臺灣博碩士論文加值系統

革蘭氏陰性菌 Xanthomonas campestris pv. campestris (Xcc) 為十字花科蔬菜黑腐病 (black rot) 的植物病原菌，造成農業上嚴重損失。此菌所產生之大量胞外多醣稱為 xanthan gum，在工業上有許多用途。本菌尚能分泌多種胞外酵素，如蛋白酶、纖維酶及果膠酶等。這些胞外多醣與胞外酵素為本菌重要的致病因子，目前已知多元調控因子 Clp (cAMP receptor protein-like protein) 蛋白與 rpf (regulation of pathogenicity factors) 基因串參與其產生與分泌。
本研究探討 Xcc 之聚半乳糖醛酸酶基因 peh，共分五方向進行。首先，利用含 polygalacturonic acid (PGA) 為受質之平板培養基測試胞外聚半乳糖醛酸酶的活性。結果發現 AU56E (clp 基因缺失突變株) 及 MH172 (peh 基因缺失突變株) 之水解 PGA 能力，明顯低於 Xc17 (野生株)，表示 clp 參與此酵素的表現，且 peh 之編碼產物為本菌主要胞外聚半乳糖醛酸酶。在生長曲線方面，MH172 及 Xc17 在 LB 和本試驗所用之其他貧瘠的培養基中，均具相似生長速率。在致病性測試上，MH172 病徵出現的速度和 Xc17 相比只具輕微差異，由此推測 Xcc 之胞外聚半乳糖醛酸酶在致病性方面可能扮演次要的角色。其次，將 peh 編碼區選殖到載體 pET30b 並於 BL21(DE3) 中進行重組蛋白表現。利用親和性管柱層析純化此重組蛋白，並以 SDS-PAGE 及 Western blot 分析，可於 51 kDa 處發現一蛋白色帶，與 peh 基因之推估胺基酸序列的分子量 (47452 Da) 相近。第三部份則利用 5’-RACE (rapid amplification of cDNA ends) 技術來訂定 peh mRNA 之 5’-端。結果顯示位於轉譯起始密碼上游 74 nt 之核甘酸 “T”，為 peh 之轉錄起始點。第四部份為 peh 上游區域之探討。將取得不同長度的 peh 上游片段，各別選殖於 pFY13-9 (為 promoter-probing vector)，所得之 Ppeh-lacZ transcriptional fusion constructs 再經電孔法送入 Xc17、AU56E 及 RM17F (rpfF 基因缺失突變株) 中，分析啟動子活性。結果指出 peh 轉錄起始點上游 -144 至 +56 這 200 bp 的區域，為 peh 完全表現所必需。透過 transcriptional fusion 分析也顯示 peh 的啟動子活性，不論在 clp 突變株或 rpfF 突變株，其活性均比野生株低，因此推測 Clp 及 RpfF 對於 peh 之轉錄屬正調控。另外，peh 的表現可因 PGA 的存在下被誘導，而此基因之轉錄也受 catabolite repression、厭氧 (oxygen limitation)、缺氮 (nitrogen starvation) 及滲透度 (osmolarity) 所影響。最後，透過 gel retardation 試驗，證實 Clp 可和 peh 啟動子形成 DNA-蛋白質複合物，亦即 Clp 對於 peh 之轉錄活化機制為直接調控。

The Gram-negative plant pathogenic Xanthomonnas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, causing tremendous loss in agriculture. This bacterium produces great amounts of an exopolysaccharide, xanthan gum, which has a variety of applications in industry. In addition, it is capable of secreting several extracellular enzymes, including proteases, cellulases, and pectinases. These enzymes and xanthan have long been considered important virulence determinants. Gene regulation of these virulence factors is only known that the global transcription factor Clp (cAMP receptor protein-like protein) and the rpf (regulation of pathogenicity factors) gene cluster regulate the production and secretion of these virulence determinants.
The aim of this study was to characterize the peh gene encoding polygalacturonase in Xcc. First, the extracellular polygalacturonase activity was analyzed by plate assays supplemented with polygalacturonic acid (PGA) as substrate. The results showed that the pectinolytic activity was dramatically reduced in AU56E (clp mutant) and MH172 (peh mutant) using Xc17 (wild-type) for comparison. It was suggested that clp involved in this extracellular enzyme expression and peh gene might coding for major polygalacturonase in Xcc. Growth curve analysis indicated that MH172 has same growth rates as Xc17 in LB or other basal medium used in this study. In pathogenicity test, the rate of development of symptoms in MH172 was slightly slower than Xc17. These findings suggested that polygalacturonase of Xcc may play minor role in pathogenesis. Second, the peh coding region was cloned in pET30b and over expressed in BL21(DE3). Recombinant Peh protein, purified by affinity chromatography, migrated as a band of 51 kDa in SDS-PAGE and Western blot, and revealed a size similar to that of the deduced amino acid sequence from peh gene (47452 Da). Third, using 5’-RACE (rapid amplification of cDNA ends) technique, nucleotide T at 74 nt upstream of the peh translation initiation codon was mapped as the 5’ end of the peh mRNA, is the transcription start site. Fourth, the upstream region of peh was characterized. Different fragments of the peh upstream region were separately cloned into the pFY13-9 (promoter-probing vector), and the resultant Ppeh-lacZ transcriptional fusion constructs were introduced into the Xc17, AU56E and RM17F (rpfF mutant) by electroporation respectively. Results of reporter assays indicated that the 200 bp region containing bp -144 to +56 relative to the peh transcription start site was required for maximal level of peh expression. Transcriptional fusion analysis also revealed that the promoter activity of peh was reduced in either clp mutant or rpfF mutant and suggested that Clp as well as RpfF positively regulate transcription of the peh gene. In addition, the peh expression was induced in the presence of PGA and affected by catabolite repression, oxygen limitation, nitrogen starvation, and osmolarity. Fifth, gel retardation assays indicated that Clp and peh promoter form a DNA-protein complex. In other words, the transcription of peh was upregulated by Clp in a directly manner.

壹. 中文摘要 1
貳. 英文摘要 3
參. 前言 5
肆. 材料 12
一、菌種及質體 12
二、一般藥品 12
三、酵素 12
四、培養基 12
1. 液體培養基 13
2. 固體培養基 13
五、引子 13
伍. 儀器 14
一、吸光值測定 14
二、桌上型微量離心機 14
三、桌上型低溫冷凍離心機 14
四、電孔儀 14
五、聚合酶鏈鎖反應儀 14
六、低溫迴轉式振盪培養箱 14
七、紫外光照相系統 14
八、電泳及轉漬設備 14
1.洋菜凝膠電泳 14
2.聚丙烯醯胺凝膠電泳 14
3.轉漬 14
九、其它 14
1.水浴機 14
2.酸鹼測定儀 14
3.振盪器 14
4.乾浴機 14
5.攪拌器 14
6.電子天枰 14
陸. 方法 15
一、 細菌之培養與保存 15
二、 胞外聚半乳糖醛酸酶之活性分析 15
三、 DNA 的製備 15
1.小量質體 DNA 抽取法 15
2.染色體 DNA 抽取法 16
四、 聚合酶鏈鎖反應 (Polymerase Chain Reaction，PCR) 16
五、 洋菜凝膠製備與電泳分析 17
1.凝膠製備 17
2.電泳分析 17
六、 質體的選殖 17
1.PCR 增幅片段之選殖 17
2.限制酶的切割分析 18
3.DNA 回收 18
4.DNA 的黏接 18
七、 轉形作用 18
1.E. coli 之轉形 18
2.Xcc 之轉形 19
八、 長曲線測定 20
九、 致病性測試 20
十、 重組蛋白之大量表現與純化 20
1.重組 Peh 蛋白之大量表現與純化 21
2.重組 Clp 蛋白之大量表現與純化 21
十一、 SDS-聚丙烯醯胺凝膠電泳 (SDS-polyacrylamide gel electrophoresis，SDS-PAGE) 22
1.10% 聚丙烯醯胺之解析凝膠 (resolving gel) 溶液 22
2.4% 聚丙烯醯胺之集膠凝膠 (stacking gel) 溶液 22
3.凝膠製備 22
4.電泳分析 22
十二、 西方點墨法 (Western blot) 23
1.電泳分離與轉漬 23
2.偵測 23
十三、 Xcc total RNA 的抽取 24
十四、 cDNA 末端的快速增幅 (Rapid Amplification of cDNA Ends，RACE) 24
1.引子設計 25
2.第一股互補 DNA (complementary DNA，cDNA) 合成 25
3.第一股 cDNA 的純化 25
4.cDNA 的 tailing 反應 26
5.dC-tailed cDNA 的 PCR 反應與選殖 26
6.巢式 PCR 增幅 26
十五、 啟動子之活性分析 26
1.培養方式 26
2.��-Galactosidase 之活性測試 27
十六、 Gel retardation 27
1.生物素標定 DNA 片段製備 27
2.結合反應 27
3.4.5% 聚丙烯醯胺凝膠製備 28
4.電泳分析與轉漬 28
5.偵測 28
柒. 結果與討論 30
一、 以生物資訊學方式，尋找 Xcc 之疑似果膠酶 (putative pectinase) 30
二、 胞外聚半乳糖醛酸酶之活性分析 31
三、 peh 突變株 MH172 之構築 31
1.質體 pOKpehG 之構築 31
2.進行同質互換 (homologous recombination) 32
3.突變株的確認 32
四、 MH172互補株 MH172(pRKpeh) 之構築 32
1.質體 pRKpeh 之構築 32
2.互補株之取得 33
五、 Xc17、MH172 及 MH172(pRKpeh) 之表型分析 33
1.胞外聚半乳糖醛酸酶活性比較 33
2.生長曲線 34
3.致病性 34
六、 重組 Peh 蛋白之誘導表現與純化 34
1.質體 pETpeh 之構築 35
2.重組 Peh 蛋白誘導與純化 35
七、 聚半乳糖醛酸酶之胺基酸序列特性 36
1.Xanthomonas 屬胞外聚半乳糖醛酸酶之推估胺基酸序列 比對 36
2.不同物種胞外聚半乳糖醛酸酶基因推估胺基酸序列比對 37
八、 peh 轉錄起始點之訂定 38
1.5’-RACE 產物之製備 38
2.巢式 PCR 產物之選殖與序列分析 38
3.有關 Xcc 啟動子序列的特性 39
九、 peh上游片段之選殖 39
十、 peh 啟動子的活性分析 40
1.以 LB 為培養基，peh 之啟動子活性 41
2.不同碳源及 PGA，對於 peh 啟動子活性的影響 42
3.其他環境因子對於 peh 啟動子活性的影響 46
十一、 peh上游片段之 gel retardation 試驗 48
1.結合反應所需 peh 上游片段之選擇 48
2.結合反應 48
十二、rpfF 上游片段啟動子活性探討 49
1.rpfF 上游片段之選殖 49
2.rpfF 上游片段啟動子的活性分析 50
捌. 結論 51
玖. 參考文獻 53
拾. 表 61
拾壹. 圖 65
拾貳. 縮寫字對照表 87

Barber, C. E., Tang, J. L., Feng, J. X., Pan, M. Q., Wilson, T. J., Slater, H., Dow, J. M., Williams, P. & Daniels, M. J. (1997). A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Mol Microbiol 24, 555-566.

Beaulieu, C., Minsavage, G. V., Canteros, B. I. & Stall, R. E. (1991). Biochemical and genetic analysis of a pectate lyase gene from Xanthomonas campestris pv. vesicatoria. Mol Plant Microbe Interact 4, 446-451.

Becker, A., Katzen, F., Puhler, A. & Ielpi, L. (1998). Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50, 145-152.

Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.

Blanco, P., Sieiro, C. & Villa, T. G. (1999). Production of pectic enzymes in yeasts. FEMS Microbiol Lett 175, 1-9.

Blin, N. & Stafford, D. W. (1976). A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 3, 2303-2308.

Chan, J. W. & Goodwin, P. H. (1999). The molecular genetics of virulence of Xanthomonas campestris. Biotechnol Adv 17, 489-508.

Chang, K. W., Weng, S. F. & Tseng, Y. H. (2001). UDP-glucose dehydrogenase gene of Xanthomonas campestris is required for virulence. Biochem Biophys Res Commun 287, 550-555.

Cohen, S. N., Chang, A. C. & Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A 69, 2110-2114.

da Silva, A. C., Ferro, J. A., Reinach, F. C., Farah, C. S., Furlan, L. R., Quaggio, R. B., Monteiro-Vitorello, C. B., Van Sluys, M. A., Almeida, N. F., Alves, L. M., do Amaral, A. M., Bertolini, M. C., Camargo, L. E., Camarotte, G., Cannavan, F., Cardozo, J., Chambergo, F., Ciapina, L. P., Cicarelli, R. M., Coutinho, L. L., Cursino-Santos, J. R., El-Dorry, H., Faria, J. B., Ferreira, A. J., Ferreira, R. C., Ferro, M. I., Formighieri, E. F., Franco, M. C., Greggio, C. C., Gruber, A., Katsuyama, A. M., Kishi, L. T., Leite, R. P., Lemos, E. G., Lemos, M. V., Locali, E. C., Machado, M. A., Madeira, A. M., Martinez-Rossi, N. M., Martins, E. C., Meidanis, J., Menck, C. F., Miyaki, C. Y., Moon, D. H., Moreira, L. M., Novo, M. T., Okura, V. K., Oliveira, M. C., Oliveira, V. R., Pereira, H. A., Rossi, A., Sena, J. A., Silva, C., de Souza, R. F., Spinola, L. A., Takita, M. A., Tamura, R. E., Teixeira, E. C., Tezza, R. I., Trindade dos Santos, M., Truffi, D., Tsai, S. M., White, F. F., Setubal, J. C. & Kitajima, J. P. (2002). Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417, 459-463.

Daniels, M. J., Barber, C. E., Turner, P. C., Cleary, W. G. & Sawczyc, M. K. (1984a). Isolation of mutants of Xanthomonas campestris pv. campestris showing altered pathogenicity. J Gen Microbiol 130, 2447-2455.

Daniels, M. J., Collinge, D. B., Dow, J. M., Osbourn, A. E. & Roberts, I. N. (1987). Molecular biology of the interaction of Xanthomonas campestris with plant. Plant Physiol Biochem 25, 353-359.

Daniels, M. J., Barber, C. E., Turner, P. C., Sawczyc, M. K., Byrde, R. J. & Fielding, A. H. (1984b). Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. Embo J 3, 3323-3328.

de Crecy-Lagard, V., Glaser, P., Lejeune, P., Sismeiro, O., Barber, C. E., Daniels, M. J. & Danchin, A. (1990). A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity. J Bacteriol 172, 5877-5883.

Dong, Q. & Ebright, R. H. (1992). DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein. J Bacteriol 174, 5457-5461.

Dow, J. M., Scohield, G., Trafford, K., Turner, P. C. & Daniels, M. J. (1987). A gene cluster in Xanthomonas campestris pv. campestris required for pathogenicity controls the excretion of polygalacturonate lyase and other enzymes. Physiol Mol Plant Pathol 31, 261-271.

Dow, J. M., Milligan, D. E., Jamieson, L., Barber, C. E. & Daniels, M. J. (1989). Molecular cloning of a polygalacturonate lyase gene from Xanthomonas campestris pv. campestris and role of the gene product in pathogenicity. Physiol Mol Plant Pathol 35, 113-120.

Dow, J. M., Feng, J. X., Barber, C. E., Tang, J. L. & Daniels, M. J. (2000). Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris. Microbiology 146, 885-891.

Dow, J. M., Crossman, L., Findlay, K., He, Y. Q., Feng, J. X. & Tang, J. L. (2003). Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proc Natl Acad Sci U S A 100, 10995-11000.

Frohman, M. A. (1993). Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol 218, 340-356.

Fu, J. F. & Tseng, Y. H. (1990). Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey. Appl Environ Microbiol 56, 919-923.

Giovane, A., Servillo, L., Balestrieri, C., Raiola, A., D''Avino, R., Tamburrini, M., Ciardiello, M. A. & Camardella, L. (2004). Pectin methylesterase inhibitor. Biochim Biophys Acta 1696, 245-252.

Gough, C. L., Dow, J. M., Barber, C. E. & Daniels, M. J. (1988). Cloning of two endoglucanase genes of Xanthomonas campestris pv. campestris: Analysis of the role of the major endoglucanase in pathogenesis. Mol Plant Microbe Interact 1, 275-581.

Hagerman, A. E., Blau, D. M. & McClure, A. L. (1985). Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores. Anal Biochem 151, 334-342.

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.

Harding, N. E., Cleary, J. M., Cabanas, D. K., Rosen, I. G. & Kang, K. S. (1987). Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris. J Bacteriol 169, 2854-2861.

He, Y. W., Xu, M., Lin, K., Ng, Y. J., Wen, C. M., Wang, L. H., Liu, Z. D., Zhang, H. B., Dong, Y. H., Dow, J. M. & Zhang, L. H. (2006). Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions. Mol Microbiol 59, 610-622.

Herlache, T. C., Hotchkiss, A. T., Jr., Burr, T. J. & Collmer, A. (1997). Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum. Appl Environ Microbiol 63, 338-346.

Herron, S. R., Benen, J. A., Scavetta, R. D., Visser, J. & Jurnak, F. (2000). Structure and function of pectic enzymes: virulence factors of plant pathogens. Proc Natl Acad Sci U S A 97, 8762-8769.

Hsiao, Y. M. & Tseng, Y. H. (2002). Transcription of Xanthomonas campestris prt1 gene encoding protease 1 increases during stationary phase and requires global transcription factor Clp. Biochem Biophys Res Commun 295, 43-49.

Hsiao, Y. M., Liao, H. Y., Lee, M. C., Yang, T. C. & Tseng, Y. H. (2005). Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites. FEBS Lett 579, 3525-3533.

Huang, J. H. & Schell, M. A. (1990). DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum. J Bacteriol 172, 3879-3887.

Huang, P. H. (2002).Transcriptional analysis of pilBA2A1CD cluster in Xanthomonas campestris pv. campestris. Master thesis, National Chung Hsing University.

Hugouvieux-Cotte-Pattat, N., Dominguez, H. & Robert-Baudouy, J. (1992). Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937. J Bacteriol 174, 7807-7818.

Hugouvieux-Cotte-Pattat, N., Shevchik, V. E. & Nasser, W. (2002). PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases. J Bacteriol 184, 2664-2673.

Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W. & Reverchon, S. (1996). Regulation of pectinolysis in Erwinia chrysanthemi. Annu Rev Microbiol 50, 213-257.

Jhoncon, J., Sakai, F. & Tsuyumu, S. (1994). Determination of nucleotide sequence of a region producing multiple polygalacturonases from Erwinia carotovora subsp. carotovora EC1. Ann. Phytopathol. Soc. Jpn. 60, 202-215.

Katzen, F., Becker, A., Zorreguieta, A., Puhler, A. & Ielpi, L. (1996). Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing biosynthesis of the xanthan polysaccharide. J Bacteriol 178, 4313-4318.

Kazemi-Pour, N., Condemine, G. & Hugouvieux-Cotte-Pattat, N. (2004). The secretome of the plant pathogenic bacterium Erwinia chrysanthemi. Proteomics 4, 3177-3186.

Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988). Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191-197.

Lang, C. & Dornenburg, H. (2000). Perspectives in the biological function and the technological application of polygalacturonases. Appl Microbiol Biotechnol 53, 366-375.

Lee, T. C., Lee, M. C., Hung, C. H., Weng, S. F. & Tseng, Y. H. (2001a). Sequence, transcriptional analysis and chromosomal location of the Xanthomonas campestris pv. campestris uvrB gene. J Mol Microbiol Biotechnol 3, 519-528.

Lee, T. C., Chen, S. T., Lee, M. C., Chang, C. M., Chen, C. H., Weng, S. F. & Tseng, Y. H. (2001b). The early stages of filamentous phage phiLf infection require the host transcription factor, Clp. J Mol Microbiol Biotechnol 3, 471-481.

Liao, C. H., Gaffney, T. D., Bradley, S. P. & Wong, L. C. (1996). Cloning of a pectate lyase gene from Xanthomonas campestris pv. malvacearum and comparison of its sequence relationship with pel genes of soft-rot Erwinia and Pseudomonas. Mol Plant Microbe Interact 9, 14-21.

Liu, Y., Chatterjee, A. & Chatterjee, A. K. (1994). Nucleotide sequence and expression of a novel pectate lyase gene (pel-3) and a closely linked endopolygalacturonase gene (peh-1) of Erwinia carotovora subsp. carotovora 71. Appl Environ Microbiol 60, 2545-2552.

Marin-Rodriguez, M. C., Orchard, J. & Seymour, G. B. (2002). Pectate lyases, cell wall degradation and fruit softening. J Exp Bot 53, 2115-2119.

Miller, J. H. (1972). Experiments in molecular genetics. Cold Spring Habor Laboratory, Cold Spring Harbor, N.Y.

Monteiro-Vitorello, C. B., Camargo, L. E., Van Sluys, M. A., Kitajima, J. P., Truffi, D., do Amaral, A. M., Harakava, R., de Oliveira, J. C., Wood, D., de Oliveira, M. C., Miyaki, C., Takita, M. A., da Silva, A. C., Furlan, L. R., Carraro, D. M., Camarotte, G., Almeida, N. F., Jr., Carrer, H., Coutinho, L. L., El-Dorry, H. A., Ferro, M. I., Gagliardi, P. R., Giglioti, E., Goldman, M. H., Goldman, G. H., Kimura, E. T., Ferro, E. S., Kuramae, E. E., Lemos, E. G., Lemos, M. V., Mauro, S. M., Machado, M. A., Marino, C. L., Menck, C. F., Nunes, L. R., Oliveira, R. C., Pereira, G. G., Siqueira, W., de Souza, A. A., Tsai, S. M., Zanca, A. S., Simpson, A. J., Brumbley, S. M. & Setubal, J. C. (2004). The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli. Mol Plant Microbe Interact 17, 827-836.

Nasser, W., Shevchik, V. E. & Hugouvieux-Cotte-Pattat, N. (1999). Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator. Mol Microbiol 34, 641-650.

Osbourn, A. E., Clarke, B. R., Stevens, B. J. & Daniels, M. J. (1990). Use of oligonucleotide probes to identify members of two-component regulatory system in Xanthomonas campestris pathovar campestris. Mol Gen Genet 222, 145-151.

Qian, W., Jia, Y., Ren, S. X., He, Y. Q., Feng, J. X., Lu, L. F., Sun, Q., Ying, G., Tang, D. J., Tang, H., Wu, W., Hao, P., Wang, L., Jiang, B. L., Zeng, S., Gu, W. Y., Lu, G., Rong, L., Tian, Y., Yao, Z., Fu, G., Chen, B., Fang, R., Qiang, B., Chen, Z., Zhao, G. P., Tang, J. L. & He, C. (2005). Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris. Genome Res 15, 757-767.

Ryan, R. P., Fouhy, Y., Lucey, J. F., Crossman, L. C., Spiro, S., He, Y. W., Zhang, L. H., Heeb, S., Camara, M., Williams, P. & Dow, J. M. (2006). Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl Acad Sci U S A 103, 6712-6717.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a laboratory manual, 2nd. Cold Spring Habor Press, Cold Spring Harbor. N. Y.

Sawada, K., Ogawa, A., Ozawa, T., Sumitomo, N., Hatada, Y., Kobayashi, T. & Ito, S. (2000). Nucleotide and amino-acid sequences of a new-type pectate lyase from an alkaliphilic strain of Bacillus. Eur J Biochem 267, 1510-1515.

Schweizer, H. D. (1993). Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques 15, 831-834.

Shevchik, V. E. & Hugouvieux-Cotte-Pattat, N. (1997). Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937. Mol Microbiol 24, 1285-1301.

Shevchik, V. E. & Hugouvieux-Cotte-Pattat, N. (2003). PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937. J Bacteriol 185, 3091-3100.

Spok, A., Stubenrauch, G., Schorgendorfer, K. & Schwab, H. (1991). Molecular cloning and sequencing of a pectinesterase gene from Pseudomonas solanacearum. J Gen Microbiol 137, 131-140.

Sutton, J. C. & Williams, P. H. (1970). Comparison of extracellular polysaccharide of Xanthomonas campestris from culture and from infected cabbage leaves. Can J Bot 48, 645-651.

Tang, J. L., Gough, C. L., Barber, C. E., Dow, J. M. & Daniels, M. J. (1987). Molecular cloning of protease gene(s) from Xanthomonas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity. Mol Gen Genet 1987, 443-448.

Tang, J. L., Liu, Y. N., Barber, C. E., Dow, J. M., Wootton, J. C. & Daniels, M. J. (1991). Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol Gen Genet 226, 409-417.

Thieme, F., Koebnik, R., Bekel, T., Berger, C., Boch, J., Buttner, D., Caldana, C., Gaigalat, L., Goesmann, A., Kay, S., Kirchner, O., Lanz, C., Linke, B., McHardy, A. C., Meyer, F., Mittenhuber, G., Nies, D. H., Niesbach-Klosgen, U., Patschkowski, T., Ruckert, C., Rupp, O., Schneiker, S., Schuster, S. C., Vorholter, F. J., Weber, E., Puhler, A., Bonas, U., Bartels, D. & Kaiser, O. (2005). Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence. J Bacteriol 187, 7254-7266.

Thorne, L., Tansey, L. & Pollock, T. J. (1987). Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris. J Bacteriol 169, 3593-3600.

Tseng, Y. H., Choy, K. T., Hung, C. H., Lin, N. T., Liu, J. Y., Lou, C. H., Yang, B. Y., Wen, F. S., Weng, S. F. & Wu, J. R. (1999). Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation. J Bacteriol 181, 117-125.

Vieira, J. & Messing, J. (1991). New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100, 189-194.

Wang, T. W. & Tseng, Y. H. (1992). Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage phi Lf. Lett Appl Microbiol 14, 65-68.

Wengelnik, K. & Bonas, U. (1996). HrpXv, an AraC-type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria. J Bacteriol 178, 3462-3469.

Willats, W. G., Orfila, C., Limberg, G., Buchholt, H. C., van Alebeek, G. J., Voragen, A. G., Marcus, S. E., Christensen, T. M., Mikkelsen, J. D., Murray, B. S. & Knox, J. P. (2001). Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion. J Biol Chem 276, 19404-19413.

Williams, P. H. (1980). Black rot: A continuing threat to world crucifers. Plant Dis 64, 736-742.

Wilson, T. J., Bertrand, N., Tang, J. L., Feng, J. X., Pan, M. Q., Barber, C. E., Dow, J. M. & Daniels, M. J. (1998). The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase. Mol Microbiol 28, 961-970.

Yang, B. Y. & Tseng, Y. H. (1988). Productin of exopolysaccharide and levels of protease and pectinase activity in pathogenic and non-pathogenic strains of Xanthomonas campestris pv. campestris. Bot Bull Acad Sin 29, 93-99.

Yoshida, A., Matsuo, Y., Kamio, Y. & Izaki, K. (1992). Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er. Biosci Biotechnol Biochem 56, 1596-1600.