Xanthomonas axonopodis pv. glycines 8ra로부터 분리한 모자이크형 박테리오신 GlyR의 유전체 및 생화학적 연구
Genomic and Biochemical Studies of the Large Mosaic Bacteriocin GlyR from Xanthomonas axonopodis pv. glycines 8ra
- 주제(키워드) bacteriocin
- 발행기관 아주대학교
- 지도교수 문은표
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 박사
- 학과 및 전공 일반대학원 생영과학과
- 본문언어 영어
초록/요약
Xanthomonas axonopodis pv. glycines 8ra is known to strongly inhibit the growth of closely related xanthomonas species by producing bacteriocin named glycinecin. In this study a clone containing a novel glycinecin of glyR gene product, glycinecin R, was isolated from a pG35, cosmid genomic library, of X. axonopodis pv. glycines 8ra. A series of deletion and transposon-insertion mutagenesis revealed that the 5 kb genomic region in the clone is responsible for the full bacteriocin activity. Inhibitory activity of glycinecin R is specific to X. campestris. pv. vesicatoria and X. oryzae pv. oryzae, pathogens of pepper and rice, respectively. The partially purified glycinecin R was stable under the wide range of pH from 2.0 up to 9.0, and it was also found to be moderately stable to heat, as no loss of activity was observed after incubation for 20 minutes at 50 ºC. Series of Southern hybridization analysis revealed that the glyR homologs exist in all the xanthomonads up to three copies, but not in other bacteria species examined. Thus, the glyR may be useful as the genus-specific DNA marker for the identification of xanthomonads. Furthermore, the multiple copies of glyR homologs exhibit pathovar-specific patterns in the pathovars glycines, dieffenbachiae, citri and vesicatoria. The unique pathovar-specific patterns of the glyR homologs are also useful as the critical criteria in identifying various pathovars of xanthomonas species. The glyR gene is 4,422 bp in length capable of producing the high molecular weight glycinecin R. The glycinecin R protein consists of two domains, core and core extension. The core is made up of a motif, GXXXXYXYDXXGRLT, repeated 31 times. Homologs of the glyR core domain at the amino acid level were also identified in a wide spectrum of unrelated bacterial genomes by BLAST search and the 60-80 amino acids in the C-terminal end of their cores were aligned using ClUSTAL algorithm. Extensive sequence analysis revealed that the core domain of the glycinecin R shows homology to the cores of Rhs element of unknown function in E. coli and toxin complex (tc) gene of Photorhabdus luminescens. Even though the function of the core domain in those proteins is not known so far, our result implies that the core domain is associated with killing activity since the domain is common in two cytotoxic proteins, glycinecin R and tc toxin. It is also important to emphasize these toxins are active in the target-specific manner. The hypervariable core extension might be one of the major components for the recognition of their own target cells. The second cosmid clone, pG11, containing a distinct genomic region carrying the glyR homolog and also active in production of glycinecin R was isolated and characterized as well. The results confirm the existence of multiple copies of glyR homologs shown by genomic Southern analysis. Therefore, glycinecin R is a mosaic bacteriocin encoded by a gene family.
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Abstract-------------------------------------------------------------------------------5
Contents------------------------------------------------------------------------------8
List of figures------------------------------------------------------------------------11
List of Tables------------------------------------------------------------------------13
Ⅰ. Introduction--------------------------------------------------------------------14
Ⅱ. Materials and Methods-----------------------------------------------------26
Ⅱ. A. Bacterial strains and plasmids-------------------------------------26
Ⅱ. B. Culture media, antibiotics and bacterial growth--------------26
Ⅱ. C. Construction and Screening of cosmid library of X. axonopodis pv. glycines 8ra----------------------------28
Ⅱ. D. Enzymes and Chemicals--------------------------------------28
Ⅱ. E. Plasmid DNA isolation-----------------------------------------28
Ⅱ. F. Genomic DNA large preparation from G-bacteria---------30
Ⅱ. G. General recombinant DNA techniques-------------------------32
Ⅱ. H. Agarose gel electrophoresis----------------------------------------32
Ⅱ. I. DNA fragment elution-----------------------------------------------32
Ⅱ. J. Transformation--------------------------------------------------------33
Ⅱ. K. Nucleotide Sequencing---------------------------------------------33
Ⅱ. L. Transposon mutagenesis--------------------------------------------34
Ⅱ. M. Bacteriocin production and preparing cell crude extraction-----35
Ⅱ. N. Assay for bacteriocin activity--------------------------------------36
1. Spot-on-lawn method---------------------------------------------36
2. Agar diffusion methods------------------------------------------36
3. Serial dilution method-on broth media-----------------------37
Ⅱ. O. Effects of heat, pH and degradative enzyme on bacteriocin activity--------37
Ⅲ. P. Production of the bacteriocin, glyR in different growth media----------------38
Ⅱ. Q. Southern hybridization---------------------------------------------39
1. Cross hybridization analysis of cosmid clones--------------39
2. Southern hybridization analysis of bacterial genomes with glyR gene as a probe---------------40
Ⅱ. R. Total RNA isolation-----------------------------------------------41
Ⅱ. S. Reverse transcription and PCR (RT-PCR)-----------------------42
1. First strand cDNA synthesis-------------------------------------42
2. PCR--------------------------------------------------------------------43
Ⅱ. T. DNA alignment-----------------------------------------------------4
Ⅲ. Results
Part1. Molecular and Biochemical characterization of glycinecin R --45
Ⅲ. A. Screening of cosmid library clones for bacteriocin producing genes------45
Ⅲ. B. Cross hybridization analysis of pG11, pG13, pG35 clones--46
Ⅲ. C. Cloning of the DNA region responsible for glycinecin R from pG35---------------------46
Ⅲ. D. Deletion constructs of glycinecin R and complementation test ------------------------------47
Ⅲ. E. Transposon mutagenesis and screening of mutant clones--47
Ⅲ. F. Antimicrobial spectrum of glycinecin R-----------------------48
Ⅲ. G. Nucleotide sequencing of pGly63-------------------------------49
Ⅲ. H. Transcription analysis----------------------------------------------50
Ⅲ. I. Biochemical characterization of glycinecin R-------------------50
1. Growth phase dependent glycinecin R production--------50
2. Biochemical characterization of glycinecin R----------------52
Ⅳ. Results
Part2. Genomic studies of glyR homologs--------------------------------72
Ⅳ. A. Genus-specific distribution of glyR homologs in Xanthomonas genomes------------------72
Ⅳ. B. Pathovar-specific distribution pattern of glyR homologs in three other pathovars of Xanthomonas strains--------------73
Ⅳ. C. Analysis of amino-acid sequence of glyR-----------------------75
Ⅳ. D. Internal repetition in the glyR core peptide---------------------76
Ⅳ. E. Blast search - glyR homologs and Rhs elements---------------77
Ⅳ. F. Comparison structure of Glycinecin (core/ext.) with other glyR-like protein---------------------------------------------------78
Ⅳ. G. Alignment and phylogeny of glyR core-like sequences from both Gram-negative and Gram-positive bacteria--81
Ⅴ. Discussion and Conclusion------------------------------------------------102
References---------------------------------------------------------110
