BVB.Comparative proteome analysis of psychrophilic versus mesophilic bacterial species:insights into the molecular basis of cold adaptation of proteins.BMCGenomics,2009,10:11.
[27]Arpigny JL,Feller G,Gerday C.Cloning,sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrobacter immobilis B10.Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,1993,1171(3):331-333.
[28]Hallberg KB,Lindstr?m EB.Characterization of Thiobacillus caldus sp.nov.,a moderately thermophilic acidophile.Microbiology,1994,140(12):3451-3456.
[29]Acu?a LG,Cárdenas JP,Covarrubias PC,Haristoy JJ,Flores R,Nu?ez H,Riadi G,Shmaryahu A,Valdés J,Dopson M,Rawlings DE,Banfield JF,Holmes DS,Quatrini R.Architecture and gene repertoire of the flexible genome of the extreme acidophile Acidithiobacillus caldus.PLo S One,2013,8(11):e78237-e78237.
[30]Travisany D,Cortés MP,Latorre M,Di Genova A,Budinich M,Bobadilla-Fazzini RA,Parada P,González M,Maass A.Anew genome of Acidithiobacillus thiooxidans provides insights into adaptation to a bioleaching environment.Research in Microbiology,2014,165(9):743-752.
[31]Hottes AK,Freddolino PL,Khare A,Donnell ZN,Liu JC,Tavazoie S.Bacterial adaptation through loss of function.PLo S Genetics,2013,9(7):e1003617.
[32]Murata M,Fujimoto H,Nishimura K,Charoensuk K,Nagamitsu H,Raina S,Kosaka T,Oshima T,Ogasawara N,Yamada M.Molecular strategy for survival at a critical igh Temperature in Eschierichia coli.PLo S One,2011,6(6):e20063.
[2]Meng CY,Jing QK,Ma J,Liu WY,Liu XY,Wen JK.Overview of microbiological technology for recovery of rare metal resources.Chinese Journal of Rare Metals,2015,39(4):371-378.(in Chinese)孟春瑜,荆乾坤,马骏,刘文彦,刘兴宇,温建康.微生物技术在稀有金属资源利用中的研究概况.稀有金属,2015,39(4):371-378.
[3]Dopson M,Halinen AK,Rahunen N,?zkaya B,Sahinkaya E,Kaksonen AH,Lindstr?m EB,Puhakka JA.Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms.Biotechnology and Bioengineering,2007,97(5):1205-1215.
[4]Rawlings DE.Characteristics and adaptability of iron-and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates.Microbial Cell Factories,2005,4:13.
[5]Lenski RE.Bacterial evolution and the cost of antibiotic resistance.International Microbiology,1998,1(4):265-270.
[6]Sonderegger M,Sauer U.Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose.Appllied and Environmental Microbiology,2003,69(4):1990-1998.
[7]Kuyper M,Toirkens MJ,Diderich JA,Winkler AA,van Dijken JP,Pronk JT.Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain.FEMS Yeast Research,2005,5(10):925-934.
[8]Wang QL,Hu EM,Yu RL,Zhang XW,Qiu GZ.Low-temperature bacteria domestication and leaching of sandstone-type refractory uranium ore.Mining and Metallurgical Engineering,2011,31(6):6-8,12.(in Chinese)王清良,胡鄂明,余润兰,张晓文,邱冠周.细菌低温驯化及其浸出砂岩型铀矿试验研究.冶矿工程,2011,31(6):6-8,12.
[9]Chen ZW,Liu YY,Wu JF,She Q,Jiang CY,Liu SJ.Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates.Appllied Microbiology and Biotechnology,2007,74(3):688-698.
[10]You XY,Guo X,Zheng HJ,Zhang MJ,Liu LJ,Zhu YQ,Zhu BL,Wang SY,Zhao GP,Poetsch A,Jiang CY,Liu SJ.Unraveling the Acidithiobacillus caldus complete genome and its central metabolisms for carbon assimilation.Journal of Genetics and Genomics,2011,38(6):243-252.
[11]Russell RJM,Gerike U,Danson MJ,Hough DW,Taylor GL.Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium.Structure,1998,6(3):351-361.
[12]Kube M,Chernikova TN,Al-Ramahi Y,Beloqui A,Lopez-Cortez N,Guazzaroni ME,Heipieper HJ,Klages S,Kotsyurbenko OR,Langer I,Nechitaylo TY,Lünsdorf H,Fernández M,Juárez S,Ciordia S,Singer A,Kagan O,Egorova O,Petit PA,Stogios P,Kim Y,Tchigvintsev A,Flick R,Denaro R,Genovese M,Albar JP,Reva ON,Martínez-Gomariz M,Tran H,Ferrer M,Savchenko A,Yakunin AF,Yakimov MM,Golyshina OV,Reinhardt R,Golyshin PN.Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.Nature Communications,2013,4:2156.
[13]Ferrer M,Chernikova TN,Yakimov MM,Golyshin PN,Timmis KN.Chaperonins govern growth of Escherichia coli at low temperatures.Nature Biotechnology,2003,21(11):1266-1267.
[14]Hunger K,Beckering CL,Wiegeshoff F,Graumann PL,Marahiel MA.Cold-induced putative DEAD box RNAhelicases Csh A and Csh B are essential for cold adaptation and interact with cold shock protein B in Bacillus subtilis.Journal of Bacteriology,2006,188(1):240-248.
[15]Chintalapati S,Kiran MD,Shivaji S.Role of membrane lipid fatty acids in cold adaptation.Cellular and Molecular Biology,2004,50(5):631-642.
[16]Yancey PH.Organic osmolytes as compatible,metabolic and counteracting cytoprotectants in high osmolarity and other stresses.Journal of Experimental Biology,2005,208(15):2819-2830.
[17]Kandror O,De Leon A,Goldberg AL.Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures.Proceedings of the National Academy of Science of the United States of America,2002,99(15):9727-9732.
[18]Zhang MJ,Jiang CY,You XY,Liu SJ.Construction and application of an expression vector from the new plasmid p LAtc1 of Acidithiobacillus caldus.Appllied Microbiology and Biotechnology,2014,98(9):4083-4094.
[19]Marmur J.A procedure for the isolation of deoxyribonucleic acid from micro-organisms.Journal of Molecular Biology,1961,3(2):208-218.
[20]Jones PG,Krah R,Tafuri SR,Wolffe AP.DNA gyrase,Cs7.4,and the cold shock response in Escherichia coli.Journal of Bacteriology,1992,174(18):5798-5802.
[21]Mizushima T,Kataoka K,Ogata Y,Inoue R,Sekimizu K.Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock.Molecular Microbiology,1997,23(2):381-386.
[22]Esparza M,Cárdenas JP,Bowien B,Jedlicki E,Holmes DS.Genes and pathways for CO2 fixation in the obligate,chemolithoautotrophic acidophile,Acidithiobacillus ferrooxidans,carbon fixation in A.ferrooxidans.BMC Microbiology,2010,10:229.
[23]Vashisht AA,Tuteja N.Stress responsive DEAD-box helicases:a new pathway to engineer plant stress tolerance.Journal of Photochemistry and Photobiology B:Biology,2006,84(2):150-160.
[24]Watanabe T,Kojima H,Fukui M.Draft Genome sequence of a psychrotolerant sulfur-oxidizing bacterium,Sulfuricella denitrificans sk B26,and proteomic insights into cold adaptation.Appllied and Environmental Microbiology,2012,78(18):6545-6549.
[25]Mazzon RR,Lang EAS,Braz VS,Marques MV.Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance.FEMS Microbiology Letters,2008,288(2):178-185.
[26]Metpally RPR,Reddy BVB.Comparative proteome analysis of psychrophilic versus mesophilic bacterial species:insights into the molecular basis of cold adaptation of proteins.BMCGenomics,2009,10:11.
[27]Arpigny JL,Feller G,Gerday C.Cloning,sequence and structural features of a lipase from the antarctic facultative psychrophile Psychrobacter immobilis B10.Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,1993,1171(3):331-333.
[28]Hallberg KB,Lindstr?m EB.Characterization of Thiobacillus caldus sp.nov.,a moderately thermophilic acidophile.Microbiology,1994,140(12):3451-3456.
[29]Acu?a LG,Cárdenas JP,Covarrubias PC,Haristoy JJ,Flores R,Nu?ez H,Riadi G,Shmaryahu A,Valdés J,Dopson M,Rawlings DE,Banfield JF,Holmes DS,Quatrini R.Architecture and gene repertoire of the flexible genome of the extreme acidophile Acidithiobacillus caldus.PLo S One,2013,8(11):e78237-e78237.
[30]Travisany D,Cortés MP,Latorre M,Di Genova A,Budinich M,Bobadilla-Fazzini RA,Parada P,González M,Maass A.Anew genome of Acidithiobacillus thiooxidans provides insights into adaptation to a bioleaching environment.Research in Microbiology,2014,165(9):743-752.
[31]Hottes AK,Freddolino PL,Khare A,Donnell ZN,Liu JC,Tavazoie S.Bacterial adaptation through loss of function.PLo S Genetics,2013,9(7):e1003617.
[32]Murata M,Fujimoto H,Nishimura K,Charoensuk K,Nagamitsu H,Raina S,Kosaka T,Oshima T,Ogasawara N,Yamada M.Molecular strategy for survival at a critical igh Temperature in Eschierichia coli.PLo S One,2011,6(6):e20063.