Open AccessComplementation of Escherichia coli sigma 54 (NtrA)-dependent formate hydrogenlyase activity by a cloned Thiobacillus ferrooxidans ntrA gene
Author(s) -
David Kenneth Berger,
David R. Woods,
Douglas E. Rawlings
Publication year - 1990
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.172.8.4399-4406.1990
Subject(s) - biology , rpon , complementation , rhodobacter , genetics , gene , open reading frame , peptide sequence , microbiology and biotechnology , promoter , mutant , gene expression
The ntrA gene of Thiobacillus ferrooxidans was cloned by complementation of an Escherichia coli ntrA mutant that was unable to produce gas via the sigma 54 (NtrA)-dependent formate hydrogenlyase pathway. Analysis of the DNA sequence showed that the T. ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, 52,972). The T. ferrooxidans NtrA protein had 49, 44, 33, and 18% amino acid similarity with the NtrA proteins of Klebsiella pneumoniae, Azotobacter vinelandii, Rhizobium meliloti, and Rhodobacter capsulatus, respectively. The ability of the T. ferrooxidans NtrA protein to direct transcription from sigma 54-dependent promoters was demonstrated in E. coli by using fdhF-lacZ and nifH-lacZ fusions. An open reading frame coding for a protein of 241 amino acids (calculated Mr, 27,023) was situated 12 base pairs upstream of the T. ferrooxidans ntrA gene. Comparison of this protein with the product of the open reading frame ORF1, located upstream of the R. meliloti ntrA gene, showed that the two proteins had 55% amino acid similarity. The cloned T. ferrooxidans ntrA gene was expressed in E. coli from a promoter located within the ORF1 coding region.