Fnr mutants that activate gene expression in the presence of oxygen.

The regulatory protein Fnr is required for anaerobic expression of several anaerobic respiratory enzymes in Escherichia coli. To gain insight into how Fnr activity is regulated by oxygen, we have isolated Fnr mutants that increase expression of the nitrate reductase operon in the presence of oxygen (Fnr* mutants). Seven single-amino-acid substitutions that mapped within two regions of Fnr have been characterized. Two mutants mapped adjacent to two Cys residues in the N-terminal Cys cluster. Five Fnr* substitutions mapped to a region of Fnr that is similar to the cyclic AMP-binding domain of the catabolite activator protein (CAP). Within this group, four mutants were clustered in a region analogous to the CAP C helix, which is important in CAP dimer subunit interactions. Taken together, these data implicate regions in Fnr that may be important either in sensing oxygen deprivation or in the conformational change proposed to be necessary for Fnr activation under anaerobic conditions.     

Aspartate 141 is the fourth ligand of the oxygen-sensing [4Fe-4S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr

The Bacillus subtilis redox regulator Fnr controls genes of the anaerobic metabolism in response to low oxygen tension. An unusual structure for the oxygen-sensing [4Fe-4S](2+) cluster was detected by a combination of genetic experiments with UV-visible and M?ssbauer spectroscopy. Asp-141 was identified as the fourth iron-sulfur cluster ligand besides three Cys residues. Exchange of Asp-141 with Ala abolished functional in vivo complementation of an fnr knock-out strain by the mutagenized fnr gene and in vitro DNA binding of the recombinant regulator FnrD141A. In contrast, substitution of Asp-141 with Cys preserved [4Fe-4S](2+) structure and regulator function.     

Use of phi(glp-lac) in studies of respiratory regulation of the Escherichia coli anaerobic sn-glycerol-3-phosphate dehydrogenase genes (glpAB).

Expression of the glpA operon encoding the extrinsic membrane anaerobic sn-glycerol-3-phosphate dehydrogenase complex of Escherichia coli K-12 was studied in five strains carrying independent glpA-lac operon fusions. The location of the fusions was confirmed by transduction. Two of the strains produced an enzymatically active anaerobic sn-glycerol-3-phosphate dehydrogenase that accumulated in the cytoplasmic fraction of the cells. This suggests the loss of a specific membrane anchor subunit encoded by a distal gene, glpB, which was disrupted by the insertion. beta-Galactosidase in all five strains carrying phi(glpA-lac) was highly inducible by glycerol only anaerobically. A mutation in fnr, a pleiotropic activator gene, prevented full induction of the phi(glpA-lac), demonstrating that the Fnr protein is a positive regulator of the primary dehydrogenase as well as of the terminal reductases of anaerobic respiratory chains. Low concentrations of the respiratory poison KCN had a permissive effect on aerobic expression of phi(glpA-lac). Aerobic expression of the hybrid operon was also enhanced in isogenic derivatives of the fusion strains deficient in protoporphyrin biosynthesis (hemA). Thus, heme proteins may play a role in mediating aerobic repression of the anaerobic respiratory chain.     

Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-1. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S. putrefaciens etrA is able to complement an fnr mutant of E. coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etrA mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S. putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.