克雷白氏肺炎桿菌kvgAII基因之選殖與表現分析

Abstract

克雷白氏肺炎桿菌（Klebsiella pneumonia）是一種伺機性的病原菌，常在免疫機能不全的病人引起菌血症和尿道感染。 本實驗室先前利用基因體刪除雜交法，在一高毒性的臨床菌株CG43得到一段被命名為kvgASQR的二階段的訊號傳遞系統基因組。 利用此基因序列與美國華盛頓大學的克雷白氏肺炎桿菌 MGH78578的基因庫進行比對，找到了一段與kvgAS 有高相似性的基因序列。 我們依據其基因序列設計引子，以克雷白氏肺炎桿菌CG43 染色體為模板進行聚合酉每連鎖反應，選殖出這段基因並進一步分析核酸序列，雖然此基因序列與kvgAS相似，但是卻不相同，我們將這段基因組命名為kvgASⅡ。 利用墨點雜交法調查本實驗室收集的克雷白氏肺炎桿菌臨床菌株發現，相對於在臨床菌株中只佔了13 ﹪的kvgASQR基因，我們發現kvgASⅡ存在於所有菌株，顯示kvgASⅡ對克雷白氏肺炎桿菌的生長可能扮演必需的角色。 我們接著利用primer extension找出kvgAⅡ基因轉錄起始點，分別位在開讀骨架(open reading frame)第一個ATG前4及58 bp，但分別在這兩個可能的啟動子區域沒有典型的-10及-35序列。 我們進一步在大腸桿菌中大量表現KvgAⅡ蛋白，並純化此蛋白質，由電泳膠遲滯實驗發現KvgAⅡ蛋白會與kvgAⅡ基因可能的啟動子序列結合，顯示可能具有自我調控的能力。

Klebsiella pneumoniae is a common cause of septicemia and urinary tract infection in immunocompromised patients. Our laboratory had previously identified a gene cluster encoding a two-component system in the bacterium that we termed kvgASQR. We have used the sequence to search K. pneumoniae strain MGH78578 database (Genome Sequencing Center of Washington University) and found a sequence exhibits highly homology with the kvgAS. Then we cloned the genes by PCR with K. pneumoniae CG43 DNA as the template and the cloned genes were sequence-determined. The nucleotide sequences of the genes are different from those of the kvgAS, and therefore named as kvgASII. In contrast to the kvgASQR genes which were shown to present in approximately 13% of the bacteriumic isolates, the kvgASII gene was found to present in all strains analyzed using dot-blotting hybridization. The result suggests an essential role of the kvgASII genes for K. pneumoniae. Two primer extension products were found which are 4 and 58 base pair, respectively, upstream to the ATG codon of the major open reading frame. Moreover, we have cloned the kvgAII gene into a pET30c expression vector, and purified the recombinant KvgAII protein with His-Tag purification kit. The purified KvgAII protein was found to be able to bind its putative promoter, suggesting that the expression of KvgAII protein is autoregulated.