利用氧化亚铁硫杆菌nifH - lacZ融合体分离氧化亚铁硫杆菌ntrBC基因。

Isolation of the Thiobacillus ferrooxidans ntrBC genes using a T. ferrooxidans nifH-lacZ fusion.

作者信息

Kilkenny C A, Berger D K, Rawlings D E

机构信息

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

出版信息

Microbiology (Reading). 1994 Oct;140 ( Pt 10):2543-53. doi: 10.1099/00221287-140-10-2543.

DOI:10.1099/00221287-140-10-2543

PMID:8000526

Abstract

An agar plating technique was developed in which the activation of expression of a Thiobacillus ferrooxidans nifH-lacZ gene fusion was used to isolate the ntrBC genes from a T. ferrooxidans gene library. An Escherichia coli ntrC mutant containing the nifH-lacZ fusion was transformed and plated on a low-nitrogen medium so that on flooding with ONPG, the production of yellow colonies indicated the presence of the cloned T. ferrooxidans ntrBC genes. A 4.47 kb region from the T. ferrooxidans chromosome was sequenced. Analysis of the sequence revealed that the ntrB and ntrC genes were closely linked to a third ORF of unknown function. Analysis of the 900 bp region upstream of the T. ferrooxidans ntrBC genes and Southern hybridization experiments confirmed that in T. ferrooxidans ATCC 33020, the glnA and ntrBC genes are unlinked. Expression of the T. ferrooxidans nifH-lacZ fusion in E. coli was activated in the presence of the T. ferrooxidans ntrBC genes and regulated by nitrogen.

摘要

开发了一种琼脂平板培养技术，其中利用氧化亚铁硫杆菌nifH-lacZ基因融合表达的激活从氧化亚铁硫杆菌基因文库中分离出ntrBC基因。将含有nifH-lacZ融合体的大肠杆菌ntrC突变体进行转化，并接种在低氮培养基上，以便在用ONPG淹没时，黄色菌落的产生表明克隆的氧化亚铁硫杆菌ntrBC基因的存在。对氧化亚铁硫杆菌染色体的一个4.47 kb区域进行了测序。序列分析表明，ntrB和ntrC基因与一个功能未知的第三个开放阅读框紧密相连。对氧化亚铁硫杆菌ntrBC基因上游900 bp区域的分析和Southern杂交实验证实，在氧化亚铁硫杆菌ATCC 33020中，glnA和ntrBC基因不连锁。氧化亚铁硫杆菌nifH-lacZ融合体在大肠杆菌中的表达在氧化亚铁硫杆菌ntrBC基因存在的情况下被激活，并受氮的调节。