GlnB and its homologs are integrators of signals of nitrogen and carbon status in almost all prokaryotic cells, including all major pathogens. GlnB can exist in two extreme forms, one that signals nitrogen sufficiency and one that signals nitrogen deficiency. The equlibrium between those forms is affected by binding (carbon-rich) 2-ketoglutarate and ATP, and by covalent modification of GlnB in response to low levels of (nitrogen-rich) glutamine. These various forms of GlnB then interact differentially with a critical set of receptor proteins that control nitrogen metabolism. The signal transduction mechanism within GlnB that controls this process is poorly understood and we will gain information about this process through the biochemical and physiological analysis of some striking GlnB variants that we have obtained. These GlnB variants include (i) variants that are shifted toward the form that signals nitrogen deficiency; (ii) variants that are shifted toward the form that signals nitrogen sufficiency; and (iii) variants that are differentially altered in their affinities for ATP and 2- ketoglutarate. These variants will be examined by in vitro assays for small molecule binding, interaction with receptors and conformational changes. The variants will be examined in vivo for to determine the precise physiological impact of the biochemical properties measured in vitro. All of these effects will be interpreted in light of existing and proposed analysis of GlnB conformation and structure. The approach has a very high likelihood of success because the interesting variants are already in hand and the biochemical and genetic tools are available. We will also examine a related aspect of GlnB regulation, namely its removal from the cytoplasm by interaction with the membrane-associated AmtB. We have developed the tools for the direct in vitro analysis of this interaction and the information will be integrated with the above data on GlnB itself. The result of the proposed experiments will be a dramatically improved description of how GlnB homologs in all organisms integrate signals of nitrogen, carbon and energy status at the molecular level. The information is broadly important because such signal integration is critical for both beneficial (antibiotic production) and harmful (toxin production) microbial processes. We will therefore understand better how both beneficial and pathogenic organisms sense and respond to their environment.
