青藏高原土壤中一株原油降解菌的作用机制探究
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摘要
分别采用原油和丙酮酸作为碳源培养Pedobacter steynii DX4细胞,采用Illumina高通量测序技术对两种碳源条件下细胞的转录组进行测序,测序数据拼接组装后共得到7 693个Unigene,其中有5 017个Unigene获得CDS注释。对Unigene进行筛选共获得1 195个差异表达基因,约占Unigene总数的15. 5%,对差异表达基因进行功能分析发现,在原油为碳源的生长条件下,DX4细胞中Luciferase-like monooxygenase基因和卤代烷脱氢酶基因表达水平显著提高,这两个基因可能在DX4细胞中催化烷烃末端氧化反应。KEGG分析显示,以原油为碳源的DX4细胞中的脂肪酸降解途径中发现了8个UPDEGs、苯酸盐降解途径中发现了10个UPDEGs,揭示了细胞对原油中烷烃和芳香烃类物质的降解通路。此外,关于信号处理、物质转运、胞外多糖合成以及细胞趋向运动代谢途径的基因表达水平上调,表明DX4细胞可能依赖细胞趋向运动和合成生物表面活性物质来辅助原油降解。
Crude oil and pyruvate as carbon source were used respectively to cultivate Pedobacter steynii DX4 cells,using Illumina high-throughput sequencing technologies to the cells of two kinds of carbon sources under the condition of the transcriptome sequencing. Sequencing data were spliced and assembled into 7 693 unigene,among them there were 5 017 unigene identified as CDS annotation. Filtering the unigene had received 1 195 differentially expressed genes,accounts for about 15. 5% of total unigene,of which functional analyses had found that under the condition of growth of the crude oil as carbon source,DX4 cells luciferase( like monooxygenase genes and halogenated dehydrogenase gene) expression level increased significantly,the two genes may catalyze the ends of alkane oxidation reaction in DX4 cells. The differently expressed genes were annotated according to the COG and KEGG databases. The cells,which fed on crude oil,expression levels of genes coding for luciferaselike monooxygenase and haloalkane dehalogenase were significantly up-regulated. The luciferase-like monooxygenase and haloalkane dehalogenase were deduced to be the key enzymes catalyzing alkane terminal oxidations in DX4. KEGG annotation showed that there were 8 UPDEGs in fatty acid degradation pathway and 10 UPDEGs in benzoate degradation pathway,indicating that the petroleum components were bio-degraded through these pathways. In addition,many genes were up-regulated in oil treated cells,including genes related to signal transduction,material transport,extracellular polysaccharide synthesis and chemotaxis. The chemotaxis and biosurfactant may be useful assisted mechanisms in DX4 when cells degrade crude oil.
Crude oil and pyruvate as carbon source were used respectively to cultivate Pedobacter steynii DX4 cells,using Illumina high-throughput sequencing technologies to the cells of two kinds of carbon sources under the condition of the transcriptome sequencing. Sequencing data were spliced and assembled into 7 693 unigene,among them there were 5 017 unigene identified as CDS annotation. Filtering the unigene had received 1 195 differentially expressed genes,accounts for about 15. 5% of total unigene,of which functional analyses had found that under the condition of growth of the crude oil as carbon source,DX4 cells luciferase( like monooxygenase genes and halogenated dehydrogenase gene) expression level increased significantly,the two genes may catalyze the ends of alkane oxidation reaction in DX4 cells. The differently expressed genes were annotated according to the COG and KEGG databases. The cells,which fed on crude oil,expression levels of genes coding for luciferaselike monooxygenase and haloalkane dehalogenase were significantly up-regulated. The luciferase-like monooxygenase and haloalkane dehalogenase were deduced to be the key enzymes catalyzing alkane terminal oxidations in DX4. KEGG annotation showed that there were 8 UPDEGs in fatty acid degradation pathway and 10 UPDEGs in benzoate degradation pathway,indicating that the petroleum components were bio-degraded through these pathways. In addition,many genes were up-regulated in oil treated cells,including genes related to signal transduction,material transport,extracellular polysaccharide synthesis and chemotaxis. The chemotaxis and biosurfactant may be useful assisted mechanisms in DX4 when cells degrade crude oil.
引文
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[19]Kumari B,Singh S N,Singh D P. Characterization of two biosurfactant producing strains in crude oil degradation[J]. Process Biochemistry,2012,47(12):2463-2471.
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[25]Andersen M R,Salazar M P,Schaap P J,et al. Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015versus enzyme-producing CBS 513. 88[J]. Genome Research,2011,21(6):885-897.
[26]Wang Liping,Wang Wanpeng,Lai Qiliang,et al. Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic Ocean[J]. Environmental M icrobiology,2010,12(5):1230-1242.
[27]Li Liu,Liu Xueqian,Yang Wen,et al. Crystal structure of longchain alkane monooxygenase(LadA)in complex w ith coenzyme FM N:unveiling the long-chain alkane hydroxylase[J]. Journal of M olecular Biology,2008,376(2):453-465.
[28]Van Berkel W J H,Kamerbeek N M,Fraaije M W. Flavoprotein monooxygenases,a diverse class of oxidative biocatalysts[J].Journal of Biotechnology,2006,124(4):670-689.
[29]Bowman J S,Deming J W. Alkane hydroxylase genes in psychrophile genomes and the potential for cold active catalysis[J].BM C Genomics,2014,15(1):1120.
[30]Verschueren K H G,Seljée F,Rozeboom H J,et al. Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase[J]. Nature,1993,363(6431):693-698.
[31] Mason O U,Hazen T C,Borglin S,et al. Metagenome,metatranscriptome and single-cell sequencing reveal microbial response to Deepw ater Horizon oil spill[J]. The ISM E Journal,2012,6(9):1715.
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[33] Gao R,Stock A M. Biological insights from structures of twocomponent proteins[J]. Annual Review of M icrobiology,2009,63:133-154.
[34]Yuste L,Rojo F. Role of the crc gene in catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathw ay[J].Journal of Bacteriology,2001,183(21):6197-6206.
[35]Whyte L G,Smits T H M,Labbe D,et al. Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531[J]. Applied and Environmental M icrobiology,2002,68(12):5933-5942.
[36]Liu Yichen,Zhou Tiantian,Zhang Jian,et al. Molecular characterization of the alk B gene in the thermophilic Geobacillus sp.strain M H-1[J]. Research in microbiology,2009,160(8):560-566.
[37] Wang Wanpeng,Shao Zongze. Enzymes and genes involved in aerobic alkane degradation[J]. Frontiers in M icrobiology,2013,4:116.
[38]Satpute S K,Banat I M,Dhakephalkar P K,et al.Biosurfactants,bioemulsifiers and exopolysaccharides from marine microorganisms[J]. Biotechnology Advances,2010,28(4):436-450.
[39]Smits T H M,Balada S B,Witholt B,et al. Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria[J]. Journal of Bacteriology,2002,184(6):1733-1742.
[2] Das N,Chandran P. Microbial degradation of petroleum hydrocarbon contaminants:an overview[J]. Biotechnology research international, 2011, 2011:941810. DOI:10. 4061/2011/941810.
[3] Megharaj M,Ramakrishnan B,Venkateswarlu K,et al. Bioremediation approaches for organic pollutants:a critical perspective[J]. Environment International,2011,37(8):1362-1375.
[4] Maeng J H,Sakai Y,Tani Y,et al. Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1[J]. Journal of Bacteriology,1996,178(13):3695-3700.
[5] Whyte L G,Smits T H M,Labbe D,et al. Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531[J]. Applied and Environmental M icrobiology,2002,68(12):5933-5942.
[6] Van Beilen J B,Funhoff E G. Alkane hydroxylases involved in microbial alkane degradation[J]. Applied M icrobiology and Biotechnology,2007,74(1):13-21.
[7] Sajna K V,Sukumaran R K,Gottumukkala L D,et al. Crude oil biodegradation aided by biosurfactants from Pseudozyma sp.NII 08165 or its culture broth[J]. Bioresource Technology,2015,191:133-139.
[8] Zhang Wen,Li Jianbing,Huang Guohe,et al. An experimental study on the bio-surfactant-assisted remediation of crude oil and salt contaminated soils[J]. Journal of Environmental Science and Health,Part A,2011,46(3):306-313.
[9] Bourret R B,Hess J F,Simon M I. Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY[J]. Proceedings of the National Academy of Sciences,1990,87(1):41-45.
[10]Lanfranconi M P,Alvarez H M,Studdert C A. A strain isolated from gas oil‐contaminated soil displays chemotaxis tow ards gas oil and hexadecane[J]. Environmental M icrobiology,2003,5(10):1002-1008.
[11]Hassanshahian M,Emtiazi G,Kermanshahi R K,et al. Comparison of oil degrading microbial communities in sediments from the Persian Gulf and Caspian Sea[J]. Soil and Sediment Contamination,2010,19(3):277-291.
[12]Feng Lu,Wang Wei,Cheng Jiansong,et al. Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir[J].Proceedings of the National Academy of Sciences,2007,104(13):5602-5607.
[13]Guo Dongnan,Zang Shuying,Zhao Guangying. Effect of freezing and thaw ing cycles on soil microbial activity and organic carbon density in forest sw amp w etland w ith various drainage afforestation years[J]. Journal of Glaciology and Geocryology,2017,39(1):175-184.[郭冬楠,臧淑英,赵光影.冻融交替对不同年代排水造林湿地土壤微生物活性及有机碳密度的影响[J].冰川冻土,2017,39(1):175-184.]
[14] Long Haozhi,Wang Yilin,Chang Sijing,et al. Diversity of crude oil-degrading bacteria and alkane hydroxylase(alk B)genes from the Qinghai-Tibet Plateau[J]. Environmental M onitoring and Assessment,2017,189(116):1-14.
[15]Yang Ruiqi. Diversity analysis and degradation characteristics of low temperature crude oil-degrading bacteria from the soils in the Qinghai-Tibet Plateau[D]. Lanzhou:Lanzhou Jiaotong University,2016.[杨蕊琪.青藏高原土壤中原油降解细菌的多样性及降解特性研究[D].兰州:兰州交通大学,2016.]
[16]Yang Ruiqi,Xue Lingui,Chang Sijing,et al. Elevation-distribution characteristics of the crude oil degrading bacterium in the Qilian M ountains[J]. Journal of Glaciology and Geocryology,2016,38(3):785-793.[杨蕊琪,薛林贵,常思静,等.祁连山不同海拔低温原油降解菌群的分布特性研究[J].冰川冻土,2016,38(3):785-793.]
[17]Chang Sijing,Zhang Gaosen,Chen Ximing,et al. The complete genome sequence of the cold adapted crude-oil degrader:Pedobacter steynii DX4[J]. Standards in Genomic Sciences,2017,12(1):45.
[18]WangYilin,Ai Xue,Li Shiweng,et al. Isolation,identification and degradation characteristics of a low temperature crude oil degrading bacteria strain from the soil of Tibetan Plateau[J]. Journal of Glaciology and Geocryology,2015,37(2):528-537.[王艺霖,艾雪,李师翁,等.青藏高原土壤中一株低温原油降解菌的分离鉴定及其原油降解特性[J].冰川冻土,2015,37(2):528-537.]
[19]Kumari B,Singh S N,Singh D P. Characterization of two biosurfactant producing strains in crude oil degradation[J]. Process Biochemistry,2012,47(12):2463-2471.
[20]Wentzel A,Ellingsen T E,Kotlar H K,et al. Bacterial metabolism of long-chain n-alkanes[J]. Applied M icrobiology and Biotechnology,2007,76(6):1209-1221.
[21]Sabirova J S,Becker A,Lünsdorf H,et al. Transcriptional profiling of the marine oil-degrading bacterium Alcanivorax borkumensis during grow th on n-alkanes[J]. FEM S M icrobiology Letters,2011,319(2):160-168.
[22]Grabherr M G,Haas B J,Yassour M,et al. Trinity:reconstructing a full-length transcriptome w ithout a genome from RNA-Seq data[J]. Nature Biotechnology,2011,29(7):644.
[23]Oyola S O,Otto T D,Gu Y,et al. Optimizing Illumina nextgeneration sequencing library preparation for extremely AT-biased genomes[J]. BM C Genomics,2012,13(1):1.
[24] Mortazavi A,Williams B A,Mc Cue K,et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq[J]. Nature M ethods,2008,5(7):621.
[25]Andersen M R,Salazar M P,Schaap P J,et al. Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015versus enzyme-producing CBS 513. 88[J]. Genome Research,2011,21(6):885-897.
[26]Wang Liping,Wang Wanpeng,Lai Qiliang,et al. Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic Ocean[J]. Environmental M icrobiology,2010,12(5):1230-1242.
[27]Li Liu,Liu Xueqian,Yang Wen,et al. Crystal structure of longchain alkane monooxygenase(LadA)in complex w ith coenzyme FM N:unveiling the long-chain alkane hydroxylase[J]. Journal of M olecular Biology,2008,376(2):453-465.
[28]Van Berkel W J H,Kamerbeek N M,Fraaije M W. Flavoprotein monooxygenases,a diverse class of oxidative biocatalysts[J].Journal of Biotechnology,2006,124(4):670-689.
[29]Bowman J S,Deming J W. Alkane hydroxylase genes in psychrophile genomes and the potential for cold active catalysis[J].BM C Genomics,2014,15(1):1120.
[30]Verschueren K H G,Seljée F,Rozeboom H J,et al. Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase[J]. Nature,1993,363(6431):693-698.
[31] Mason O U,Hazen T C,Borglin S,et al. Metagenome,metatranscriptome and single-cell sequencing reveal microbial response to Deepw ater Horizon oil spill[J]. The ISM E Journal,2012,6(9):1715.
[32]Peixoto R S,Vermelho A B,Rosado A S. Petroleum-degrading enzymes:bioremediation and new prospects[J]. Enzyme Research,2011,2011:475193.
[33] Gao R,Stock A M. Biological insights from structures of twocomponent proteins[J]. Annual Review of M icrobiology,2009,63:133-154.
[34]Yuste L,Rojo F. Role of the crc gene in catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathw ay[J].Journal of Bacteriology,2001,183(21):6197-6206.
[35]Whyte L G,Smits T H M,Labbe D,et al. Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531[J]. Applied and Environmental M icrobiology,2002,68(12):5933-5942.
[36]Liu Yichen,Zhou Tiantian,Zhang Jian,et al. Molecular characterization of the alk B gene in the thermophilic Geobacillus sp.strain M H-1[J]. Research in microbiology,2009,160(8):560-566.
[37] Wang Wanpeng,Shao Zongze. Enzymes and genes involved in aerobic alkane degradation[J]. Frontiers in M icrobiology,2013,4:116.
[38]Satpute S K,Banat I M,Dhakephalkar P K,et al.Biosurfactants,bioemulsifiers and exopolysaccharides from marine microorganisms[J]. Biotechnology Advances,2010,28(4):436-450.
[39]Smits T H M,Balada S B,Witholt B,et al. Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria[J]. Journal of Bacteriology,2002,184(6):1733-1742.