克雷白氏肺炎桿菌CG43中RstA/RstB雙分子訊息傳遞系統的特性分析

Abstract

因為克雷白氏肺炎桿菌( Klebsiella pneumoniae )的RstA/RstB之基因組成和大腸桿菌、沙門氏菌不盡相同，為了了解克雷白氏肺炎桿菌中RstA/RstB的功能：首先，我們利用同源互換的方式取得rstA及rstB缺損株，其外型和生長曲線和野生株並沒有差異；另外，分析RstA/RstB對細菌抗藥性的影響時發現，大量表現RstA會增加細菌對cefotaxime的感受性；進一步，我們發現克雷白氏肺炎桿菌asr（acid shock RNA）的啟動子活性在rstA或rstB缺損株中皆明顯下降，因此，我們推測RstA/RstB可能和抗酸作用有關。然而，rstA或rstB缺損株在酸性環境的生長曲線或酸逆境下的存活率和野生株差異不大。更意外的，asr的基因缺損對細菌在酸性環境的生長或酸逆境下存活的影響也不明顯。不過，藉由西方墨點法分析，我們發現克雷白氏肺炎桿菌的Asr在生成過程中也會有被切割的情形；另外，藉由啟動子活性分析，我們也發現rstA的基因缺損不會影響其它和酸有關的hdeD、yfdX、yfiD的啟動子活性表現。分析rstA啟動子序列，可以找到PhoP及RstA可能的鍵結序列，而以啟動子活性測試，輔以實驗室EMSA分析的結果，證實PhoP/PhoQ會調控RstA/RstB的表現。而為了解RstA/RstB所感應的訊息，我們以添加不同金屬離子來分析啟動子活性表現，結果發現銅、鋅離子可以使rstA、rstB、asr啟動子活性明顯減少。另外，分析rstB啟動子序列，發現類似Fur的鍵結序列，而由啟動子活性測試顯示fur的基因缺損，除了影響rstB啟動子活性外，asr啟動子的活性也會明顯減少。

Abstract

Gene organization of the two-component signal transduction system RstA/RstB in Klebsiella pneunomiae is different from that of Salmonella enterica and Escherichia coli. In order to investigate if different functional role exerted by the RstA/RstB, deletion mutants of rstA or rstB were firstly generated by allelic exchange via homologous recombination. Neither deletion has apparent effect on the phenotype or growth. The drug susceptibility analysis revealed that the overexpression of RstA rendered the E. coli an increase of susceptibility to cefotaxime. Deletion of rstA or rstB was found to decrease dramatically the promoter activity of the acid response regulator Asr. Hence, we presumed RstA/RstB may involve in the acid resistance. However, deletion of rstA or rstB did not change the acid survival rate or affect the growth in acidic condition. Intriguingly, neither the asr deletion had apparent effect on the acid survival rate or the growth in acidic condition. Nevertheless, we found that the K. pneumoniae Asr was also susceptible to the protease digestion and hence two fragments were found in the cytosol after the overexpression of the Asr in the recombinant E. coli. Moreover, the deletion of rstA did not influence the promoter activity of the acid resistance-related genes hdeD, yfdX and yfiD. Analyses of the putative promoter of rstA revealed a conserved PhoP and RstA binding box, suggesting a PhoP/PhoQ-dependent expression of the RstA/RstB. In order to determine the signal sensory molecules for the expression of RstA/RstB, different metal ions were added into the medium for the effect on the promoter activity. The results showed that copper or zinc ion could reduce the promoter activity of rstA, rstB and asr. A putative Fur binding box was found in the rstB promoter, and therefore fur deletion effect was studied. Interestingly, deletion of fur not only reduced the promoter activity of rstB but also the asr promoter activity.