標題: 克雷白氏肺炎桿菌CG43中雙分子調控系統KvgAS、KvhR以及KvhAS的功能分析
Functional analysis of the two-component systems KvgAS, KvhR and KvhAS in Klebsiella pneumoniae CG43
作者: 林靖婷
Ching-Ting Lin
彭慧玲
Hwei-Ling Peng
生物科技學系
關鍵字: 克雷白氏肺炎桿菌;雙分子調控系統;KvgAS;KvhR;KvhAS;莢膜多醣類;RpoS;fosfomycin;啟動子誘捕系統;cDNA表現異型分析;蛋白質體分析;Klebsiella pneumoniae;Two-component system;KvgAS;KvhR;KvhAS;capsule polysaccharide;RpoS;fosfomycin;promoter trapping system;cDNA substractive hybridization analysis;proteomic approach
公開日期: 2006
摘要: 克雷白氏肺炎桿菌是一伺機性的病原菌,我們在一株具有高毒性的菌株CG43中發現兩組相似度高的雙分子調控系統基因組,分別命名為kvgAS-kvhR和kvhAS。根據序列的分析,KvgS和KvhS是組氨酸激酉每可以讓細菌感應外在的環境變化,而KvgA、KvhR以及KvhA則是感應蛋白可以反應由組氨酸激酉每所傳達的訊息,進而調節下游基因的表現。為了了解這兩套雙分子調控系統在細菌體內所扮演的角色,本研究在克雷白氏肺炎桿菌中建立了以lacZ為報導基因的系統,並構築了一系列突變株: kvgA- (AZ18),kvhA- (AhZ01), kvhR- (RZ01), kvhA-kvgA- (AAh01), kvhA-kvhR- (AhR01), kvgA-kvhR- (AR01), 以及kvgA-kvhA-kvhR- (AAhR01)。根據這一系列突變株的表現型,把它們分成兩群: 第一群帶有kvgA或者kvhR的基因突變,和野生株比較起來生長較快、菌體的黏性降低、腹腔注射小鼠的半致死率升高;相反的,第二群kvhA-以及kvhA-kvhR-突變株在上述的表現型分析則和野生株呈現一樣的結果。我們進一步發現第一群菌體的黏性降低是因為莢膜多醣類合成量的減低,經由測試莢膜合成基因組中的三個啟動子活性發現: kvgA基因的突變會造成orf16~17的啟動子活性下降,而kvhR基因的突變則造三個啟動子的活性都下降。而在營養缺乏的環境下,kvgS基因的突變不僅會降低kvgAS本身啟動子的活性,同時也會使kvhAS啟動子的活性降低。經由截短啟動子做活性測試和電泳膠遲滯實驗,本研究除了證明這兩套訊息傳遞系統可以有交互調控外,還分別在kvgAS和kvhAS的啟動子區域找到KvgA可能的結合片段。進一步也利用5’-RACE的實驗確認KvgAS和KvhAS的轉錄起始點,而分別在這兩個啟動子序列的-35上游都發現有RpoS可能的結合位置。進一步在rpoS突變株中,發現kvgAS啟動子的活性會明顯降低,而 kvhAS啟動子的活性反而升高,而截除了RpoS可能結合的序列後,rpoS突變對於kvgAS或kvhAS啟動子活性的影響也消失了,顯示這各序列經由RpoS結合後進而調控這兩套基因組的表現。同時,本研究也發現kvgA的基因缺損會降低sodC和katG的啟動子表現,這暗示著KvgAS是RpoS調控網路的一員。而大量表現KvhA,則會造成菌體對fosfomycin的敏感度升高,並使菌體內UDP-N-acetylglucosamine enolpyruvyl transferase活性上升,這暗示著KvhA在細菌對抗生素抗性上扮演調控的角色。最後,本研究分別利用啟動子誘捕系統、cDNA表現異型分析以及蛋白質體分析,找出可能受這兩套訊息基因調控組的目標基因,希望未來進一步的實驗確認後,可以畫出這兩套訊息傳遞系統的調控途徑。
Klebsiella pneumoniae is a common opportunistic pathogen. Two-component system (2CS) gene clusters including kvgAS, kvhR and kvhAS, have previously been isolated from a highly virulent strain K. pneumoniae CG43. According to sequence analysis, KvgS and KvhS are sensory histidine kinases which allow bacteria to sense the signal changes in their environment. KvgA, KvhA and KvhR are the response regulators responding to the signal relaid by the cognate sensor thereby regulating the downstream genes expression. To identify the functional roles of the 2CSs, a LacZ reporter system CG43S3-Z01 and a series of the derived mutants including kvgA- (AZ18), kvhA- (AhZ01), kvhR- (RZ01), kvhA-kvgA- (AAh01), kvhA-kvhR- (AhR01), kvgA-kvhR- (AR01), and kvgA-kvhA-kvhR- (AAhR01) were constructed. Comparative analysis of their growth and phenotype allowed the classification of the mutants into two groups: group I bacteria carrying either kvgA or kvhR mutation displayed less mucoidy, a faster growth rate and an increase of LD50 by comparing to the parental strain Z01. In contrast, the group II bacteria including kvhA- and kvhA-kvhR- mutants exert a similar phenotype with that of the parental strain. Decreasing amount of glucuronic acid, the core component of Klebsiella capsule polysaccharide (CPS), was found for the group I mutants. Comparing the promoter activity of the three K2-cps transcription units of wild type, AZ18, and RZ01 revealed that deletion of kvgA decreased the promoter activity of orf16-17, while deletion of kvhR reduced the promoter activities of all three cps transcripts. Deletion of kvgS appeared to reduce not only the expression of kvgAS, but also the expression of kvhAS in minimal medium. Both promoter activity measurement and EMSA analysis allowed localization of the binding elements of KvgA on both putative promoters PkvgAS and PkvhAS, which indicating an interacting regulation between the two 2CSs. Using 5’-RACE analysis, both identified promoters of kvgAS and kvhAS appeared to possess a relatively conserved –10 and –35 sequences for Sigma-70 regulation. In addistion, a close-to-consensus RpoS binding element as identified upstream of both promoters, suggested an RpoS-dependent regulation. Deletion of rpoS affected negatively the expression of kvgAS but positively the kvhAS expression, supporting an RpoS-dependent regulation of the two 2CSs. Promoter activity measurement further help to confirm that the RpoS binding sites are contained respectively in the consensus sequences of the two promoters. Moreover, deletion of kvgA was shown to affect the expression of the antioxidant defense genes katG and sodC, which are components of the RpoS regulon, at transcriptional level. Overexpression of KvhA altered the susceptibility of the bacteria to fosfomycin and resulted in an increased activity of UDP-N-acetylglucosamine enolpyruvyl transferase, the target protein of fosfomycin. Taken together, these indicated that the two 2CSs probably belong to different regulatory circuits of the RpoS regulon. Finally, we also employed a promoter trapping system, cDNA substractive hybridization analysis, and proteomic approach to identify the target sequences under the control of the 2CSs. However, more experiments will need to be carried out to demonstrate the regulation of the 2CSs on the expression of the target genes. Till then, a regulatory network of what could be determined completely.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009028801
http://hdl.handle.net/11536/38324
Appears in Collections:Thesis


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