茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究
作者:
基金项目:

国家自然科学基金(31570063,31870020);山东省重点研发计划(2017GSF17129);山东省重大科技创新工程(2017CXGC0303);山东省农业科学院农业科技创新工程(+CXGC2016B10)


Prediction and functional analysis of GGDEF/EAL domain-containing proteins in Azorhizobium caulinodans ORS571
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [41]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    [目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白(AZC_3085、AZC_3226和AZC_4658)进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。

    Abstract:

    [Objective] c-di-GMP, an important second messenger regulating multiple functions of bacteria, is generally synthesized and hydrolysed by proteins containing GGDEF or EAL domain. In this study, we analyzed the genome-wide GGDEF/EAL domain-containing proteins of Azorhizobium caulinodans ORS571, and selected three GGDEF-EAL composite proteins (AZC_3085, AZC_3226 and AZC_4658) for functional analysis. [Methods] SMART and CLUSTALW were used for prediction and multi-alignment of GGDEF/EAL domain-containing proteins. Mutants were constructed by homologous recombination. Phenotypes including cell motility, exopolysaccharide (EPS) production, biofilm formation and nodulation with legume host were investigated. [Results] There were 37 GGDEF/EAL domain-containing proteins in A. caulinodans ORS571. Mutant △4658 showed deficiency in cell motility, while its EPS production and biofilm formation were higher than that of wild type. Mutant △4658 showed stronger competitiveness than wild type in competitive nodulation assay. The loss of AZC_4658 led to the increase of intracellular c-di-GMP level. Mutants △3085 and △3226 did not show obvious difference in comparison with wild type. [Conclusion] The vast number of GGDEF/EAL domain-containing proteins suggested that c-di-GMP may play an important role in signal transduction of ORS571. The GGDEF-EAL composite protein AZC_4658 was involved in cell motility, EPS production, biofilm formation and nodulation of A. caulinodans ORS571.

    参考文献
    [1] Dreyfus B, Garcia JL, Gillis M. Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. International Journal of Systematic Bacteriology, 1988, 38(1):89-98.
    [2] Goormachtig S, Capoen W, James EK, Holsters M. Switch from intracellular to intercellular invasion during water stress-tolerant legume nodulation. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(16):6303-6308.
    [3] Hengge R. Principles of c-di-GMP signalling in bacteria. Nature Reviews Microbiology, 2009, 7(4):263-273.
    [4] Chan C, Paul R, Samoray D, Amiot NC, Giese B, Jenal U, Schirmer T. Structural basis of activity and allosteric control of diguanylate cyclase. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(49):17084-17089.
    [5] Rao F, Yang Y, Qi Y, Liang ZX. Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase:a study of the EAL domain-containing RocR from Pseudomonas aeruginosa. Journal of Bacteriology, 2008, 190(10):3622-3631.
    [6] Kazmierczak BI, Lebron MB, Murray TS. Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosa. Molecular Microbiology, 2006, 60(4):1026-1043.
    [7] Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S. A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Molecular Microbiology, 2007, 65(6):1474-1484.
    [8] Tamayo R, Tischler AD, Camilli A. The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase. Journal of Biological Chemistry, 2005, 280(39):33324-33330.
    [9] Solano C, García B, Latasa C, Toledo-Arana A, Zorraquino V, Valle J, Casals J, Pedroso E, Lasa I. Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(19):7997-8002.
    [10] Kulasakara H, Lee V, Brencic A, Liberati N, Urbach J, Miyata S, Lee DG, Neely AN, Hyodo M, Hayakawa Y, Ausubel FM, Lory S. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(8):2839-2844.
    [11] Wang YW, Xu J, Chen AM, Wang YZ, Zhu JB, Yu GQ, Xu L, Luo L. GGDEF and EAL proteins play different roles in the control of Sinorhizobium meliloti growth, motility, exopolysaccharide production, and competitive nodulation on host alfalfa. Acta Biochimica et Biophysica Sinica, 2010, 42(6):410-417.
    [12] Gao SJ, Romdhane SB, Beullens S, Kaever V, Lambrichts I, Fauvart M, Michiels J. Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli. Applied Microbiology and Biotechnology, 2014, 98(10):4589-4602.
    [13] Nakajima A, Aono T, Tsukada S, Siarot L, Ogawa T, Oyaizu H. Lon protease of Azorhizobium caulinodans ORS571 is required for suppression of reb gene expression. Applied and Environmental Microbiology, 2012, 78(17):6251-6261.
    [14] Marx CJ, Lidstrom ME. Broad-host-range cre-lox system for antibiotic marker recycling in Gram-negative bacteria. BioTechniques, 2002, 33(5):1062-1067.
    [15] Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proceedings of the National Academy of Sciences of the United States of America, 1979, 76(4):1648-1652.
    [16] Liu W, Yang JB, Sun Y, Liu XL, Li Y, Zhang ZP, Xie ZH. Azorhizobium caulinodans transmembrane chemoreceptor TlpA1 involved in host colonization and nodulation on roots and stems. Frontiers in Microbiology, 2017, 8:1327.
    [17] Wei C, Jiang WD, Zhao MR, Ling JJ, Zeng X, Deng J, Jin DL, Dow JM, Sun WX. A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola. Scientific Reports, 2016, 6:23769.
    [18] Russell MH, Bible AN, Fang X, Gooding JR, Campagna SR, Gomelsky M, Alexandre G. Integration of the second messenger c-di-GMP into the chemotactic signaling pathway. mBio, 2013, 4(2):e00001-13.
    [19] Liu XL, Liu W, Sun Y, Xia CL, Elmerich C, Xie ZH. A cheZ-like gene in Azorhizobium caulinodans is a key gene in the control of chemotaxis and colonization of the host plant. Applied and Environmental Microbiology, 2017, 84(3):e01827-17.
    [20] Jiang N, Liu W, Li Y, Wu HL, Zhang ZH, Alexandre G, Elmerich C, Xie ZH, Voordouw G. A chemotaxis receptor modulates nodulation during the Azorhizobium caulinodans-Sesbania rostrata symbiosis. Applied and Environmental Microbiology, 2016, 82(11):3174-3184.
    [21] Lee KB, de Backer P, Aono T, Liu CT, Suzuki S, Suzuki T, Kaneko T, Yamada M, Tabata S, Kupfer DM, Najar FZ, Wiley GB, Roe B, Binnewies TT, Ussery DW, D'Haeze W, den Herder J, Gevers D, Vereecke D, Holsters M, Oyaizu H. The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571. BMC Genomics, 2008, 9:271.
    [22] Schirmer T, Jenal U. Structural and mechanistic determinants of c-di-GMP signalling. Nature Reviews Microbiology, 2009, 7(10):724-735.
    [23] Christen M, Christen B, Folcher M, Schauerte A, Jenal U. Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. Journal of Biological Chemistry, 2005, 280(35):30829-30837.
    [24] Schmidt AJ, Ryjenkov DA, Gomelsky M. The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase:enzymatically active and inactive EAL domains. Journal of Bacteriology, 2005, 187(14):4774-4781.
    [25] Tarutina M, Ryjenkov DA, Gomelsky M. An unorthodox bacteriophytochrome from Rhodobacter sphaeroides involved in turnover of the second messenger c-di-GMP. Journal of Biological Chemistry, 2006, 281(46):34751-34758.
    [26] Bharati BK, Sharma IM, Kasetty S, Kumar M, Mukherjee R, Chatterji D. A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis. Microbiology, 2012, 158:1415-1427.
    [27] Taylor BL. Aer on the inside looking out:paradigm for a PAS-HAMP role in sensing oxygen, redox and energy. Molecular Microbiology, 2007, 65(6):1415-1424.
    [28] Bourret RB. Receiver domain structure and function in response regulator proteins. Current Opinion in Microbiology, 2010, 13(2):142-149.
    [29] Heikaus CC, Pandit J, Klevit RE. Cyclic nucleotide binding GAF domains from phosphodiesterases:structural and mechanistic insights. Structure, 2009, 17(12):1551-1557.
    [30] Henry JT, Crosson S. Ligand-binding PAS domains in a genomic, cellular, and structural context. Annual Review of Microbiology, 2011, 65(1):261-286.
    [31] Romling U, Galperin MY, Gomelsky M. Cyclic di-GMP:the first 25 years of a universal bacterial second messenger. Microbiology and Molecular Biology Reviews, 2013, 77(1):1-52.
    [32] Jenal U, Malone J. Mechanisms of cyclic-di-GMP signaling in bacteria. Annual Review of Genetics, 2006, 40(1):385-407.
    [33] Galperin M, Nikolskaya A, Koonin EV. Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiology Letter, 2001, 203(1):11-21.
    [34] Christen B, Christen M, Paul R, Schmid F, Folcher M, Jenoe P, Meuwly M, Jenal U. Allosteric control of cyclic di-GMP signaling. Journal of Biological Chemistry, 2006, 281(42):32015-32024.
    [35] Barends TR, Hartmann E, Griese JJ, Beitlich T, Kirienko NV, Ryjenkov DA, Reinstein J, Shoeman RL, Gomelsky M, Schlichting I. Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase. Nature, 2009, 459(7249):1015-1018.
    [36] Chen MW, Kotaka M, Vonrhein C, Bricogne G, Rao F, Chuah MLC, Svergun D, Schneider G, Liang ZX, Lescar J. Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic di-GMP signaling. Journal of Bacteriology, 2012, 194(18):4837-4846.
    [37] Purcell EB, McKee RW, McBride SM, Waters CM, Tamayo R. Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile. Journal of Bacteriology, 2012, 194(13):3307-3316.
    [38] Pérez-Mendoza D, Sanjuán J. Exploiting the commons:cyclic diguanylate regulation of bacterial exopolysaccharide production. Current Opinion in Microbiology, 2016, 30:36-43.
    [39] Hickman JW, Harwood CS. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Molecular Microbiology, 2008, 69(2):376-389.
    [40] Pérez-Mendoza D, Bertinetti D, Lorenz R, Gallegos MT, Herberg FW, Sanjuán J. A novel c-di-GMP binding domain in glycosyltransferase BgsA is responsible for the synthesis of a mixed-linkage β-glucan. Scientific Reports, 2017, 7:8997.
    [41] Schäper S, Krol E, Skotnicka D, Kaever V, Hilker R, Søgaard-Andersen L, Becker A, Stock AM. Cyclic di-GMP regulates multiple cellular functions in the symbiotic alphaproteobacterium Sinorhizobium meliloti. Journal of Bacteriology, 2016, 198(3):521-535.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

孙雨,解志红,刘卫,郭洪恩. 茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究[J]. 微生物学报, 2019, 59(10): 2000-2012

复制
分享
文章指标
  • 点击次数:911
  • 下载次数: 1121
  • HTML阅读次数: 1202
  • 引用次数: 0
历史
  • 收稿日期:2018-12-04
  • 最后修改日期:2019-01-17
  • 在线发布日期: 2019-10-10
文章二维码