Erwinia herbicola Eho10類葫蘿蔔素生合成基因結構的分析

Abstract

Erwinia herbicola Eho10是從植物葉面分離出的革蘭氏陰性菌，屬於腸內菌科。此菌能表現黃色素。前人實驗證明此黃色素?類葫蘿蔔素（carotenoids）的混合物。其色素生合成基因的最小選殖體pPL376能在E. coli中表現。本論文是針對此色素生合成基因的結構及其調控機制進行分析。以轉位子Tn1000(γδ)插入實驗，可使色素生合成基因產生突變，得到不同顏色的E． coli菌落，進而決定影響色素生合成基因表現的重要區域。其中四個區域對色素的生合成有重要的影響。Region I及Region IV經轉位子插入後，形成白色的菌落，由此可知此二區域扮演重要的角色，可能此區域的基因產物催化早期的色素中間產物，或扮演基因調控的角色，一旦此區域被破壞，就會影響色素的生合成。而Tn1000插入Region II，則使得E. coli表現淡黃色；插入Region III時，則使得E. coli由黃色變?粉紅色。這可能是色素生合成步驟中的某個酵素被破壞，導致色素中間產物累積的結果。除此之外，也針對色素生合成的三個基因（crtH, crtG和ORF2）進行核酸序列分析。並發現此crtH及crtG合成之蛋白，和E. uredovora所合成之蛋白具有相似性。利用promoter-probe vector pKM005偵測在色素生合成基因上，具有起動子活性的DNA片段，結果顯示0.9-kb的BamHI以及2.3-kb的SalI-BglII的DNA片段能帶動lacZ的表現，而使在MacConkey agar上的菌落呈現紅色。並以primer extension決定轉錄起始點，且發現其中具有cAMP-CRP作用之位置。

Carotenoids comprise one of the most wide-spread classes of pigments found in nature. All photosynthetic prokaryotes and eukaryotes, as well as certain fungi, yeasts, and nonphotosynthetic bacteria, synthesize carotenoids. Erwania herbicola Eho 10 is a carotenoid-producing bacterium. The clone carrying the pigment biosynthesis genes (pPL376) were identified and is able to form yellow colonies in E. coli. The main theme of this thesis concerns the functional organization of a set of genes specifying the synthesis of the yellow pigment of Erwinia herbicola Eho 10 and the regulation of these genes. Insertion mutagenesis with transposon Tn1000(γδ) resulted in pink, light-yellow and nonpigmented colonies. Four regions (Region I, II, III, IV) for the expression of the pigment -biosynthesis genes were defined. The DNA fragments containing pigment biosynthesis genes crtH, crtG, ORF2 were subcloned into vector pGEM3Zf(+) and pGEM3. These clones were then sequenced using the T7 and Sp6 primers, and gaps in the sequence were sequenced by primer walking. DNA sequencing study has determined the sequences of crtH, crtG, and an open reading frame (1044 bp) of unknown function. The similarity of the proteins encoded by crtH and crtG was 61% and 47% with the crtZ and crtX of Erwinia uredovora, respectively. DNA fragment containing promoter activiity in the gene cluster was identified by transcriptional fusion with a promoter-probe vector pKM005. The promoter clones were selected on MacConkey agar. Results revealed that the activity was localized in a 0.9-kb BamHI fragment and on a 2.3-kb SalI-BglII fragment which were located in front of crtH and crtE, respectively. The transcription-start site were determined by primer extension. The putative cAMP-CRP binding sites in these two promoters were also identified.