細菌當面對環境的轉變,會藉著RNA聚合酶和所含的不同σ因子及相關的調控蛋白來活化或關閉一群受其調控的基因。σ54所辨認基因啟動子的區域是在轉錄起始位-24/-12兩個區域的保守序列(5''-TGGC-N8- TGCA/T-3''),其辨認基因啟動子區域及轉錄的機制與主要σ因子─σ70不同。 十字花科黑腐病菌為一革蘭氏陰性菌,會感染十字花科植物造成黑腐病。此菌在農業上會造成嚴重的災害,但是所產生的胞外黏多醣在工業上卻是有多重用途,可在食品加工、化妝品、製造業上作為膠化劑、乳化劑、塑化劑、安定劑之用。十字花科黑腐病菌基因體中含有兩套rpoN基因― rpoN1基因和rpoN2基因,這兩個RpoN蛋白彼此之間的相同度低(41%),和其他菌不同的是彼此可能存在個別的專一性。σ54調節子在其他的菌種曾經被研究過,但是在十字花科黑腐病菌未曾被研究過。生理學的初步研究顯示兩個 rpoN 基因的功能是獨立和不可互換的。在這次研究中,首先利用前不久發表的文獻提供完整的十字花科黑腐病菌的基因體序列,來預測基因體中所有可能受RpoN調控的基因啟動子。再以分子生物技術釐清上述搜尋到的基因啟動子是否受XCC中RpoN1或 RpoN2調控。本研究的結果發現受RpoN1調控的啟動子有glnA (麩醯胺合成酶基因)、nasA(硝酸塩轉運蛋白基因)、pilA1(纖毛蛋白基因)及prpB(丙酮烯醇磷酸轉位酶基因)四個基因的啟動子,受RpoN2調控的啟動子有flhF(鞭毛生合成基因)、flgG(鞭毛基底的桿狀物遠端結構蛋白基因)、flgB(鞭毛基底的桿狀物近端結構蛋白基因)、fliE(鞭毛MS環桿狀交界蛋白基因)及fliQ(鞭毛運輸蛋白基因)五個基因的啟動子。根據以上實驗的結果,進一步比較RpoN1或RpoN2所辨認基因啟動子保守序列的異同,發現RpoN1所辨認基因啟動子保守序列為5''-TTGGC-AN7-TGCG/T-3'',而RpoN2所辨認基因啟動子保守序列為 5''-TTTGGC-N8-TGCA -3''(粗體加底線的文字代表兩者的差異)。由此推論RpoN1或RpoN2具有專一性的決定因子。
Bacteria are able to activate or switch off specific sets of genes as they face changing environmental conditions. This can be achieved through the activities of RNA polymerases (RNAP) containing alternate sigma factors and their cognate regulatory proteins. The sigma factor σ54 recognizes a conserved sequence motif centered at -24 and -12 nucleotides from the transcriptional start site (5''-TGGC-N8- TGCA/T-3'') and confers RNAP properties different from those of the major house-keeping form of RNAP such asσ70. Xanthomonas campestris pv. campestris (XCC) is the Gram-negative bacterium causing black rot in crucifers, resulting in tremendous loss in agriculture. It produces great amounts of an exopolysaccharide, xanthan gum, which is used as viscosifying, emulsifying, plasticizer and stabilizing agent in food, cosmetics, agriculture and industry. The fully sequenced XCC genome has two homologues of the rpoN genes, rpoN1 and rpoN2. The two deduced RpoN sequences share only 41% of identity and both show different levels of homology to the RpoN proteins from other bacteria. Theσ54-dependent regulon has previously been studied in several groups of bacteria, but not in XCC. Preliminary physiological studies have demonstrated that the two rpoN genes are functionally independent and not interchangeable. In this study, I carried out searches on the recently published complete genome sequence of XCC in order to predict the possibleσ54-dependent genes. Molecular biological methods were then used to confirm whether the predicted σ54-dependent genes are indeed controlled by RpoN1 or RpoN2. The results revealed that glnA (encoding glutamine synthetase), nasA (encoding nitrate transporter), pilA1 (encoding pilin), and prpB (encoding carboxyphosphonoenolpyruvate phosphonomutase) are RpoN1-dependent, whereas flhF (encoding flagellar biosynthetic protein), flgG (encoding the distal rod protein of the flagellum basal body), flgB (encoding the proximal rod protein of the basal body), fliE (encoding MS-ring rod junction protein), and fliQ (encoding export component of flagellar protein) are RpoN2-dependent. Based on the RpoN-binding sequence in the upstream regions of the above genes and their homologs, the consensus RpoN1- and RpoN2-binding sites complied are 5''-TTGGC-AN7-TGCG/T- 3'' and 5''-TTTGGC-N8-TGCA-3'', respectively. In other words, the two Xcc RpoN proteins each has its specific recognition sequences.