臺灣博碩士論文加值系統

革蘭氏陰性的植物致病菌 Xanthomonas campestris pv. campestris（Xcc）是十字花科植物黑腐病的病原菌，此菌會產生的大量胞外多醣，稱為 xanthan gum，也會分泌多種胞外酵素，如蛋白&;#37238;、果膠&;#37238;以及纖維素&;#37238;等，胞外多醣與胞外酵素長期以來被視為重要的致病因子，這些重要的致病因子的生產會受到多元轉錄活化因子 Clp（cyclic AMP receptor protein-like protein）的調控，此蛋白為 Escherichia coli CRP 的同源蛋白。Clp 會與 engA、 pehA、 pelA1 及 xpsE 等基因的啟動子區域結合，對其轉錄活化進行正向調控；Clp 也會對 fliC、groESL、prt1 及 manA 等基因的轉錄進行活化，但不會與這些基因上游區域結合。
本研究的目標在於闡明 Clp 對於 Xcc 基因體的轉錄調控情形，為了達成這個目標，使用 PromScan 程式（http://www.promscan.uklinux.net） 於 Xcc strain ATCC 33913 基因體（GenBank accession no. AE008922）內搜尋可能的 Clp-binding sites（CBSs），程式所須的 frequency matrices 則分別使用彙整的典型與非典型 CBSs 製成。
首先，對 clp、 gumB 及 xpsE 等基因的上游區域進行探討。利用 electrophoretic mobility shift assay (EMSA) 和 transcriptional fusion assay，證明 Xcc clp 的轉錄受到 Clp 正向調控，Clp 會與位於其轉錄起始點（transcription initiation site）上游的典型 CBS 進行結合作用。根據 reporter assay 和 EMSA的結果，得知 Clp 會與 gum operon 上游區域中的 2 個非典型 CBSs 結合後，對 gum operon 的轉錄進行正向調控，這 2 個 CBSs 的右臂都具有較高的序列保守性，於結合作用發生時，可以彌補缺乏序列保守性的左臂所喪失的結合能力；另外，中間區域的 6 個鹼基對為高 GC 含量是有助於結合作用的，而且是迴文結構的 GC 鹼基對能增強結合能力。transcriptional fusion assays 的結果顯示：Clp 會活化 xpsEF operon 的轉錄，但對 xpsG 沒有直接調控的情形，雖然在 xpsE 上游區域中有 2 個 CBSs，不過只有鄰近 xpsE 啟動子的CBS 才是轉錄活化所必要的。
將 gumB CBS I、CBS II 與 xpsF CBS 三個 atypical CBSs 的序列比例轉換成 frequency matrix，利用 PromScan 程式找出 Xcc 基因體所有基因啟動子區域中與 atypical CBS 相似的序列，這些序列與 microarray 試驗的結果重複的基因有 21 個，但彼此推測的 CBS 並不相同，從其中挑選 5 個進行驗證，經 competitive EMSA 證實本研究的推測無誤。將此 8 個 atypical CBSs 的鹼基組合加以統計，歸納出 consensus atypical CBS，序列為 5’-AnAGGCGA-ACGCnG-TCACAnAA-3’。同時把 8 個已知的 typical CBSs 的鹼基組合加以統計，歸納出 consensus typical CBS，序列為 5’-TnGTGTGn-nnnAnn-TCnCATCG-3’，也把這 8 個 Xcc typical CBSs 序列組合比例轉換成另一個 frequency matrix，執行 PromScan 程式，也找到 30 個與 microarray 試驗的結果重複的基因，這些 CBSs 有待將來的研究加以證實。
由本研究的結果可知有 typical 與 atypical CBSs 這兩類的 CBSs 存在於 Xcc 基因上游區域中，而且 Clp 保有與這兩類 CBSs 進行結合作用的能力。本研究另有 2 個重要的發現：（一） xpsE 的轉錄受到 RpfF 的正調控；（二）透過 EMSA 證明 Clp 與 Xcc engA 上游區域片段於 in vitro 情況下的結合作用會受到 cyclic di-GMP 的抑制。

The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. It is capable of producing large amounts of an exopolysaccharide, xanthan gum, and secreting an array of extracellular enzymes, including proteases, pectinases, and endoglucanases, which have long been considered important virulence determinants. Production of these extracellular products is regulated by the global transcription factor Clp (cyclic AMP receptor protein-like protein, a homolog of the Escherichia coli CRP) and DSF whose biosynthesis involves RpfF protein. It is also known that Xcc encodes no adenylate cyclase required for the production of cyclic AMP, an effector that is involved in activation of CRP for binding to CRP-regulated promoters. Furthermore, it has previously been demonstrated in our laboratory that Clp 1) in vitro can bind to the regulated promoters in the absence of cyclic AMP, and 2) exerts transcriptional activation by binding to the promoter of some regulated genes (i.e., engA, pehA, and pelA1), but without direct binding to others (i.e., fliC, groESL, prt1, and manA). Despite these findings, prediction of the sequences for Clp binding (Clp-binding site, CBS) was merely based on similarity to the consensus E. coli CRP-binding site, which is 22-bp long (5’-AAATGTGATCTAGATCACATTT-3’) exhibiting perfect twofold sequence symmetry, because Clp has been shown to have the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. However, close examination of the above CBSs that have been characterized revealed that some of them possess sequences of arms and a central region deviated from the CRP consensus sequence. Therefore, this study was aimed to deduce consensus CBS sequence(s) on the basis of a larger number of characterized Xcc CBSs than what was already available. To achieve this end, PromScan program was used to search in Xcc genome. Several putative CBSs were identified, among which promoters of clp, gumB, and xpsE were then confirmed to be up-regulated by Clp, via deletion mapping in conjunction with electrophoretic mobility shift assay (EMSA) and transcriptional fusion assay. The results suggested that 1) clp is auto-regulated positively by Clp via binding to a typical CBS, 2) gum promoter possesses two atypical CBSs, in which a more conserved right arm compensates for the lack of conservation in the left arm, a high GC content in the central region (6 bp) is important for binding, and binding is enhanced by the palindromic GC-rich central region, and 3) two typical CBSs are present upstream of xpsE and both could bind to Clp in EMSA; however, only the one in proximity to the xpsE transcription initiation site is required for transcription activation. The three atypical CBSs (gumB CBS I, gumB CBS II, and xpsF CBS) were combined to create a frequency matrix which was then used in PromScan to perform genome-wide search. Twenty-one possible atypical CBSs were identified and five of them were confirmed by competitive EMSA. These CBS-containing genes have previously been shown to be up-regulated by Clp in microarray assay; however, most of the CBSs confirmed here are situated at different positions from those predicted by the microarray assay. Based on these eight atypical CBSs, a consensus atypical CBS, 5’-AnAGGCGAACGCnGTCACAnAA-3’, was compiled. In the meantime, by aligning 8 known typical CBSs, a more informative consensus typical CBS (5’-TnGTGTGnnnnAnnTCnCATCG-3’) was deduced. Using a frequency matrix based on this consensus sequence for search with PromScan, 30 Xcc genes, which have been previously identified by microarray assay to be regulated by Clp, were identified to possess typical CBS. Further EMSA is needed for confirmation of these typical CBSs. In conclusion, these results suggest that both typical and atypical CBSs exist in Xcc genome and that Clp has evolved to gain the capability to bind both types of CBSs. In addition to the above results, two important findings were also made here: 1) xpsE promoter is also up-regulated by RpfF, and 2) binding of Clp in vitro is inhibited by cyclic di-GMP, as demonstrated by using the Xcc engA promoter region as the probe in EMSA.

目次
縮寫字對照表 i
中文摘要 iv
英文摘要 vi
目次 ix
壹、前言 1
一、Xanthomonas campestris pv. campestris clp 的簡介 1
二、E. coli CRP 的轉錄調控與自我調控機 1
三、CRP 族群成員參與轉錄調控 2
四、Xcc Clp 參與轉錄調控的情形 3
五、Xcc xanthan 的合成與調控 4
六、第二型蛋白分泌系統（T2SS）的轉錄調控 5
七、論文研究目的 6
貳、材料 7
一、菌種、質體與引子 7
二、藥品 7
三、培養基 7
四、抗生素濃度 7
五、試劑與緩衝溶液 8
1.抽取染色體的試劑 8
2.DNA 電泳試劑 8
3.SDS-聚丙烯醯胺膠體電泳試劑 8
4.beta-galactosidase 活性測試所需的試劑 8
5.透析及保存 Clp 的緩衝溶液 8
6.純化 native form Clp 所需的緩衝溶液 8
7.西方轉漬法（Western blot）所需的緩衝溶液 9
8.Electrophoretic mobility shift assay（EMSA）所需的試劑 9
參、實驗方法 10
一、細菌的培養與保存 10
二、DNA 的製備 10
1.質體 DNA 的抽取 10
2.染色體 DNA 的抽取 10
三、電泳分析 11
1.洋菜凝膠電泳分析 11
2.SDS-聚丙烯醯胺膠體電泳分析 11
3.聚丙烯醯胺膠體電泳分析 11
四、聚合酶鏈鎖反應（PCR） 11
五、Rapid amplification of cDNA ends (RACE) 12
1.設計引子 12
2.第一股互補 DNA 合成與去除 RNA 12
3.純化第一股互補 DNA 12
4.互補 DNA 的 TdT（terminal deoxynucleotidyl transferase）tailing 反應 13
5.dC-tailed互補 DNA 的 PCR 與選殖 13
6.Nested PCR 擴增 13
六、質體的選殖 13
1.PCR 擴增片段的選殖 13
2.限制酶切割 13
3.DNA 片段的回收 13
4.DNA 的黏接 14
七、轉形作用 14
1.E. coli 的轉形 14
2.Xcc 的轉形 15
八、beta-galactosidase 活性測試 15
九、生長曲線測定 16
十、Total RNA 的純化 16
十一、Clp 的大量表現與純化 16
十二、Electrophoretic mobility shift assay (EMSA) 17
1.DNA 探針的製備 17
2.結合反應 17
3.電泳分析與轉漬 17
4.探針訊號偵測 17
十三、西方轉漬法 18
1.蛋白質轉漬 18
2.呈色 18
十四、利用 His‧BindTM column 取得 Clp binding DNA 片段 18
肆、結果與討論 20
一、clp上游區域的選殖與 Clp 自我調控的研究 20
(一)利用表現載體 pFY13-9 分析 clp 啟動子的活性 20
(二) DSF 對 clp 的調控 21
(三) Clp 與 clp-36+65 片段進行 EMSA 21
(四) Clp 自我調控 22
二、gumB 上游區域的選殖與受 Clp 轉錄調控的研究 22
(一) gumB 上游區域中 CBS 的分析 22
(二) 利用表現載體 pFY13-9 分析 gumB 啟動子的活性 23
(三) Clp 與 gumB 上游不同區域的片段進行 EMSA 24
(四) Clp 對 gumB 的轉錄調控 25
三、xpsE 上游區域的選殖與受 Clp 轉錄調控的研究 26
(一) xpsE TIS 的訂定 26
(二) xpsE 上游區域中 CBS 的分析 27
(三) 利用表現載體 pFY13-9 分析 xpsE 啟動子的活性 27
(四) Clp 與 xpsE-114+5 片段進行 EMSA 28
(五)Clp 對 xpsE 的轉錄調控 28
四、xpsF 上游區域的選殖與受 Clp 轉錄調控的研究 28
(一) xpsF TIS 的訂定 28
(二) xpsF 上游區域中 CBS 的分析 29
(三) Clp 與 xpsF-140-40 片段進行 EMSA 29
(四) 利用表現載體 pFY13-9分析 xpsF 啟動子的活性 29
(五) Clp 對 xpsE/F 的轉錄調控 30
五、xpsG上游區域受 Clp 轉錄調控的研究 30
(一) xpsG TIS 的訂定 31
(二) xpsG上游區域中 CBS 的分析 31
(三) Clp 與 xpsG-224+23 片段進行 EMSA 31
(四) Clp 對 xpsG 的轉錄調控 32
六、xyl 和 pglA 上游區域的選殖與受 Clp 轉錄調控的研究 32
(一) 使用 PromScan 搜尋 CBS 32
(二) xyl 上游區域中 CBS 的分析 32
(三) Clp 與 xyl-132+63 片段進行 EMSA 33
(四) 利用表現載體 pFY13-9 分析 xyl 啟動子的活性 33
(五) pglA 上游區域中 CBS 的分析 33
(六) Clp 與 pglA-519-259進行 EMSA 33
(七) Clp 對 xyl與 pglA 的轉錄調控 34
七、Xcc 基因體 CBS 分析 34
(一)使用 PromScan 程式搜尋 Xcc 基因體內的 atypical CBS 34
(二)以 competitive EMSA 驗證 PromScan 搜尋的結果 34
(三) Xcc 典型 CBS（typical CBS）的分析 35
(四) Clp 對 Xcc 基因的轉錄調控 36
伍、結論 37
陸、參考文獻 38
柒、表 48
捌、圖 66
玖、附錄 86
一、利用His•bind resin親和管柱找出Xcl7基因體中含有CBS的DNA片段86
二、Cyclic di-GMP 對Xcc Clp結合作用的影響 86

參考文獻
Aiba, H., 1983. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Cell. 32: 141-149.
Aiba, H., 1985. Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein. J. Biol. Chem. 260: 3063-3070.
Albus, A.M., Pesci, E.C., Runyen-Janecky, L.J., West, S.E., Iglewski, B.H., 1997. Vfr controls quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179: 3928-3935.
Barber, C.E., Tang, J.L., Feng, J.X., Pan, M.Q., Wilson, T.J., Slater, H., Dow, J.M., Williams, P., et al., 1997. A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Mol. Microbiol. 24: 555-566.
Barrios, H., Valderrama, B., Morett, E., 1999. Compilation and analysis of sigma(54)-dependent promoter sequences. Nucleic Acids Res. 27: 4305-4313.
Becker, A., Katzen, F., Puhler, A., Ielpi, L., 1998. Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl. Microbiol. Biotechnol. 50: 145-152.
Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J., et al., 1990. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res. 18: 7243-7250.
Belyaeva, T.A., Rhodius, V.A., Webster, C.L., Busby, S.J., 1998. Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits. J. Mol. Biol. 277: 789-804.
Berg, O.G., von Hippel, P.H., 1988. Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J. Mol. Biol. 200: 709-723.
Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H., Tommassen, J., 1998. Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol. Microbiol. 27: 209-219.
Blattner, F.R., Plunkett, G., 3rd, Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., et al., 1997. The complete genome sequence of Escherichia coli K-12. Science. 277: 1453-1462.
Busby, S., Ebright, R.H., 1999. Transcription activation by catabolite activator protein (CAP). J. Mol. Biol. 293: 199-213.
Capage, M.A., Doherty, D.H., Betlach, M., Vanderslice, R.W., 1987. Recombinant-DNA mediated production of xanthan gum. International patent WO87/05938.
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.
Chang, W.H., Lee, M.C., Yang, M.T., Tseng, Y.H., 2005. Expression of heat-shock genes groESL in Xanthomonas campestris is upregulated by CLP in an indirect manner. FEMS Microbiol. Lett. 243: 365-372.
Chapon-Herve, V., Akrim, M., Latifi, A., Williams, P., Lazdunski, A., Bally, M., 1997. Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa. Mol. Microbiol. 24: 1169-1178.
Chen, C.H. 1998. Cloning and analysis of the clp gene in Xanthomonas campestris pv. campestris. Master’s thesis, Institute of Molecular Biology, National Chung Hsing University, Taichung.
Chen, L.Y., Chen, D.Y., Miaw, J., Hu, N.T., 1996. XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J. Biol. Chem. 271: 2703-2708.
Chen, Y., Hu, N.T., Chan, N.L., 2004. Crystallization and preliminary X-ray crystallographic analysis of the N-terminal domain of XpsE protein from Xanthomonas campestris, an essential component of the type II protein-secretion machinery. Acta. Crystallogr. D. Biol. Crystallogr. 60: 129-131.
Chiang, S.H. 2004. The specificity study of the two rpoN genes in Xanthomonas campestris pv. campestris. Master’s thesis, Department of Biotechnology and Bioinformatics, Asia University, Taichung.
Chin, K.H., Lee, Y.C., Tu, Z.L., Chen, C.H., Tseng, Y.H., Yang, J.M., Ryan, R.P., McCarthy, Y., et al., 2010. The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris. J. Mol. Biol. 396: 646-662.
Chou, F.L., Chou, H.C., Lin, Y.S., Yang, B.Y., Lin, N.T., Weng, S.F., Tseng, Y.H., 1997. The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot. Biochem. Biophys. Res. Commun. 233: 265-269.
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. USA. 69: 2110-2114.
Collmer, A., Keen, N.T., 1986. The Role of Pectic Enzymes in Plant Pathogenesis. Annual Review of Phytopathology. 24: 383-409.
Cossart, P., Gicquel-Sanzey, B., 1985. Regulation of expression of the crp gene of Escherichia coli K-12: in vivo study. J. Bacteriol. 161: 454-457.
Cossart, P., Groisman, E.A., Serre, M.C., Casadaban, M.J., Gicquel-Sanzey, B., 1986. crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli. J. Bacteriol. 167: 639-646.
Costerton, J.W., Lappin-Scott, H.M., 1995. Introduction to microbial biofilms. Cambridge University Press. Cambridge.
Costerton, J.W., Stewart, P.S., Greenberg, E.P., 1999. Bacterial biofilms: a common cause of persistent infections. Science. 284: 1318-1322.
Crossman, L., Dow, J.M., 2004. Biofilm formation and dispersal in Xanthomonas campestris. Microbes. Infect. 6: 623-629.
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., et al., 2002. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature. 417: 459-463.
da Silva, F.R., Vettore, A.L., Kemper, E.L., Leite, A., Arruda, P., 2001. Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity. FEMS Microbiol. Lett. 203: 165-171.
Daniels, M.J., Collinge, D.B., Dow, J.M., Osbourn, Roberts, A.E., 1987. Molecular biology of the interaction of Xanthomonas campestris with plants. Plant Physiol. Biochem. 25: 353-359.
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., 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. USA. 100: 10995-11000.
Dow, J.M., Daniels, M.J., 1994. Pathogenicity determinants and global regulation of pathogenicity of Xanthomonas campestris pv. campestris. Curr. Top Microbiol. Immunol. 192: 29-41.
Dow, J.M., Daniels, M.J., 2000. Xylella genomics and bacterial pathogenicity to plants. Yeast. 17: 263-271.
Dums, F., Dow, J.M., Daniels, M.J., 1991. Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Mol. Gen. Genet. 229: 357-364.
Fath, M.J., Kolter, R., 1993. ABC transporters: bacterial exporters. Microbiol. Rev. 57: 995-1017.
Finlay, B.B., Falkow, S., 1997. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61: 136-169.
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.
Fuqua, C., Winans, S.C., Greenberg, E.P., 1996. Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol. 50: 727-751.
Ge, C., He, C., 2008. Regulation of the type II secretion structural gene xpsE in Xanthomonas campestris Pathovar campestris by the global transcription regulator Clp. Curr. Microbiol. 56: 122-127.
Ghosaini, L.R., Brown, A.M., Sturtevant, J.M., 1988. Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides. Biochemistry. 27: 5257-5261.
Griggs, D.W., Kafka, K., Nau, C.D., Konisky, J., 1990. Activation of expression of the Escherichia coli cir gene by an iron-independent regulatory mechanism involving cyclic AMP-cyclic AMP receptor protein complex. J. Bacteriol. 172: 3529-3533.
Hanahan, D., 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
Hanamura, A., Aiba, H., 1991. Molecular mechanism of negative autoregulation of Escherichia coli crp gene. Nucleic Acids Res. 19: 4413-4419.
Hanamura, A., Aiba, H., 1992. A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation. Mol. Microbiol. 6: 2489-2497.
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.
Harding, N.E., Raffo, S., Raimondi, A., Cleary, J.M., Ielpi, L., 1993. Identification, genetic and biochemical analysis of genes involved in synthesis of sugar nucleotide precursors of xanthan gum. J. Gen. Microbiol. 139: 447-457.
He, Y.W., Ng, A.Y., Xu, M., Lin, K., Wang, L.H., Dong, Y.H., Zhang, L.H., 2007. Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network. Mol. Microbiol. 64: 281-292.
Henderson, I.R., Navarro-Garcia, F., Desvaux, M., Fernandez, R.C., Ala''Aldeen, D., 2004. Type V protein secretion pathway: the autotransporter story. Microbiol. Mol. Biol. Rev. 68: 692-744.
Hsiao, Y.M., Fang, M.C., Sun, P.F., Tseng, Y.H., 2009. Clp and RpfF up-regulate transcription of pelA1 gene encoding the major pectate lyase in Xanthomonas campestris pv. campestris. J. Agric. Food Chem. 57: 6207-6215.
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.
Hsiao, Y.M., Liu, Y.F., Fang, M.C., Tseng, Y.H., 2010. Transcriptional regulation and molecular characterization of the manA gene encoding the biofilm dispersing enzyme mannan endo-1,4-beta-mannosidase in Xanthomonas campestris. J. Agric. Food Chem. 58: 1653-1663.
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., Zheng, M.H., Hu, R.M., Yang, T.C., Tseng, Y.H., 2008. Regulation of the pehA gene encoding the major polygalacturonase of Xanthomonas campestris by Clp and RpfF. Microbiology. 154: 705-713.
Hu, N.T., Hung, M.N., Liao, C.T., Lin, M.H., 1995. Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris. Microbiology. 141: 1395-1406.
Hueck, C.J., 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62: 379-433.
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.
Ielpi, L., Couso, R.O., Dankert, M.A., 1993. Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris. J. Bacteriol. 175: 2490-2500.
Jansson, P.E., Kenne, L., Lindberg, B., 1975. Structure of extracellular polysaccharide from Xanthomonas campestris. Carbohydr. Res. 45: 275-282.
Kanack, K.J., Runyen-Janecky, L.J., Ferrell, E.P., Suh, S.J., West, S.E., 2006. Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein. Microbiology. 152: 3485-3496.
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.
Katzen, F., Ferreiro, D.U., Oddo, C.G., Ielmini, M.V., Becker, A., Puhler, A., Ielpi, L., 1998. Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. J. Bacteriol. 180: 1607-1617.
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.
Koplin, R., Arnold, W., Hotte, B., Simon, R., Wang, G., Puhler, A., 1992. Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J. Bacteriol. 174: 191-199.
Langdon, R.C., Hochschild, A., 1999. A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators. Proc. Natl. Acad. Sci. USA. 96: 12673-12678.
Leduc, J.L., Roberts, G.P., 2009. Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri. J. Bacteriol. 191: 7121-7122.
Lee, M.C., Weng, S.F., Tseng, Y.H., 2003. Flagellin gene fliC of Xanthomonas campestris is upregulated by transcription factor Clp. Biochem. Biophys. Res. Commun. 307: 647-652.
Lee, T.C., Chen, S.T., Lee, M.C., Chang, C.M., Chen, C.H., Weng, S.F., Tseng, Y.H., 2001. The early stages of filamentous phage phiLf infection require the host transcription factor, Clp. J. Mol. Microbiol. Biotechnol. 3: 471-481.
Lee, T.C., Lee, M.C., Hung, C.H., Weng, S.F., Tseng, Y.H., 2001. Sequence, transcriptional analysis and chromosomal location of the Xanthomonas campestris pv. campestris uvrB gene. J. Mol. Microbiol. Biotechnol. 3: 519-528.
Marzocca, M.P., Harding, N.E., Petroni, E.A., Cleary, J.M., Ielpi, L., 1991. Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris. J. Bacteriol. 173: 7519-7524.
Miller, J.H., 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press. New York.
Murakami, K., Owens, J.T., Belyaeva, T.A., Meares, C.F., Busby, S.J., Ishihama, A., 1997. Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors. Proc. Natl. Acad. Sci. USA. 94: 11274-11278.
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.
Newman, M.A., Conrads-Strauch, J., Scofield, G., Daniels, M.J., Dow, J.M., 1994. Defense-related gene induction in Brassica campestris in response to defined mutants of Xanthomonas campestris with altered pathogenicity. Mol. Plant Microbe Interact. 7: 553-563.
Niu, W., Kim, Y., Tau, G., Heyduk, T., Ebright, R.H., 1996. Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase. Cell. 87: 1123-1134.
Niu, W., Zhou, Y., Dong, Q., Ebright, Y.W., Ebright, R.H., 1994. Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis. J. Mol. Biol. 243: 595-602.
Nouwen, N., Stahlberg, H., Pugsley, A.P., Engel, A., 2000. Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. Embo J. 19: 2229-2236.
Okamoto, K., Freundlich, M., 1986. Mechanism for the autogenous control of the crp operon: transcriptional inhibition by a divergent RNA transcript. Proc. Natl. Acad. Sci. USA. 83: 5000-5004.
Okamoto, K., Hara, S., Bhasin, R., Freundlich, M., 1988. Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA. J. Bacteriol. 170: 5076-5079.
Paulsen, I.T., Beness, A.M., Saier, M.H., Jr., 1997. Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria. Microbiology. 143: 2685-2699.
Pirhonen, M., Flego, D., Heikinheimo, R., Palva, E.T., 1993. A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. Embo J. 12: 2467-2476.
Planet, P.J., Kachlany, S.C., DeSalle, R., Figurski, D.H., 2001. Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc. Natl. Acad. Sci. USA. 98: 2503-2508.
Polayes, D.A., Rice, P.W., Garner, M.M., Dahlberg, J.E., 1988. Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli. J. Bacteriol. 170: 3110-3114.
Pollock, T.J., Thorne, L., Yamazaki, M., Mikolajczak, M.J., Armentrout, R.W., 1994. Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides. J. Bacteriol. 176: 6229-6237.
Possot, O., Pugsley, A.P., 1994. Molecular characterization of PulE, a protein required for pullulanase secretion. Mol. Microbiol. 12: 287-299.
Pugsley, A.P., 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57: 50-108.
Pugsley, A.P., Francetic, O., Possot, O.M., Sauvonnet, N., Hardie, K.R., 1997. Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria--a review. Gene. 192: 13-19.
Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W.C., Heidelberg, J.F., Mekalanos, J.J., 2006. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. USA. 103: 1528-1533.
Puls, J., Schuscil, J., 1993. Chemistry of Hemicelluloses: Relationship Between Hemicelluloses Structure and Enzymes Required for Hydrolysis. Portland Press. London.
Py, B., Loiseau, L., Barras, F., 1999. Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL. J. Mol. Biol. 289: 659-670.
Ray, S.K., Rajeshwari, R., Sonti, R.V., 2000. Mutants of Xanthomonas oryzae pv. oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase. Mol. Plant Microbe Interact. 13: 394-401.
Rhodius, V.A., Busby, S.J., 2000. Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3. J. Mol. Biol. 299: 295-310.
Rouanet, C., Nomura, K., Tsuyumu, S., Nasser, W., 1999. Regulation of pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: involvement of the main transcriptional factors. J. Bacteriol. 181: 5948-5957.
Russel, M., 1998. Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. J. Mol. Biol. 279: 485-499.
Runyen-Janecky, L.J., Sample, A.K., Maleniak, T.C., West, S.E., 1997. A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp. J. Bacteriol. 179: 2802-2809.
Salmond, G.P., Reeves, P.J., 1993. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem. Sci. 18: 7-12.
Sandkvist, M., 2001. Biology of type II secretion. Mol. Microbiol. 40: 271-283.
Sandkvist, M., Bagdasarian, M., Howard, S.P., DiRita, V.J., 1995. Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. Embo J. 14: 1664-1673.
Schweizer, H.P., 1993. Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes. Gene. 134: 89-91.
Shiue, S.J., Kao, K.M., Leu, W.M., Chen, L.Y., Chan, N.L., Hu, N.T., 2006. XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. Embo J. 25: 1426-1435.
Slater, H., Alvarez-Morales, A., Barber, C.E., Daniels, M.J., Dow, J.M., 2000. A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol. Microbiol. 38: 986-1003.
Studholme, D.J., Dixon, R., 2003. Domain architectures of sigma54-dependent transcriptional activators. J. Bacteriol. 185: 1757-1767.
Suh, S.J., Runyen-Janecky, L.J., Maleniak, T.C., Hager, P., MacGregor, C.H., Zielinski-Mozny, N.A., Phibbs, P.V., Jr., West, S.E., 2002. Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa. Microbiology. 148: 1561-1569.
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.
Tseng, Y.H., Choy, K.T., Hung, C.H., Lin, N.T., Liu, J.Y., Lou, C.H., Yang, B.Y., Wen, F.S., et al., 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.
Tseng, Y.H., Lo, M.C., Lin, K.C., Pan, C.C., Chang, R.Y., 1990. Characterization of filamentous bacteriophage phi Lf from Xanthomonas campestris pv. campestris. J Gen Virol. 71: 1881-1884.
Tseng, Y.H., Ting, W.Y., Chou, H.C., Yang, B.Y., Chen, C.C., 1992. Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris. Lett. Appl. Microbiol. 14: 43-46.
Turner, L.R., Lara, J.C., Nunn, D.N., Lory, S., 1993. Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J. Bacteriol. 175: 4962-4969.
Ushida, C., Aiba, H., 1990. Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity. Nucleic Acids Res. 18: 6325-6330.
Van Gijsegem, F., Genin, S., Boucher, C., 1993. Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria. Trends Microbiol. 1: 175-180.
Vanderslice, R.W., Doherty, D.H., Capage, M.A., Betlach, M.R., Hassler, R.A., Henderson, N.M., Ryan-Graniero, J., Tecklenburg, M. 1990. Genetic engineering of polysaccharide structure in Xanthomonas campestris. In Biomedical and Biotechnological Advances in Industrial Polysaccharides. p. 145-156. Edited by Crescenzi, V., Dea, I.C.M., Paoletti, S., Stivala, S.S., Sutherland, I.W. New York: Gordon & Breach.
Vieira, J., Messing, J., 1991. New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene. 100: 189-194.
Vojnov, A.A., Slater, H., Daniels, M.J., Dow, J.M., 2001. Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta. Mol. Plant Microbe Interact. 14: 768-774.
Vojnov, A.A., Zorreguieta, A., Dow, J.M., Daniels, M.J., Dankert, M.A., 1998. Evidence for a role for the gumB and gumC gene products in the formation of xanthan from its pentasaccharide repeating unit by Xanthomonas campestris. Microbiology. 144: 1487-1493.
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.
Wei, C.L., Lin, N.T., Weng, S.F., Tseng, Y.H., 1996. The gene encoding UDP-glucose pyrophosphorylase is required for the synthesis of xanthan gum in Xanthomonas campestris. Biochem. Biophys. Res. Commun. 226: 607-612.
West, S.E., Sample, A.K., Runyen-Janecky, L.J., 1994. The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J. Bacteriol. 176: 7532-7542.
Williams, R.M., Rhodius, V.A., Bell, A.I., Kolb, A., Busby, S.J., 1996. Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters. Nucleic Acids Res. 24: 1112-1118.
Wolfgang, M.C., Lee, V.T., Gilmore, M.E., Lory, S., 2003. Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. Dev. Cell. 4: 253-263.
Yang, B.Y., Tseng, Y.H., 1988. Production 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.