Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/96258
標題: Cyclic-di-GMP and Cyclic-AMP receptor co-regulate the genes expression of low-molecular-weight bacteriocin in Pectobacterium carotovorum subsp. carotovorum
Pectobacterium carotovorum subsp. carotovorum中Cyclic-di-GMP與Cyclic-AMP receptor 低分子量細菌素基因表現之調控機制
作者: Wan-Ying Jhuo
卓琬瑩
關鍵字: 細菌素
CRP調控因子
Carocin
CRP regulation factor
引用: 1. LUND, B.M., Formation of Reducing Sugars from Sucrose by Erwinia Species. Microbiology, 1975. 88(2): p. 367-371. 2. LUND, B.M. ., et al., The nature of reducing compounds formed from sucrose by Erwinia carotovora var. atroseptica. Journal of General Microbiology, 1973. 78(2): p. 331. 3. Pe'rombelon, M.C.M., Potato diseases caused by soft-rot erwinias: an overview of pathogenesis. The role of pectic enzymes in plant pathogenesis. Plant Pathol, 2002. 51. 4. Malcolmson, J.F., A study of Erwinia isolates obtained from soft rots and blackleg of potatoes. Transactions of the British Mycological Society, 1959. 42(2): p. 261-269. 5. Kloepper, J.W., et al., The association ofErwinia carotovora var.atroseptica andErwinia carotovora var.carotovora with insects in Colorado. American Potato Journal, 1979. 56(7): p. 351-361. 6. Preston, J., et al., Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16. Journal of bacteriology, 1992. 174(6): p. 2039-2042. 7. Hoondal, G., et al., Microbial alkaline pectinases and their industrial applications: a review. Applied microbiology and biotechnology, 2002. 59(4-5): p. 409-418. 8. Mukai, K ., et al., Kinetics and mechanism of heterogeneous hydrolysis of poly [(R)-3-hydroxybutyrate] film by PHA depolymerases. International journal of biological macromolecules, 1993. 15(6): p. 361-366. 9. Huang, J., et al.,, DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum. Journal of bacteriology, 1990. 172(7): p. 3879-3887. 10. Chatterjee, A.K., et al., Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia, and Klebsiella species. Canadian journal of microbiology, 1979. 25(1): p. 94-102. 11. Bergey, H., et al., Relationship of pectolytic clostridia and Erwinia carotovora strains to decay of potato tubers in storage. Plant Disease, 1982: p. 543. 12. Cotter, P.D., et al., Bacteriocins [mdash] a viable alternative to antibiotics? Nat Rev Micro, 2013. 11(2): p. 95-105. 13. Michel-Briand, Y. ., et al., The pyocins of Pseudomonas aeruginosa. Biochimie, 2002. 84(5-6): p. 499-510. 14. RodrÍGuez, E.V.A., et al., Control of Listeria monocytogenes by bacteriocins and monitoring of bacteriocin-producing lactic acid bacteria by colony hybridization in semi-hard raw milk cheese. Journal of Dairy Research, 2001. 68(1): p. 131-137. 15. Griffiths, G.L., et al., Vibriobactin, a siderophore from Vibrio cholerae. J Biol Chem, 1984. 259(1): p. 383-5. 16. S?rensen, K.I., et al., A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis. Applied and Environmental Microbiology, 2000. 66(4): p. 1253-1258. 17. Bennik, M.H., et al., Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity. Appl Environ Microbiol, 1997. 63(9): p. 3628-36. 18. Itoh, T., et al., Inhibition of food-borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri. Lett Appl Microbiol, 1995. 21(3): p. 137-41. 19. Strauch, E., et al., Characterization of Enterocoliticin, a Phage Tail-Like Bacteriocin, and Its Effect on Pathogenic Yersinia enterocolitica Strains. Applied and Environmental Microbiology, 2001. 67(12): p. 5634-5642. 20. Bradley, D.E., Ultrastructure of bacteriophage and bacteriocins. Bacteriol Rev, 1967. 31. 21. Clark, S., et al., Trypsin enhancement of rotavirus infectivity: mechanism of enhancement. Journal of virology, 1981. 39(3): p. 816-822. 22. Expert, D., et al., Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity. J Bacteriol, 1985. 163(1): p. 221-7. 23. Cambell, P.a.E., E, Bacteriocin production in Erwinia carotovora. Phytopathology. Phyotopathology, 1997: p. 69:526. 24. Tagg, J., et al., Assay system for bacteriocins. Applied microbiology, 1971. 21(5): p. 943. 25. Radman, M., SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. Basic Life Sci, 1975: p. 355-67. 26. Gillor, O., et al., The role of SOS boxes in enteric bacteriocin regulation. Microbiology (Reading, England), 2008. 154(Pt 6): p. 1783-1792. 27. Little, J., Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie, 1991. 73(4): p. 411-421. 28. Harmon, F.G., et al., Interaction of Escherichia coli RecA protein with LexA repressor. II. Inhibition of DNA strand exchange by the uncleavable LexA S119A repressor argues that recombination and SOS induction are competitive processes. J Biol Chem, 1996. 271(39): p. 23874-83. 29. Konisky, J., Colicins and other bacteriocins with established modes of action. Annu Rev Microbiol, 1982. 36: p. 125-44. 30. Roberts, J.W ., et al., Two mutations that alter the regulatory activity of E. coli recA protein. Nature, 1981. 290(5805): p. 422-4. 31. Little, J.W., et al., Cleavage of the Escherichia coli lexA protein by the recA protease. Proc Natl Acad Sci U S A, 1980. 77(6): p. 3225-9. 32. Kolb, A., et al., Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem, 1993. 62: p. 749-95. 33. Scott, S., et al., Transcriptional co-activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem. Mol Microbiol, 1995. 18(3): p. 521-31. 34. Zhang, X., et al., Catabolite gene activator protein mutations affecting activity of the araBAD promoter. J Bacteriol, 1998. 180(2): p. 195-200. 35. Brown, N.L., et al., The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 2003. 27(2): p. 145-163. 36. Kumar, M., et al., Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis. Microbiology, 2008. 154(Pt 10): p. 2942-55. 37. Stella, N.A., et al., Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites. J Bacteriol, 2015. 197(15): p. 2468-78. 38. McKay, D.B., et al., Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP. J Biol Chem, 1982. 257(16): p. 9518-24. 39. Popovych, N., et al., Structural basis for cAMP-mediated allosteric control of the catabolite activator protein. Proc Natl Acad Sci U S A, 2009. 106(17): p. 6927-32. 40. Chen, Y., et al., Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis. Journal of Bacteriology, 2012. 194(18): p. 5080-5090. 41. Römling, U., et al., Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger. Microbiology and Molecular Biology Reviews, 2013. 77(1): p. 1-52. 42. Caly, D.L., et al., Targeting cyclic di-GMP signalling: a strategy to control biofilm formation? Curr Pharm Des, 2015. 21(1): p. 12-24. 43. Ross, P., et al., Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature, 1987. 325(6101): p. 279-281. 44. Barraud, N., et al., Nitric Oxide Signaling in Pseudomonas aeruginosa Biofilms Mediates Phosphodiesterase Activity, Decreased Cyclic Di-GMP Levels, and Enhanced Dispersal. Journal of Bacteriology, 2009. 191(23): p. 7333-7342. 45. Krasteva, P.V., et al., Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP. Science, 2010. 327(5967): p. 866-868. 46. Jenal, U., et al., Mechanisms of Cyclic-di-GMP Signaling in Bacteria. Annual Review of Genetics, 2006. 40(1): p. 385-407. 47. Simm, R., et al., GGDEF and EAL domains inversely regulate cyclic di‐GMP levels and transition from sessility to motility. Molecular microbiology, 2004. 53(4): p. 1123-1134. 48. Chou, S.H., et al., Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms. J Bacteriol, 2016. 198(1): p. 32-46. 49. Fazli, M., et al., The CRP/FNR family protein Bcam1349 is ac‐di‐GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia. Molecular microbiology, 2011. 82(2): p. 327-341. 50. Takahara, Y., Development of the microbial pesticide for soft-rot disease. PSJ Biocont. Rept, 1994. 4: p. 1-7. 51. Parret, A.H., et al., Bacteria killing their own kind: novel bacteriocins of Pseudomonas and other γ-proteobacteria. Trends in microbiology, 2002. 10(3): p. 107-112. 52. Chan, Y.-c., et al., Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum. BMC microbiology, 2009. 9(1): p. 181. 53. Roh, E., et al., Diverse antibacterial activity of Pectobacterium carotovorum subsp. carotovorum isolated in Korea. J. Microbiol. Biotechnol, 2009. 19(1): p. 42-50. 54. Chan, Y.-C., et al., Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum. BMC Microbiology, 2011. 11(1): p. 99. 55. 陳楷茵, Pectobacterium carotovorum subsp. carotovorum 低分子量細菌素 Carocin S3的基因選殖、純化及蛋白分析2010, 國立中興大學化學系. 56. 徐志豪, Erwinia carotovora 低分子量細菌素Carocin S3基因選殖與表現2009, 國立中興大學化學系. 57. Chuang, D.-y., et al.,Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1. Journal of bacteriology, 2007. 189(2): p. 620-626. 58. Morris, D.R., et al., Upstream open reading frames as regulators of mRNA translation. Molecular and cellular biology, 2000. 20(23): p. 8635-8642. 59. Duport, C., et al., Molecular characterization of pyocin S3, a novel S-type pyocin from Pseudomonas aeruginosa. Journal of Biological Chemistry, 1995. 270(15): p. 8920-8927. 60. Roh, E., et al., Characterization of a new bacteriocin, Carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21. Applied and environmental microbiology, 2010. 76(22): p. 7541-7549. 61. Chin, K.-H., et al., The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell–cell signaling to virulence gene expression in Xanthomonas campestris. Journal of molecular biology, 2010. 396(3): p. 646-662. 62. Park, T.-H., et al., Genome sequence of Pectobacterium carotovorum subsp. carotovorum strain PCC21, a pathogen causing soft rot in Chinese cabbage. Journal of bacteriology, 2012. 194(22): p. 6345. 63. 陳彥君, Pectobacterium carotovorum subsp. carotovorum低分子量細菌素基因受c-di-GMP與cyclic AMP Receptor Protein調控作用之探討2011, 國立中興大學化學系所. 64. Sambrook, J., et al., Molecular cloning: a laboratory manual1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 65. Blin, N., et al., A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic acids research, 1976. 3(9): p. 2303-2308. 66. Ho, S.N., et al., Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989. 77(1): p. 51-59. 67. Hanahan, D., Studies on transformation of Escherichia coli with plasmids. J Mol Biol, 1983. 166. 68. Simms, D., et al., TRIzol: A new reagent for optimal single-step isolation of RNA. Focus, 1993. 15(4): p. 532-535. 69. Boedtker, H., Conformation independent molecular weight determinations of RNA by gel electrophoresis. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 1971. 240(3): p. 448-453. 70. Metzger, M., et al., Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells. Nucleic acids research, 1992. 20(9): p. 2265-2270. 71. Fredericq, P., Colicins. Annu Rev Microbiol, 1957. 11: p. 7-22. 72. Sambrook, J., et al., SDS-polyacrylamide gel electrophoresis of proteins. CSH Protoc, 2006. 1: p. 4. 73. Mahmood, T., et al., Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 2012. 4(9): p. 429. 74. Aslanidis, C., et al., Ligation-independent cloning of PCR products (LIC-PCR). Nucleic acids research, 1990. 18(20): p. 6069-6074. 75. Chiu, J., et al., Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Research, 2004. 32(21): p. e174-e174. 76. Hanahan, D., Studies on transformation of Escherichia coli with plasmids. Journal of molecular biology, 1983. 166(4): p. 557-580. 77. Schägger, H., Tricine-sds-page. Nature protocols, 2006. 1(1): p. 16. 78. 賴瑋婷, Pectobacterium carotovorum subsp. carotovorum 之 c-di-GMP 與 CRP 之協同作用對低分子量細菌素 carocin 基因之調控機制研究2013, 國立中興大學 化學系 79. 林佳德, 低分子量細菌素Carocin S2抗生蛋白質CaroS2K抗生活性最小化區域分離與功能分析2012, 國立中興大學化學系所. 80. Tao, F., et al., The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors. Journal of bacteriology, 2010. 192(4): p. 1020-1029. 81. Pommer, A.J., et al., Homing in on the role of transition metals in the HNH motif of colicin endonucleases. Journal of Biological Chemistry, 1999. 274(38): p. 27153-27160. 82. Pachuk, C.J., et al., Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments. Gene, 2000. 243(1): p. 19-25. 83. Tsuge, K., et al., One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic acids research, 2003. 31(21): p. e133-e133. 84. Olsen, R.L., et al., Alkaline phophatase from the hepatopancreas of shrimp (Pandalus borealis): a dimeric enzyme with catalytically active subunits. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1991. 99(4): p. 755-761. 85. 陳彥君, Pectobacterium carotovorum subsp. carotovorum低分子量細菌素基因受c-di-GMP與cyclic AMP Receptor Protein調控作用之探討. 2011, 國立中興大學化學系所. 86. Reusch, R., et al., Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli. Journal of bacteriology, 1986. 168(2): p. 553-562. 87. Chang, A.C., et al., Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. Journal of bacteriology, 1978. 134(3): p. 1141-1156.
摘要: Pectobacterium carotovorum subsp. carotovorum (Pcc) is a phytopathogenic enterobacterium responsible for soft rot disease, which characterized by extensive maceration of the affected plant tissue. Pcc also produces one or more antibacterial substances called bacteriocins, which enhance their competitiveness with other related rival species. Some of the Pcc strains produce low-molecular-weight bacteriocin (LMWB). The LMWB of Pectobacterium species was induced by Ultraviolet exposure and glucose. To date, little is known about the regulation mechanism of bacteriocin production. Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. c-di-GMP is synthesized by diguanylate cyclases (DGCs) from two molecules of GTP. The cAMP receptor protein (CRP) complex (cAMP-CRP) is a global regulator of gene expression. The CRP of Escherichia coli is a dimer made up of identical subunits. It influences transcription from a number of promoters in Escherichia coli. The previous reports from our laboratory the regulation factors, DGC and CRP, play an important role in expression mechanism of Carocin in Pcc.In this study, we want to known that how is the regulatory mechanism of carocin gene throgh cyclic dimeric guanosine monophosphate (c-di-GMP) and cAMP receptor protein (CRP) cooperation. First, dgc gene was amplified by PCR and subcloned into pGEM®-T Easy vector, and to from the plasmid pG-DGC. The plasmid pG-CRP was made by the same way. Both of dgc-defective and crp-defective were made by using dephosphorylation of vector DNA method. the dgc or crp mutant was acquired from homologues recombination method. And then, we analyzed the mRNA expression level by RT-PCR experiment and bactericion assay. In bacteriocin assay, the size of inhibition zone around strain 3F3/Δcrp was smaller than that around the wild-type strain 3F3. In mRNA level, the presence of the 925-bp amplicon revealed that caroS2K was being transcribed in the cell. The 3F3/Δcrp strain, which was an ampicillin insertional mutant, still could transcribe caroS2K, but the presence of the 267-bp amplicon revealed that caroS3I was being transcribed in the cell . The 3F3/Δcrp strain could not transcribe caroS3I. Based on the data presented in this study, we think maybe crp is a novel effecter for caroS3K transcription. It indicate that Carocin S2 and Carocin S3 are different gene to regulate. It will be of interest to study the role of dgc in the carocin gene in the future.
Pectobacterium carotovorum subsp. carotovorum (Pcc)屬於腸道菌科中革蘭氏陰性桿狀菌。此菌株感染植物時會產生果膠酶使植物組織成為濕黏狀態而引起軟腐病。為了與其他親屬相近的菌種競爭會產生細菌素(Bacteriocin),Pcc會生產低分子量細菌素(LMWB),透過紫外光的刺激和葡萄糖的濃度會誘導carocin基因表現,但是到目前為止在低分子量細菌素carocin基因調控的部分,尚無相關報導發表。 cyclic-di-GMP是細菌內普遍存在的二級訊號,調控細菌內ㄧ系列的生理功能,像是生物運動性、生物膜形成、細胞分化等,diguanylate cyclases (DGC)會催化兩分子的GTP產生cyclic-di-GMP;而cAMP receptor protein (CRP)在大腸桿菌中通常會與cAMP形成複合體而調控基因,影響了許多啟動子的轉錄作用。在本實驗室先前的報導中指出,crp突變株和dgc突變株的低分子量細菌素carocin基因皆無法表現,因此本研究目的在於探討c-di-GMP與CRP之協同作用對低分子量細菌素carocin基因的調控機制。 在實驗研究中,將dgc與crp基因分別構築於pGEM®-T Easy載體中,命名為pG-DGC和pG-CRP。利用插入抗藥性基因的方式形成了dgc或crp重組基因片段後,利用同質互換的方式將3F3菌株中的dgc或是crp基因阻斷。我們透過RT-PCR及細菌素方法分析3F3野生株與3F3/Δcrp突變株。在細菌素測試中,3F3/Δcrp突變株的抑制半徑小於野生株3F3菌株;而透過RT-PCR的實驗中,在野生株中可以看到caroS2K基因(925-bp)存在,而caroS2K基因在3F3/Δcrp突變株時,仍然正常轉錄,但是caroS3I基因(267-bp)在3F3/Δcrp突變株中,無法正常轉錄。 根據實驗結果我們認為在Pcc菌株中crp基因可能是調控caroS3K細菌素表現,因此我們推斷細菌素Carocin S2和Carocin S3的應為不同基因所調控。
URI: http://hdl.handle.net/11455/96258
文章公開時間: 2017-08-17
Appears in Collections:化學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.