Escherichia vulneris Eho10溶磷基因之研究

Abstract

Escherichia vulneris Eho10中溶解非水溶性磷酸鹽(IMPS)基因已被選殖到Escherichia coli內，並於其中表現。這些含有溶磷基因的E． coli可在無磷培養基中，溶解hydroxyapatite或dicalcium phosphate。利用轉位子Tn1000誘導基因突變的結果顯示IMPS基因至少有1.4 kb。如同前人研究結果，Tn1000的嵌入造成一5 kb的直接重複?生。本實驗同時證實以Tn1000雙端特殊序列合成適當引子進行DNA定序反應的可行性。以SDS-PAGE電泳分析細胞內全部蛋白質以及內、外膜間蛋白質的結果顯示：IMPS基因製造一32 Kd多勝?。Minicell實驗結果證實此一發現。本實驗同時發現hyroxyapatite的溶解與pH值的下降有關，觀察無磷培養基中hydroxyapatite溶解情況發現其可能由於酸類的形成亦或培養基中缺乏緩衝能力，而導致溶磷現象的發生，換言之，基因的表現可能不受磷的控制。包含IMPS基因的DNA片段也已開始進行定序工作；然而，必需進一步的分析，方可得到正確的IMPS基因序列。

The insoluble mineral phosphate solubilization (IMPS) gene of Escherichia vulneris Eho10 has been cloned into Escherichia coli. The E. coli strains containing IMPS gene were able to solubilize hydroxyappatite or dicalcium phosphate on phosphate-free media. Transposon Tn1000 mutagenesis study revealed that the size of the IMPS gene is at least 1.4 kb long. As previously reported, the insertion of Tn1000 generated a 5-bp direct repeat. This study also demonstrated that it is feasible to use two 16 mer primers homologous to the two ends of Tn1000 for the sequencing of DNA sequence adjacent to the two ends of Tn1000 insertion sites. SDS-gel electrophoresis of total proteins or periplasmic proteins isolated from the E. coli clones containing IMPS gene or from E. vulneris Eho10 revealed that the IMPS gene encodes a 32-Kdalton polypeptide. E. coli minicell study confirmed this finding. This study also showed that the solubilization of hydroxyappatite on phosphate-free media was related to the synthesis of acid(s) or/and due to the lack of buffering capacity of the media. In other words, the expression of the gene was probably not regulated by phosphate. The DNA fragment containing the IMPS gene has been sequenced. However, further sequencing work will still be needed to obtain an accurate sequence of IMPS gene.