克雷白氏肺炎桿菌CG43兩套尿素酶操縱組表現之比較分析

Abstract

細菌可藉分解尿素取得其生長所需的氮源，此反應產生的氨更可與酸中和而避免細菌受酸壓力的傷害，這個過程需要由多單位合成的尿素酶來完成。克雷白氏肺炎桿菌CG43具有兩套素酶基因組，相較臨床菌株NTUH-K2044 與MGH78578、或土壤分離株342多了一套基因組，我們將這幾株克雷白氏肺炎桿菌共有的尿素酶基因組命名為ure1 (ureD1-A1-B1-C1-E1-F1-G1) ，而CG43特有的則命名為 ure2 (ureA2-B2-C2-E2-F2-G2-D2)。為了確認兩組尿素酶基因組的功能性，我們先比較將ureA1和ureA2基因缺失的影響，結果發現∆ureA1、∆ureA2或 ∆ureA1∆ureA2培養於LB 培養液中，其生長情形與野生株並沒有差異；當以尿素為氮源的M9 培養基培養時，剔除ureA1明顯的影響細菌生長； ∆ureA2突變株於尿素酶活性測試的Christensen’s 尿素培養基 或Stuart’s 尿素培養液時都表現與野生株相當的尿素酶活性；相對的，∆ureA1突變株在生長36小時後表現尿素酶活性，顯示Ure2在生長後期的作用。進一步，為了尋找影響這兩套尿素酶基因組表現的環境因子與調控蛋白，以啟動子活性分析來比較二者表現的差異，結果顯示在M9培養基中，兩組基因組皆受6 mM尿素誘導表現；ure1表現會受弱酸pH 5誘導、但被高量的鐵與鎳離子抑制； ure2在室溫27度培養的表現較37度高；另外，在ure1及ure2啟動子區域序列，分別預測到可能的調控蛋白包括Fur、CRP、CpxR和 Hfq的結合位，因此，分別在各調控蛋白及其調控相關基因剔除的菌株中量測比較二者啟動子活性，結果顯示Fur基因缺失會降低ure1表現，而將fur與ryhB同時剔除，可回復其尿素酶活性；而在∆ureA1∆ryhB∆fur或∆ure1∆hfq株中，則失去了生長後期延遲表現的Ure2尿素酶活性，在ure1∆hfq中大量表現RyhB時，可以回復其Ure2尿素酶活性，然而將RyhB基因上與Hfq結合位點突變後，則失去其回復作用，這結果暗示RyhB在生長後期可活化Ure2尿素酶活性，而此活化作用需要Hfq的輔助。最後，剔除尿素酶基因使第三型纖毛主要蛋白MrkA的生成被抑制，而增加第一型纖毛主要蛋白FimA生成，此暗示尿素的累積，可抑制第三型纖毛的合成卻啟動第一型纖毛的表現。

Ureolysis helps bacteria acquire nitrogen sources and also prevent from acid damage by neutralizing the acidic environments. The process requires proteins encoded by the operon for a functional multicomponent urease. Klebsiella pneumoniae CG43 harbors two urease operons namely ure1 (ureD1-A1-B1-C1-E1-F1-G1) and ure2 (ureA2-B2-C2-E2-F2-G2-D2), different from that only ure1 operon is contained in the genome of the clinical isolates NTUH-K2044 and MGH78578 or the soil isolate KP 342. The ureA1 and ureA2 deletion effects were firstly compared to determine for their functional roles. Removal of ureA1 or ureA2 gene conferred no changes on the bacterial growth in LB, while ∆ureA1 and ∆ureA1∆ureA2 exerted an impaired growth in M9 with urea as the N source. The mutant ∆ureA2 cultured on Christensen’s urea plate or in Stuart’s urea broth carried urease activity. By contrast, the urease activity of ∆ureA1 could only be detected after 36-h culture in Stuart’s urea broth suggesting a late-onset of the ure2 enzymatic activity. Subsequently, the promoter activity was measured to investigate what environmental signals and regulatory proteins involved in influencing the urease expression. The results showed that both operons were activated when 6 mM urea added as nitrogen source into M9 medium. The expression of ure1 was induced by weak acid (pH 5) but repressed by high levels of nickel or ferric ion, whereas the ure2 expression was increased at room temperature 27℃compared to the expression level measured at 37℃. Several regulatory protein binding elements could be predicted in the putative promoter region of ure1 and ure2, and hence the promoter activity was also determined in each of the regulatory gene deletion mutants. The ure1 expression was found to be markedly decreased by fur deletion but was restored by removing ryhB gene from the fur deletion mutant. On the other hand, deletion of either hfq or ryhB from ∆ureA1 diminished the delayed type Ure2 activity. The urease activity could only be complemented by overexpression of RyhB but the Hfq-binding-residue-mutated RyhB in ∆hfq∆ureA1. This suggests that RyhB requires Hfq for the activation of Ure2 expression. Finally, deletion of either ureA1 or ureA2 abolished the production of type 3 fimbriae major pilin MrkA but increased the type 1 fimbriae major pilin FimA production. This implies that the accumulation of urea may inhibit type 3 fimrbiae expression but induce the expression of type 1 fimbriae.