摘要
本文利用生物信息学手段找出了鱼腥蓝细菌PCC7120基因组中所有的丝氨酸/苏氨酸/酪氨酸激酶和磷酸酶基因,对其结构和染色体上的遗传排列作了一定的分析。并对该细菌中部分与信号转导相关的基因进行中断,筛选得到突变体,并进行验证及初步表型分析。
1.鱼腥蓝细菌PCC7120基因组中丝氨酸/苏氨酸/酪氨酸激酶和磷酸酶基因的查找分析
经鱼腥蓝细菌PCC7120基因组的查找分析发现鱼腥蓝细菌PCC7120中共有52个丝氨酸/苏氨酸/酪氨酸激酶基因(STPK)和14个磷酸酶基因(PP)。按照STPKs结构的不同可划分为五个亚类:STK-Ⅰ亚类只含有一个催化区域;STK-Ⅱ亚类的C端含有WD40重复序列;STK-Ⅲ亚类的C端含有不同的调节区域;STK-Ⅳ亚类的催化区域位于C端,HSTK-Ⅰ亚类则既含有丝氨酸/苏氨酸激酶的催化区域,又含有组氨酸激酶催化区域。14个PPs共分为3族:属PP2C族的有8个,属PTP族的有3个,属PP1/2A/2B族的有3个。与单细胞集胞藻蓝细菌Synechocystis PCC6803基因组比较,只有11个基因可以在该基因组中找到相似的基因。经遗传排列分析发现,有12个基因在染色体上组成了6对基因簇(gene cluster)。
2.信号转导相关基因突变体的构建及表型分析
本研究全部通过插入失活获得突变体。首先将PCR扩增片段(~1.5kb)利用PstⅠ和XhoⅠ插入常规克隆载体pBluescriptSK,利用基因内部合适的酶切位点插入Sp/Sm抗性片段,再利用PstⅠ和XhoⅠ位点将已插入抗性基因的PCR片段,转入整合载体pRL271中获得体外基因中断,最后利用三亲本杂交获得体内突变。本研究共构建了91个体外基因中断结构,获得23个可能的单交换突变体,53个可能的双交换突变体,对所得的双交换突变体进行PCR验证发现有6个为野生型,4个为单交换突变体,其余43个均为真正的双交换突变体。将验证的真突变体在缺氮条件下观察其异型胞的发育以及生长状况,发现有2种突变体All2898~-,All1731~-在缺氮条件下不能生长也不产异型胞,另有3种突变体All4141~-,All1171~-,All4761~-在缺氮条件下可以产生异型胞,但仍不能维持正常生长,这可能是异型胞的结构被破坏或是异形胞中的固氮酶失活。所有这些基因在异形胞的发育过程中起重要作用。
This dissertation searched for Ser/Thr and Tyr kinases and phosphatases genes by bioinformation tools in Anabaena PCC7120, inactivated the genes involved in protein phosphorylation/dephosphorylation, and analyze the phenotypes of mutants. 1. Search for Ser/Thr and Tyr kinases and phosphateses in Anabaena PCC7120
We found 52 genes encoding Ser/Thr and Tyr kinases (STPK) and 14 genes encoding ser/thr and Tyr phosphateses (PP). We divided all STPKs into 5 subfamilies according to their own structure characterizes. STK-I only possess regions similar to the typical catalytic domain of STPKs, STK-II contain WD40 repeats, STK-III contain all kinds of regulator domain in their C-terminal region, STK-IV have their catalytic domains located in the N-terminal region, While HSTK-I also have a his kinase domain of two-component system. Among 14 PPs genes, 8 of them belong to PP2C, 3 belong to PTPs and only 3 genes encoding PP1/PP2A/PP2B could be found. Only 11 genes can be found similar genes in the Synechocystis PCC6803 genome by genomic analysis. And via the analysis of genetic array, 12 genes can be classified into 6 gene clusters in the chromosome.
2. Inactivation of genes which involved in signal transduction and analyze the phenotypes of mutants
All the mutants in this study were inactivated by inserting a resistance gene. PCR products were inserted into pBluescriptSK using PstI and XhoI. The element bearing resistance to streptomycin(Sm) and spectinomycin(Sp) was excised from pHP45 and inserted into gene coding region. The disrupted gene was isolated as a Pstl and Xhol fragment and cloned into pRL271. Triparental mating was then performed. As a result, we obtained 91 recombined plasmids, 23 possible single-cross mutants and 53 possible double-cross mutants. PCR analysis of the genomic DNA from the resulting possible double-cross mutants indicated that 6 of them were wild-type, 4 of them were single-cross mutants and the remainder ones were true double-cross mutants. Observe the heterocysts and growing states of these mutants after nitrogen depletion and found that A112898-, All 1731- can not different heterocyst, A114141-, A111171- and A114761- can different heterocysts but still can not maintain normal growth. The results indicated that all these genes play roles in hetetocyst differention.
引文
1.何家菀,王业勤,黎尚豪.鱼腥藻7120的DNA转化作用。遗传学报,1984.11:100-105
2. Alex L A and Simon M I. Protein kinase and signal transduction in prokaryotes and eukaryotes. Trends in Genet, 1994. 10:133-138
3. Appleby J L, Parkinson J S and Bourret R B. Signal transduction via the multi-step phosphorelay: not necessarily a road less travelled. Cell, 1996. 86: 845-848.
4. Bakal C J and Davies J E. No longer an exclusive club: eukaryotic signalling domains in bacteria. Trends in Cell Biol, 2000. 10: 32-38.
5. Bancroft I, Wolk C P, Oren E V. Physical and genetic maps of the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC7120. J. Bact, 1989. 171(1): 5940-5948
6. Barford D. Molecular mechanisms of the protein ser/thr phosphatases. Trends in Biochem Sci, 1996. 21: 407-412.
7. Barik S. Protein phosphorylation and signal transduction. Subcell Biochem. 1996.26:115-64.
8. Barton G J, Cohen P T W and Barford D. Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. Eur J Biochem. 1994. 220: 225-237.
9. Bilwes A M, Alex L A, Crane B R, Simon M I. Structure of CheA, a signal-transducing histidine kinase. Cell. 1999. 96(1): 131-41.
10. Bork P, Brown N P, Hegyi H and Schultz J. The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues. Protein Sci, 1996. 5:1421-1425.
11. Bourret R B, Hess J F, and Simon M I. Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY. Proc Natl Acad Sci USA, 1990. 87:41-45
12. Brissette R E, Tsung K L, and Inouye M. Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli. J.Bacteriol, 1991. 173: 601-608
13. Buikema W J, and Haselkom R. Molecular genetics of cyanobacterial development. Annu Rev Plant Physiol Plant Mol Biol, 1993.44:33-52
14. Chang C, Kwok S F, Bleecker A B and Meyerowitz E M. Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science, 1993. 262: 539-544.
15. Chauvat F, De Vrics L, Van der Ende A and Van Arkel G A. A host-vector system for gene cloning in the cyanobacterium Synechicytis sp. PCC6803. M.G.G. 1986. 204:185-191
16. Cho H S, Pelton J G, Yan D, Kustu S, Wemmer D E. Phosphoaspartates in bacterial signal transduction. Curr Opin Struct Biol. 2001.11(6): 679-84.
17. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem, 1989. 58:453-508.
18. Cozzone A J. Diversity and specificity of protein-phosphorylating systems in bacteria. Folia Microbiol. 1997.42(3): 165-70.
19. Das A K, Helps N R, Cohen P T W and Barford D. Crystal structure of the protein serine/threonine phosphatase2C at 2.0 A resolution. EMBO J, 1996. 15:6798-6809.
20. Drobak B K. Phosphoinositides and protein phosphorylation in plant signal transduction. Semin Cell Biol. 1993.4(2): 123-30.
21. Eckstein J W, Beer-Romero P and Berdo I. Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases. Protein Sci, 1996. 5: 5-12.
22. Fauman E B and Saper M A. Structure and function of the protein tyrosinephosphatases. Trends in Biochem Sci, 1996.21: 413-417.
23. Galyov E E, Hankansson S, Forsberg A and Wolf-Watz H. A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature, 1993. 361:730-732
24. Hakansson S E, Galyov E E, Rosqvist R, Wolf-Watz H. The Yersinia YpkA Ser/Thr kinaseis translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol Microbiol, 1996.20:593-603
25. Hanks S K and Hunter H. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEBJ, 1995.9:576-596
26. Hanks S K, Quinn A M and Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science, 1988. 241: 42-52.
27. Hanlon W A, Inouye M, Inouye S. Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xantbus. Mol Micobiol, 1997. 23: 459-471
28. Hoch J A. Two-component and phosphorelay signal transduction. Curr Opin Microbiol, 2000. 3:165-170.
29. Horinouchi S. and Beppu T. A-factor as a microbial hormone that controls cellular differentation and secondary metabolism in Streptomyces griseus. Mol Microbiol, 1994. 12:859-864
30. Hunter T. Protein kinases and phosphatases: the yin and yang of proteinphosphorylation and signalling. Cell, 1995. 80: 225-236.
31. Ingebritsen T S and Cohen P. Protein phosphatase: properties and role in cellar regulation. Science, 1983.221: 331-338
32. Inouye S, Jain R, Ueki T, Nariya H, Xu C Y, Hsu M Y, Femandez-Luque B A, Munoz-Dorado J, Farez-Vidal E, Inouye M. A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium. Microb Comp Genomics. 2000. 5(2): 103-20.
33. Jia Z, Barford D, Flint A J and Tonks N K. Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. Science. 1995. 268, 1754-1758.
34. Kennelly P J and Ports M. Fancy meeting you here: a fresh look at 'prokaryotic' protein phosphorylation. J. Bacteriol. 1996. 178:4759-4764
35. Kieber J J, Rothenberg M, Roman G, Feldmann K A and Ecker J R. CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinase. Cell, 1993.72:427-441
36. Maeda T, Wurgler-Murphy S M and Saito H. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature, 1994. 369:242-245
37. Matsumoto A, Hong S K, Ishizuka H, Horinouchi S, Beppu T. Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase. Gene. 1994. 146:47-56
38. Munoz-Dorado J, Inouye M and Inouye S. A gene encoding protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium. Cell, 1991. 67:995-569
39. Nadvornik R, Vomastek T, Janecek J, Technikova Z, Branny P. Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor. J Bactriol, 1999. 181:15-23
40. Packinson J S and Kofoid E C. Communication modules in bacterial signaling proteins, Anna Rev Genet. 1992.26:71 112.
41. Pannifer A D B, Flint A J, Tonks N K and Barford D. Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by X-ray crystallography. J Biol Chem. 1998. 273:10454-10462.
42. Parkison J S. Signal transduction schems of bacteria, Cell, 1993.73:857-871
43. Phalip V, Li J H and Zhang C C. HstK, a cyanobaeterial protein with both a Ser/Thr kinase domain and a His-kinase domain: implication for the mechanism of signal transduction. Biochem. J. 2001.360: 639-644.
44. Potts M H, Sun H, Mockaitis K, Kennelly P J, Reed D and Tonks N K. A protein-tyrosine/serine phosphatase encoded by the genome of the cyanobacterium Nostoc. J Biol Chem. 1993. 268: 7632-7635
45. Saier M H Jr. Introduction: protein phosphorylation and signal transduction in bacteria. J Cell Biochem. 1993.51(1): 1-6.
46. Schwartz S H, Black T A, Jager K, Panoff J M, Wolk C P. Regulation of an osmoticum-responsive gene in Anabaena sp. strain PCC 7120. J Bacteriol. 1998. 180(23): 6332-7.
47. Shi L, Potts M, Kennelly P J. The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev. 1998. 22:229-253
48. Stock A M, Robinson V L and Goudreau P N. Two-component signal transduction. Annu Rev Biochem, 2000.69:183-215.
49. Stock J B, Ninfa A D, and Stock A M. Protein phospborylatian and regulation of adaptive responses in bacteria. Microbiol Rev, 1989. 53:450-490.
50. Stock J B, Stock A M and Mottonen J M. Signal transduction in bacteria, Nature, 1990. 344: 395-400.
51. Stock J B, Surette M G, Levit M and Stock A M. Two-component signal transduction systems: structure-function relationships and mechanisms of catalysis. In: Hoch J A and Silhavy, T J. eds. Two-component signal transduction. Washingtion DC, American Society for Microbiology, Stock J. B, Ninfa A D and Stock A M. 1995. pp. 25-51
52. Udo H, Munoz-Dorado J, Inouye M, Inouye S. Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation. Genes Dev. 1995. 9(8): 972-83.
53. Urabe H and Ogewara H. Cloning, Sequencing and expression of Serine/threonine kinase-encoding genes from Streptomyces coehcolor A3 (2). Gene, 1995. 153:99-104
54. Villafranca J E, Kissinger C R, Parge H E. Protein serine/threonine phosphatases. Curr Opin Biotechnol, 1996. 7:397-402
55. Volz K and Matsumura P. crystal structure of Escheriehia coli CheY refined at 1.7- resolution. J Biol Chem. 1991. 266:15511-15519
56. Wang L, Sun Y P, Chen W L, Li J H, Zhang C C. Genomic analysis of protein kinases, protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120. FEMS Microbiol Lett. 2002. 217(2): 155-65.
57. West A H, Stock A M. Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem Sci. 2001.26(6): 369-76
58. Wolk C P, Vonshak A, Kehoe P and Elha J. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Prce Natl Acad Sci USA, 1984. 81: 1561-1565
59. Wolk C P. Heterocyst formation in Anabaena. In: Brun Y V and Shimkets L J, Eds., Prokaryotic Development, Washington DC. ASM Press, 2000. 83-104
60. Wolk C P. Heterocyst formation. Ann Rev. Genet, 1996. 30:59-78.
61. Wurgler-Murphy S M, Saito H. Two-component signal transducers and MAPK cascade. Trends in Biochem.Sci. 1997. 22:172-176
62. Zhang C C and Libs L. Cloning and eharacterisation of the pknD gene encoding an eukaryotic-type protein kinase in the cyanobacterium Anabaena sp. PCC 7120. Mol. Gen. Genet. 1998. 258:26-33.
63. Zhang C C, Friry A and Peng L. Molecular and genetic analysis of two closely linked genes that encode, respectively, a protein phosphatase 1/2A/2B homolog and a protein kinasehomolog in the eyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 1998. 180:2616-2622.
64. Zhang C C. A gene encoding a protein related to eukaryotic protein kinase from the filamentous heterocystous cyanobacterium Anabaena PCC7120. Proc Natl Acad Sci USA 1993. 90: 1840-11844.
65. Zhang C C. Bacterial signalling involving eukaryotic-type protein kinases. Mol. Microbio, 1996. 20: 9-15.
66. Zhang W, Inouye M, Inouye S. Reciprocal regulation of the differentiation of Myxococcus xanthus by Pkn5 and Pkn6, eukaryofic-like ser/Thr protein kinases. Mol. Microbiol, 1996. 20: 435-447
67. Zhang W, Munoz-Dorado J, Inouye M and Inouye S. Identification of a putative eukaryotic-like protein kinase family in a developmental bacterium Myxococcus xanthus. J Bacteriol, 1992. 174:5450-5453
68. Zhu J, Kong R, Wolk C P. A two-component system mediates developmental regulation of biosynthesis ofa heterocyst polysaccharide. J Biol Chem. 2003.278(22): 19939-46.