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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.

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