一种用于研究脆弱拟杆菌Bf1中谷氨酰胺合成酶调控的报告基因载体。

A reporter gene vector to investigate the regulation of glutamine synthetase in Bacteroides fragilis Bf1.

作者信息

Abratt V R, Zappe H, Woods D R

机构信息

Department of Microbiology, University of Cape Town, South Africa.

出版信息

J Gen Microbiol. 1993 Jan;139(1):59-65. doi: 10.1099/00221287-139-1-59.

DOI:10.1099/00221287-139-1-59

PMID:7680708

Abstract

The Clostridium acetobutylicum eglA gene, encoding a beta-1,4-endoglucanase (EG), was shown to be a useful reporter gene for the study of gene expression in Bacteroides fragilis. The eglA reporter gene has the advantages that it can be easily identified in both Escherichia coli and B. fragilis on agar media containing carboxymethylcellulose, and EG production can be rapidly quantified in liquid medium. Since the B. fragilis glutamine synthetase (GS) is inactivated in permeabilized cells and cell extracts, the eglA reporter gene was used to study the regulation of GS production in B. fragilis. Gene fusions containing the GS glnA promoter region fused to the promoterless eglA gene showed that glnA expression was regulated by nitrogen in B. fragilis at the transcriptional level. A glnA upstream region containing a near-perfect direct repeat sequence was essential for efficient GS expression and for regulation by nitrogen.

摘要

丙酮丁醇梭菌的eglA基因编码一种β-1,4-内切葡聚糖酶（EG），已证明它是用于研究脆弱拟杆菌基因表达的有用报告基因。eglA报告基因具有以下优点：在含有羧甲基纤维素的琼脂培养基上，它在大肠杆菌和脆弱拟杆菌中都能很容易地被鉴定出来，并且在液体培养基中可以快速定量EG的产生。由于脆弱拟杆菌的谷氨酰胺合成酶（GS）在透化细胞和细胞提取物中失活，因此eglA报告基因被用于研究脆弱拟杆菌中GS产生的调控。含有与无启动子的eglA基因融合的GS glnA启动子区域的基因融合表明，在转录水平上，脆弱拟杆菌中glnA的表达受氮的调控。一个含有近乎完美的直接重复序列的glnA上游区域对于高效的GS表达和氮调控至关重要。