摘要
本研究主要围绕构建高效表达的GFP解离载体标记苏云金芽胞杆菌而开展的研究工作,主要研究结果如下:
1.利用小质粒复制区构建解离载体
利用Tn4430的解离区和来源于库斯塔克亚种大小为2062bp的小质粒复制区ori2062构建解离载体pBMB1125。将该载体电转化到苏云金芽胞杆菌中,解离载体pBMB1125在编码解离酶的帮助质粒pBMB1200作用下发生解离,消除抗性基因DNA片段,只留下目的基因以及苏云金芽胞杆菌质粒复制区的重组质粒,将解离后的菌株在无抗性培养基培养传代40代后仍可检测到解离后的重组质粒。
2.采用不同启动子、不同质粒复制区构建GFP表达解离载体
分别将带有芽胞依赖型启动子BtⅠ-BtⅡ、非芽胞依赖型启动子pro3A和蜡状芽胞杆菌启动子proB.c的gfp片段插入到以不同质粒复制区构建的解离载体pBMB1205,pBMB1206R以及pBMB1125上,得到9株以不同启动子、不同质粒复制区的表达GFP解离载体。将得到的9种GFP解离载体电转化到苏云金芽胞杆菌无晶体突变株BMB171中,荧光显微镜镜检显示9株含GFP的苏云金芽胞杆菌重组菌在波长为300nm-510nm的激发光下可发射绿色荧光。
3.建杀虫晶体基因与gfp基因的融合基因
PCR扩增全长gfp片段融合于去掉终止结构的杀虫晶体蛋白基因的C-端,构建融合基因。首先PCR扩增去掉终止结构的pro3A+cryⅠAc10片段,用BamHⅠ、SphⅠ酶切PCR产物插入到解离载体pBMB1205上得到中间载体pBMB1205Ac。同时扩增全长gfp片段,将扩增的gfp片段用SphⅠ消化插入到pUC19上得到pUC19G,再利用SphⅠ、BglⅡ双酶切消化pUC19G,回收两端酶切位点分别为SphⅠ、BglⅡ的gfp片段,将得到的gfp片段插入到pBMB1205Ac上最终得到重组的融合基因载体pBMB0474。将pBMB0474电转化到BMB171中得到苏云金芽胞杆菌重组菌,SDS-PAGE结果显示重组菌可以表达大小约150kDa-160kDa的GFP融合蛋白。
4.不同苏云金芽胞杆菌基因工程菌的微量热变化
利用生物活性检测器LKB-2277研究杀虫晶体蛋白含量不同的两株菌YBT-833、YBT-833-2-1的热动力学变化,发现菌体合成杀虫晶体蛋白的过程是一个耗能的过程,杀虫晶体蛋白产量高的菌株向外释放的代谢热少,反之亦然。研究含质粒拷贝数目分别为4、15的GFP菌株BMB304GFP、BMB315GFP的热动力学变化,发现质粒拷贝数目高的菌株向外释放的代谢热少,反之亦然。研究含不同质粒复制区构建的GFP解离载体的菌株BMB0452B、BMB0462B、BMB0472B的微量热变化,证明含小质粒复制区ori2062构建的
解离载体携带外源基因的菌株的代谢热少于以or汀草、oril哪口两种质粒复制区构建的GFP
解离载体。
研究还发现含不同启动子驱动GFP的菌株向外释放的代谢热也不同,含芽胞依赖型
启动子Bll-BtIJ的菌株BMB0462B的代谢热少于含非芽胞依赖型的启动子pro3A菌株
BMB0463B.含蜡状芽胞杆菌启动子proB.。的菌株BMB0461B的代谢热界于两者之间。根
据蛋白表达量同菌株代谢热的关系从9种GFP表达解离载体中筛选出一种高效表达GFP的
载体pBMB0472用以标记苏云金芽胞杆菌。
5.肺基因标记苏云金芽胞杆菌野生菌
将GFP解离载体pBMB0472以及融合蛋白载体pBMB0474分别电转化到拟步行甲亚
种YBT一1765中,得到标记的野生型苏云金芽胞杆菌YBT1765一72B、YBT1765一74。PCR扩
增结果显示均可从两株标记基因工程菌中得到肺片段,且还可以从YBTI 765一74中扩增出
犯T一1765中没有的c尽]A cjo基因。SDS一队GE结果显示标记野生菌YBT1765一72B可以表
达大小为27kDa的GFP,但YBT1765一74中检测不到融合蛋白的表达。荧光显微镜观察结
果表明YBT1765一72B在激发光下可以发射绿色荧光。
The dissertation mainly concerns the construction of resolution vector with green fluorescent protein. The research results are summarized as following.
1. Construction of a resolution vector with a small plasmid origional replion
A small plasmid original replion about 2062bp come from B. thuringiensis subsp. kurstaki strain YBT-1520 and the res site of Tn4430 were used to construct a resolution vector. The resolution vector based on Tnpl-mediated site-specific recombination system of B. thuringiensis transposon Tn4430. The gene of or/2062 and other target DNA were inserted into two copy sets of res sites. The res sites have the same direction. With the help of the integrase( Tnpl) the antibiotic resistance genes and other non-5, thuringiensis DNA can be eliminated. When the engineered strain, which had the recombinant plasmid without resistance gene cultured for 40 generations the very recombinant plasmid could still be detected.
2. Construction of green fluorescent protein resolution vector with various promoters and different origional replicons
In this study, three kinds of specific promoter of B.thuringiensis: crySA promoter, Btl-BtH promoter and 5. cereus promoter were chosen to drive the expression of gfp. Three kinds of plasmid original replion of B.thuringiensis subsp. kustaki: ori44, oriW30 and ori2062 were chosen to construct the GFP resolution vector. There were 9 kinds of GFP resolution vector. All of the recombinant plasmids were introduced into crystal negative B. thuringiensis 8MB 171.The fluorescence of the 9 kinds engineered strain can be detected by the fluorescent microscope.
3. Construction of fusion genes with insecticidal protein gene and gfp
The fusion genes was constructed by PCR. The pesticidal crystal protein gene crylAc10 and gfp were chosen to construct the fusion genes. The gfp gene was fused to the C-terminate of crylAclO. The two genes were in an open reading frame. The fragment of pro3A+crylAc10 without terminated structure was inserted into plasmid pBMB1205 when it was digested by BamHI and SphI. The recombinant plasmid pBMB1205Ac was obtained. Recombinant plasmid of pUC19G was constructed when PCR product of gfp was digested by Sphl and inserted into pUC19. The gfp fragment with Sphl and BglII enzyme sites from pUC19G was inserted into pBMB1205Ac. The very plasmid pBMB0474 was constructed. When the recombinant plasmid pBMB0474 was transferred into crystal negative Bt strain BMB171 through
electroporation, the expression of the fusion genes about 150kDa-160kDa can be detected by SDS-PAGE.
4. Microcalorinetric study on B. thuringiensis
By using an LKB-2277 Bioacitivity Monitor, the thermogenic curves of different B thuringiensis strains YBT-833 and YBT-833-2-I, have been determined. The metabolism heat output revealed the heat output was correlated to the yield of the protein, the higher yield protein, the less heat output. A microcalorimetric technique based on the bacterial heat-output was explored to evaluate the effect of various promoters and different plasmid original replicons on the expression of GFP. In this study the heat output also suggested there was obvious relation between the gene copy number and heat output. The heat output rate of different strains with various copy number is BMB304GFP) BMB315GFP. The relation of the heat output rate with original replion is or/1030) or/44) or/2062. The heat output indicated that different promoter had various impact on the expression of GFP. The drive impact was promoter Btl-Btll } promoter B. cereus ) promoter cry3A.
5. Wild-type B.thuringiensis tabled with green fluorescent protein
Plasmid pBMB0472 and pBMB0474 were introduced into wild type B.thuringiensis subsp. tenebrionis YBT-1765 by electropotation. The transformams YBT1765-72B and YBT1765-74. were obtained. SDS-PAGE result suggested GFP about 27kDa was expressed in YBT1765-72. But the fusion protein could not be detected in YBT 1765-74. Fluorescent microscope observation results indicated there was fluorescence in YBT1765-72B. but there was no flu
引文
1.J.萨姆布鲁克,E F弗里奇,T曼尼阿蒂斯著。分子克隆实验指南。第二版。北京,科学出版社,1995
2.邓宏,常喜华。绿色荧光蛋白应用的研究进展。第四军医大学吉林军医学院学报.2001,23:236-243
3.刘义。微生物生命活动的微量热分析。[博士后工作报告]。武汉,武汉大学,1999
4.刘志锋,姜勇。报告基因技术的理论基础及其应用。生理科学进展.2002,33(4):361-363
5.孙明.乐超银,喻子牛。苏云金芽胞杆菌转座子整合载体的构建。农业生物技术学报,2000a,8(4):321-325
6.孙明,魏芳,刘子铎,喻子牛。苏云金芽胞杆菌质粒pBMB2062的克隆及遗传稳定载体的构建。遗传学报,2000b,27(10):932-938
7.吴岚。苏云金芽胞杆菌解离载体的构建及其特性。[博士论文]。武汉,华中农业大学,2000
8.张琼。苏云金芽胞杆菌蛋白Aⅱ及其对植物病原菌致病性的影响。[博士论文]。武汉,华中农业大学,2001
9.李林,喻子牛。不同理化处理对苏云金芽胞杆菌质粒稳定性的影响。华中农业大学学报,2000.19(1):29-32
10.李林。苏云金芽胞杆菌高频转化系统的建立及性能。[博士论文]。武汉,华中农业大学.1999
11.林晓燕,刘义,孙明.高振廷.屈松生,喻子牛。苏云金芽胞杆菌含有不同质粒和不同基因工程菌的生长代谢热动力学变化。化学学报,2001,59(5):769-773
12.侯学文.姜悦,郭勇。转基因植物中的标记基因。生物学通报,1997.32(10):19-21
13.喻子牛,孙明,刘子铎,戴经元,陈亚华,喻凌,罗曦霞。苏云金芽胞杆菌的分类及生物活性蛋白基因。中国生物防治。1996.12(2):85-89
14.温德才,刘义,赵儒明,沈萍,屈松生:量热技术在生命科学中的应用。自然杂志,2000,21(1):37-41
15.鲁松清。苏云金芽胞杆菌对鳞翅目昆虫广谱高毒基因工程菌。[博士论文]。武汉,华中农业大学,1999
16.魏芳。苏云金芽胞杆菌质粒复制子ori165及ori2062的克隆与分析。[硕士论文]。武汉,华中农业大学,2000
17. Anne K., Dunn, Jo Handelsman. A vector for promoter trapping in Bacillus cereus. Gene., 1999,226:297-305
18. Armstrong J. L., Wood, N. D., and Porteous L. A. Transconjugation between bacteria in the digestive tract of the cutworm Peridroma saucia. Appl. Environ. Microbiol., 1990, 56:1492-1493
19. Balis E.. Vatopoulos A.C., Kanelopoulou M., Mainas E,, Hatzoudis G,, Kontogianni V., Malanou-Lada H,, Kitou-Kiriakopoulou S., and Kalaothaki V. Indications of in vivo transfer of an epidemic F plasmid from Salmonella enteritidis to Escherichia coli of the normal human gut flora. J. Clin.Microiol, 1996, 34:977-979
20. Barloy F., Delecluse A., Nicolas L, and Lecadet M.M. Cloning and espression of the first anaerobic toxin gene from Clostridium bifermentans subsp. Malaysia, encoding a new mosquitocial protein with homologies to Bacillus thuringiensis delta endotoxins. J. Bacteriol., 1996,178:3099-3105
21. Baum J.A, Kakefuda M. and Gawron-Rurke, C. Engineering Bacillus thuringiensis bioinsecricides with an indigenous site-specific recombination system. Appl Environ Microbiol., 1996, 62:4367-4373
22. Baur B.. Hanselmann K., Schlimme W., and Jenni B. Genetic transformation in freshwater Escherichia coli is able to develop natural competence. Appl. Environ. Microbiol, 1996, 62:3673-3678
23. Belogurov A.A., Delver E. P, and Rodzevich O.V. Plasmid pKM101 encodes two nonhomologous antirestriction proteins whose expression is controlled by homologous regulatory sequences. J. Bacteriol. 1993,175:4843-4850
24. Ben-Dov E., Nissan G.. Pelleg N., Manasherob R., Boussiba S., Zaritsky A. Refined, circular restriction map of the Bacillus thuringiensis subsp, israelensis plasmid carrying the mosquito larvicidal genes. Plasmid. 1999,42 (3): 186-91
25. Bjokof K.. Suoniemi A., Haahtela K., and Romantschuk M. High frequency of conjugation versus plasmid segregation of RPI in epiphthic Pseudomonas syringae populations. Microbiology. 1995, 141: 2719-2727
26. Boyd D., Weiss D.S.. Chen J.C. and Beckwith J. Towards single-copy gene expression systems making gene cloning physiologically relevant: Lambda InCh, a simple Escherichia coli plasmid chromosome shuttle system. J. Bacteriol., 2000, 182: 842-847.
27. Bratoeva M. P., and John J.F. In vivo R plasmid transfer in a patient with a mixed infection of Shigella dysentery. Epidemiol. Infect., 1994, 112: 247-252.
28. Brendler T., Sawitzke J., Sergueev K. and Austin S. A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation. EMBO J, 2000', 19:6249-6258.
29. Bundock P., and Hooykaas P.J. Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc. Natl. Acad. Sci. USA, 1996, 93:15272-15275.
30. Chalfie M. Green fluorescent protein. Photochem. Photobiol., 1994, 62:651-656
31. Chamier B., Lorenz M. G., and Wackernagel W. Natural transformation of Acinetobacter calcoaceticus by plasmid DNA adsorbed to sand and groundwater aquifer material. Appl. Environ.
Microbiol., 1993, 59:1662-1667.
32. Chilley P. M., and Wilkins B. M. Distribution of the ardA family of antirestriction genes on conjugative plasmids, Microbioligy, 1905. 141 : 2157-2164.
33. Christensen B. B., Sternberg C., Andersen J. B., Eberl L., Moiler S., Givskov M., and Molin S. Establishment of new genetic traits in a microbial biofilm community. Appl. Environ. Microbiol.. 1998, 64: 2247-2255.
34. Clewell D, B., Flannagan S. E,, and Jaworski D. D. Unconstrained bacterial promiscuity: The Tn916-Tn 1545 family of conjugative transposons. Trends Microbiol., 1995, 3: 229-236.
35. Crameri A., Whitehorn E. A., Tate E. and Stemmer W. P, C. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotech, 1996, 14:315-319
36. Dahlberg C., Bergstrom M., and Hermansson M. In situ detection of high levels of horizontal plasmid transfer in marine bacterial communities.Appl. Environ.Microbiol., 1998,64: 2670-2675.
37. Davison J. Plant beneficial bacteria (review). Biotechnology. 1988, 6: 282-286.
38. Delagrave S., Hawtin R, E., Silva C, M,. Yang M, M. and Youvan D. C. Red-shifted excitation mutants of the green fluorescent protein. BioTechnology, 1995. 13:151-154.
39. Errampaui D., Leung K.. Cassidy M. D., Kostrzynska M.. Blears M.. Lee H. Application of the green fluorescent protein as a molecular marker in environmental microorganisms. J. Microbiol. Meth., 1999. 187-199
40. Gebhard F., and Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol. 1998, 64:1550-1554.
41. Georgious G., Stathopoulos C., Danguerty P. S., Nayak A. R., Iverson B, L., Curtiss R. Display of heterologous proteins on the surface of microorganisms:from the screening of combinatorial libtaries to live recombinant vaccines.Nat Biotechnol, 1997. 15:29-34
42. Goodman A. E., Hild K. C.. Marshall K. C.. and Hermansson M. Conjugative plasmid transfer between bacteria under simulated marine oligotrophic conditions, Appl.Environ Microbiol., 1993, 59:1035-1040.
43. Gotz A., and Smalla K. Manure enhances plasmid mobilization and survival in Pseudomonas putida introduced into the field soil. Appl. Environ Microbiol., 1997, 63: 1980-1986.
44. Gruzza M.. Fons M,, Ouriet M. F., Duval-Iflah Y.. and Ducluzeau R. Study of gene transfer in vitro anti in the digestive tract of gnotobiotic mice from Lactococcus lactis strains to various strains belonging to human intestinal flora. Microb. Releases.,1994, 2:183-189.
45. Haack B.L, Andrews R.E., and.Loynachan T.E. Tn916-mediated genetic exchange in soil. Soil Biol. Biochem., 1996,28:765-771
46. Hacker J., Blum-Oehler G., Muhldorfer I.. and Tschape H. Pathogenicity islands of virulent bacteria: Structure, function and impact on microbial evolution. Mol. Microbiol., 1997,23:
1089-1097.
47. Helm R., Cubitt A .B. and Tsien R,.Y. Improved green fluorescence. Nature, 1995.373:663-664
48. Heim R., Prasher D. C. and Tsien R.Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA. 1994, 91: 12501-12504
49. Herrick J. B., Stuart-Keil K. G., Ghiorse W. C., and Madsen E.L. Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site. Appl. Environ. Microbiol., 1997, 63: 2330-2337.
50. Imai Y., Ogasawara N., Ishigo-Oka D., Kadoya R., Daito T. and Moriya S. Subcellular iocalization of Dna-initiation proteins of Bacillus sublilis: evidence that chromosome replication begins at either edge of the nucleoids. Mol. Microbiol, 2000, 36: 1037-1048.
51. Jarrett P., and Stephenson M. Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis. Appl. Environ. Microbiol, 1990, 56:1608-1614.
52. Jiang S. C., and Paul J. H. Gene transfer by transduction in the marine environment. Appl. Environ. Microbiol.. 1980, 64: 2780-2787.
53. John D., Thomas I., Alun I.J., Morgan W., John M.. Whipps and Jon R.. Saunders. Plasmid transfer between Bacillus thuringiensis subsp, israelensis strains in laboratory, culture, river water, and dipteran larvae. Appl. Environ. Microbiol..2001,67: 330-338
54. Jose F., Kraner J., Klauser T., Pohiner J., Meyer T. Absence of periplasmic DsbA oxidoreductase facilityates export of cysteine-contalning passenger proteins to the Escherichia coil cell surface via the Iga autotrasporter pathway. Gene, 1996, 178:107-110
55. Kidambi S. P., Ripp S., and Miller R. V, Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane. Appl. Environ. Microbiol., 1994, 60: 496-500.
56. King N., Dreesen O., Stragier P., Pogliano K. and Losick R. Septation, dephosphorylation and the activation of cF during sporulation in Bacillus subtilis, Genes Dev, 1999, 13:1156-1167.
57. Kruse H., and Sorum H. Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments. Appl. Environ. Microbiol.. 1994, 60: 4015-4021.
58. Lacy G. H., Stromberg V. K., and Cannon N. P. Erwinia amylovora mutants and in planta derived transconjugants resistant to oxytetracycline. Can. J. Microbiol., 1984,6: 33-39.
59. Lawrence J. G., and Ochman H. Molecular archaeology of the Escherichia coli genome. Proc. Natl. Acad.Sci. USA, 1998,95: 9413-9417.
60. Lemon K.P., and Grossman A.D. Localization of bacterial DNA polymerase: evidence for a factory model of replication. Science, 1998, 282: 1516-1519.
61. Lewin B., Gene Ⅳ. New York. Oxford University Press, 1991
62. Lilley A. K., and Bailey M.J. The acquisition of indigenous plasmids by a genetically marked Pseudomonad population colonizing the sugar beet phytosphere is related to local environmental
conditions. Adv. Appl. Microbiol.. 1997.63:1577-1583.
63. Lilley A. K., Fry J. C., Day M. J., and Bailey M.J. In situ transfer of an exogenously isolated plasmid between Pseudomonas spp in the sugar beet rhizosphere. Microbiology. 1994, 140: 27-33.
64. Liu Yi, Xie Changli, Qu Songsheng. Mirocalorimetric study of metabolic inhibiton by humic acids in mitochondriaform oryctolagus cuniculus domestica liver cells. J. Chemospere. 1996, 33(1): 99-105
65. Lorenz M. G., and Wackernagel W. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev.. 1994, 58: 563-602.
66. Lorenz M. G., Reipschlager K., and Wackernagel W. Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater. Arch. Microbiol.. 1992,157: 355-360.
67. Louvrier P., Laguerre G., and Amarger N. Distribution of symbiotic genotypes in Rhizobium leguminosarum biovar viciae populations isolated directly from soils. Appl. Environ. Microbiol.. 1996,62: 4202-4205.
68. Lunsford R. D. Streptococcal transformation: Essential features and applications of a natural gene exchange system. Plasmid. 1998, 39: 10-20.
69. Marcinek H.. Wirth R., Muscholl-Silberhorn A., and Gauer M. Enterococcus faecalis gene transfer under natural conditions in municipal sewage water treatment plants. Appl Environ. Microbiol.. 1998,64: 626-632.
70. Matheson V. G., Forney L. J., Suwa Y.. Nakatsu C. H.. Sewtone A. J.. and Holben W. E. Evidence for aquisition in nature of a chromosomal 2.4-dichlorphenooxyacetic acid/ketoglutarate dioxygenase gene by different Burkholderia spp. Appl. Environ Microbiol., 1997,63: 2266-2272.
71. Matic I.. Taddei F.. and Radman M. Genetic barriers among bacteria. Trends Microbiol.. 1996, 4:69-72.
72. Mazodier P.. and Davies J. Gene transfer between distantly related bacteria. Annu Rev. Bioche.,1991.25:147-171
73. Mitsuhiro Itaya, Syed M., Shaheduzzaman, Kuniko Matsui. Akira Omori, and Takashi Tsuji. Green marker for colonies of Bacillus subtilis, Biosci. Biotechnol.Biochem.., 2001,65 (3):579-583
74. Moffatt B. A., and Studier F.W. Entry of bacteriophage T7 DNA into the cell and escape from host restriction. J. Bacteriol., 1998,170:2095-2105.
75. Muniesa M., and Jofre J. Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and 89 environmental gene transfer that carry the Shiga toxin 2 gene. Appl. Environ Microbiol., 1998.64: 2443-2448.
76. Nielsen K. M.. Bones A. M.. and van Elsas J.D. Induced natural transformation of Acinetobacter calcoaceticus in soil microcosms. Appl. Environ. Microbiol., 1997a. 63: 3972-3977.
77. Nielsen K. M., van Weerelt M. D., Berg T. N., Bones A. M., Hagler A. N., and van Elsas J. D. Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms. Appl. Environ. Microbiol.. 1997b, 63: 1945-1952.
78. Nijsten R., London N., van den Bogaard A.. and Stobberingh E. In-vivo transfer of resistance plasmids in rat, human or pig-derived intestinai flora using a rat model. J.Antimicrob. Chemother.,1995,36: 975-985.
79. Nikolich M. P., Hong G., Shoemaker N. B.. and Salyers A. Evidence for natural horizontal transfer of tetQ between bacteria that normally colonize humans and bacteria that normally colonize livestock. Appl. Environ.Microbiol.. 1994.60: 3255-3260.
80. Nuβlein K.. Maris D.. Timmis K., and Dwyer D. F. Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms. Appl. Environ. Microbiol., 1992. 3380-3386.
81. Onogi T.. Niki H., Yamozoe M. and Hiraga S. The assembly and migration of SeqA-GFP fusion in living cells of Escherichia coll. Mol. Microbiol.1999, 31: 1775-1782.
82. Park H-W, Ge B-X, Barter L.S. Optimization of Cry3A yields in Bacillus thuringiensis by use of sporalation-dependent promoters in combination with the STAB-SD mRNA squence. Appl. Envion Microbiol. 1998,64:3932-3928
83. Paul J. H.. Thurmond J. M., and Frischer M. E. Gene transfer in marine water column and sediment microcosms by natural plasmid transformation. Appl. Environ. Microbiol.. 1991,1509-1515.
84. Peters M., Heinaru E., Talpsep E., Wand H., Stottmeister U., Heinaru A., and Nurk A. Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species. Appl. Environ. Microbiol., 1997, 63: 4899-4906.
85. Prodinger W. M., Fille M.. Bauernfeind A., Stemplinger I., Amann S., Pfausler B., Lass-FIorl C., and Dierich M. P. Molecular epidemioiogy of Klebsiella pneumoniae producing SHV-5 b-lactamase: parallel outbreaks due to multiple plasmid transfer. J. Clin. Microbiol., 1996, 34:564-568.
86. Ravatn R., Zehnder A. J. B., and van der Meer J. R. Low-frequency horizontal transfer of an element containing the chiorocatechol degradation genes from Pseudoraonas sp. Strain B13 to Pseudomonas putida Fl and to indigenous bacteria in laboratory-scale activatedsludge microcosms. Appl. Environ. Microbiol., 1998,64:2126-2132.
87. Reddy A, Battisti L, Thorne C B. Identification of self-transmissible plasmids in four Bacillus thuringiensis suspecies. J. Bacteriol, 1987, 169:5263-5270
88. Ripp S., Ogunseitan O. A., and Miller R.V. Transduction of a freshwater microbial community by a new Pseudoraonas aeruginosa generalized transducing phage, UTI. Mol. Ecol.. 1994, 3:
121-126.
89. Ruan L., Liu Y., Gao Z.. Shen P. and Sheng Q.S. Microcalorimetric Study on Expression of Foreign Genes in Bacillus thuringiensis. J Thermal Analysis and Calorimetry., 2002,70:521-525
90. Salyers A. A., and Shoemaker N. B. Resistance gene transfer in anaerobes: New insights, new problems.Clin, Infect. Dis., 1996,23:S36-S43.
91. Sanchis V, Agaisse H, Chaufaux J and Lereclus D A. Recombinanase -mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains. Appl Environ Microbial, 1997, 63(2): 779-784
92. Sandaa R-A., and Enger E. Transfer in marine sediments of the naturally occurring plasmid pRAS1 encoding multiple antibiotic resistance. Appl. Environ. Microbial., 1994,60: 4234-4238.
93. Scott K. P.. and Flint H.J. Transfer of plasmids between strains of Escherichia coli under rumen conditions. J. Appl. Bacterial., 1995.78:189-193.
94. Smit E., Venne D., and van Elsas J.D. Mobilization of a IncQ plasmid between bacteria on agar surfaces and in soil via contransfer or retrotransfer. Appl. Environ.Microbial., 1993, 59: 2257-2263.
95. Smit E.. Waiters A., and van Elsas J. D. Selftransmissible mercury resistance plasmids with gene-mobilizing capacity in soil bacterial populations: Influence of wheat roots and mercury addition Appl Environ.Microbiol.. 1998.64:1210-1219.
96. Sousa C.. Kotrba P., Rumi T. Cebolta A.. de Lorenzo V. Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer memebrane protein LamB. J.Bacterial, 1998, 180:2280-2284
97. Sundin G. W., Demezas D. H., and Bender C. L. Genetic and plasmid diversity within natural populations of Pseudomonas syringae With various exposures to copper and streptomycin bactericides.Appl. Environ. Microbial., 1994.60:4421-4431.
98. Tauxe R. V., Cavanagh T. R.., and Cohen M.L. Interspecies gene transfer in viva producing an outbreak of mulyiply resistant shigellosis. J Infect Dis.. 1989, 160:1067-1070.
99. Troxler J.. Azelvandre P.. Zala M., De fago G.. and Haas D. Conjugal transfer of chromosomal genes between Pseudomonads fluorescent in the wheat rhizosphere. Appl. Environ. Microbial.,1997,63 : 213-219.
100. Van der Meet, J. R., de Vos W. M., Harayama S., and Zhender A. J.B. Molecular mechanisms of genetic adaptation to xenobiotic compounds. MicrobioL Rev., 1992, 56: 677-694.
101. van Veen J. A., van Overbeek L. S., and van Elsas J. D., Fate and activity of microorganisms introducedinto soil. Microbial. Mol. Biol Rev,. 1997.61: 121-135,
102. Veal D. A., Stokes H. W.. and Grant D. Genetic exchange in natural communities. Adv. Microb. Ecol.. 1992. 12: 383-430.
103. Waldor M. K., and Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding
cholera toxin. Science, 1996, 272: 1910-1914.
104. Watanabe K., and Sato, M. Plasmid-mediated gene trqnsfer between insect-resident bacteria. Enterobactercloacae, and plant-epiphytic bacteria. Erwinia herbicola, in guts of silkworm larvae. Curr. Microbiol,, 1998, 37: 352-355.
105. Wilcks A., Jayaswal N., Lereclus D. Characterization of plasmid pA W63, a second self-transmissible plasmid in Bacillus thuringiensis subsp, kurstaki HD73. Microbiology, 1998,144:1263-1270
106. Williams H. G., Day M., Fry. J. C., and Stewart G.J. Natural transformation in river epilithon. Appl.Environ. Microbiol. 1996. 62: 2994-2998.
107. Wilson M.. and Lindow S. E. Release of recombinant microorganisms.Annu. Rev. Microbiol.. 1993, 47: 913-944.