Nitrogen must be obtained from the environment by all living things in order to synthesize compounds like proteins and nucleic acids. The NifA protein is required to activate transcription of the nitrogen fixation operons in a wide variety of proteobacteria. In all of them NifA activity is controlled by molecular oxygen (02) but the mechanism differs with the organism. In members of the y-subgroup, NifA activity is inhibited by a second protein, NifL. NifL can also inhibit NifA activity in the presence of combined nitrogen. Thus relief of NifL inhibition requires the absence of both 02 and combined nitrogen, and how such effects are coordinated is an important regulatory problem. NifL carries a FAD cofactor associated with its N-terminal domain. Reduction of the cofactor with the powerful reductant dithionite relieves NifL inhibition of NifA activity in a purified transcription system in vitro. The working hypothesis is that an unidentified iron-containing protein may be the physiological reductant for the FAD cofactor of NifL in Klebsiella pneumoniae. There is strong evidence that the nitrogen regulatory protein GlnK, a protein allosteric effector of a sort recently identified in all three domains of life, is also required for relief of NifL inhibition. It is postulated that GlnK enables reduction of the FAD co-factor of NifL under anaerobic conditions only when combined nitrogen is also limiting, e.g. by raising the mid-point potential for reduction into the physiological range. To test this hypothesis a combination of genetic, molecular biological and biochemical methods is being used to determine: 1) whether GlnK makes reduction of the FAD co-factor of NifL easier in vitro and thereby results in relief of NifL inhibition and 2) whether GlnK interacts directly with NifL. Although there was no previous report of a growth defect associated with disruption of the amtB (ammonium-methylammonium transport B) gene in bacteria, which lies downstream of glnK and in an operon with it, amtB mutant strains of enteric bacteria grow poorly at low concentrations of NH4+ at pH values below 7. Growth studies with a variety of mutant strains in combination with studies of transport of the NH4+ analogue l4Cmethylammonium are most parsimoniously interpreted to indicate that AmtB proteins, which are found in all three domains of life, transport the uncharged species NH3 rather than the charged species NH4, and that they facilitate the rate of diffusion of this species rather than concentrating it in an energy-dependent manner. Both interpretations are at odds with previous views from a number of other laboratories. To test them further, aim 3) is to study the role of the AmtB protein of enteric bacteria in acquisition of NH3 at pH 7.0, which must be done in continuous culture; aim 4)is to determine whether AmtB mediates loss of NH3 to the medium when strains are grown on poor nitrogen sources that generate it--i.e. whether diffusion is bi-directional; and aim 5) is to determine whether apparent concentrative uptake of 14Cmethylammonium by S. cerevisiae is due to "acid trapping" in yeast vacuoles. These studies are of interest with respect to understanding the coordination of transcriptional regulatory responses to different environmental signals, that is, in understanding how environmental signals are coordinated to control decoding of particular portions of an organism's DNA. They also bear on the functions of the enteric GlnK protein, part of a sophisticated "two-tiered" regulatory system for sensing nitrogen availability, and on those of AmtB, which is essential for acquisition of ammonia at low concentrations.
