- 學位論文
靈菌表面移行行為之調控:-二元系統RssA-RssB調控flhDC進而影響表面移行-RssC, 醯基轉移脢調控靈菌表面移行
Regulation of swarming behaviour in Serratia marcescens: -The two component system RssA-RssB regulates swarming through modulating flhDC promoter activity-A potential acyltransferase, RssC, regulates S. marcescens swarming
摘要
靈菌 (Serratia marcescens) 能夠在30 oC時在固體表面上能以swarming的方式移動，但在37 oC時這種現象被抑制，已知細菌swarming需利用鞭毛並分泌biosurfactant，但調控swarming的機制並不清楚。我們利用Tn5跳躍子選擇出在37 oC也能進行Swarming的突變株，其中兩株突變株是分別被跳躍子破壞rssA基因及rssC基因。RssA和RssB為一套two-component system，而RssA為sensor kinase，RssC則預測為醯基轉移脢(acyl-transferase)。而我進一步利用Electrophoretic mobility shift assays及Modified chromatin immunoprecipitation (ChIP) assay證明不論是in vivo或in vitro，磷酸化的RssB可結合(bind)到flhDCSm的promoter， 並以DNase I footprinting assay確定RssB在flhDCSm promoter的位置。此外在Primer extension的實驗也確定flhDCSm的兩個transcriptional start sites， P1和P2，經由promoter activity assay發現RssA-RssB主要調控遠端的promoter，P2。在我第二部份的研究顯示rssC突變株表現出泳動能力增強，鞭毛增加，capsule多醣體增加，細胞的附著能力降低，以及Lipopolysaccharide pattern (脂多醣體)的改變。在電子顯微鏡下，突變株呈現不規則的表面，顯示rssC可能影響outermembrane合成。此外，分析rssC promoter region發現一段類似FlhDC 結合的區域(binding site)，將rssC promoter接luxCDABE分析promoter activity在野生株及flhDCSm突變株是否不同，發現rssC promoter活性在flhDCSm突變株比在野生株較低。綜合實驗結果，flhDCSm可直接受到RssA-RssB的負向調控，由於flhDCSm是啟動鞭毛系統的第一層基因，因此證明RssB-RssA是透過抑制flhDCSm而抑制鞭毛，另一方面也能如鞭毛系統的弟二層基因一樣受到FlhDC的活化。
並列摘要
Swarming in Serratia marcescens is a specialized form of bacterial surface translocation. S. marcescens cells swarm at 30℃ but not at 37℃. The mechanism of swarming, however, is not clear. Our previous studies showed both mutated rssA, encoded a sensor kinase, and mutated rssC, which was predicted to encode a 364 amino-acid polypeptide showing high identity to members of acyl-transferase protein family, revealed precocious swarming phenotypes at 37℃. I showed that direct interaction of phosphorylated RssB (RssB~P) with the flhDCSm promoter region in vivo by electrophoretic mobility shift assays and in vitro by modified chromatin immunoprecipitation (ChIP) assay. The DNase I footprinting assay determine the RssB~P binding site on the flhDCSm promoter region. Primer extension analysis revealed two transcriptional start sites, P1 and P2, of flhDCSm. Analysis of promoter activity was shown that RssA-RssB mainly regulated P2 promoter than P1. In the second part, rssC mutant showed pleiotropic phenotypes including increased swimming motility, flagellum production and capsule polysaccharide production and decreased cell attachment ability. Lipopolysaccharide (LPS) pattern analysis indicated rssC mutant shows a significantly more intense O antigen sugar unit band ladder. Transmission electron microscopy and atomic force microscopy observation showed altered surface morphology in rssC mutant. Further DNA sequence analysis showed a potential FlhDC binding region identified in the rssC promoter region, and the rssC transcription activity was confirmed to be up-regulated by FlhDC. In conclusion, my data showed that activated RssA-RssB signaling directly interacts with the flhDCSm promoter, and inhibits its transcriptional activity, leading to reduced flagellum expression and swarming in S. marcescens. Furthermore FlhDC also activates transcriptional level of rssC. Deficiency of RssC leads to precocious swarming in S. marcescens.