Studies on the regulatory mechanisms controlling nitrogenase synthesis and ammonia assimilation in Azotobacter vinelandiiand Sinorhizobium meliloti

Abstract

Biological nitrogen fixation (BNF) is the nitrogenase-catalyzed conversion of dinitrogen to ammonia by a select group of Bacteria and Archaea called diazotrophs. In turn, plants and other microbes assimilate ammonia during the synthesis of nucleic acids, proteins and other biomolecules. BNF is of special interest in agriculture where it replenishes soil nitrogen lost during repetitive farming. Basic knowledge of BNF might eventually lead to less dependence on expensive and polluting chemical fertilizers. For the studies presented here, two model diazotrophs, the free-living Azotobacter vinelandii , and the alfafa symbiont, Sinorhizobium meliloti, were used to investigate mechanisms controlling nitrogen fixation and nitrogen metabolism. In A. vinelandii, ammonia inhibits nitrogenase expression by limiting activity of the two-component activator, NifA; this involves the negatively acting sensor protein, NifL. Groundwork indicated that a global nitrogen-sensing system, present in many bacteria might control NifA activity since glnD mutants were unable to fix nitrogen. In other organisms, nitrogen limitation signals GlnD-mediated uridylylation of PII-like signal transduction proteins, which signals activation of a suite of genes involved in nitrogen source utilization. The goals of the current study were to characterize the operon encoding a PII-like protein in A. vinelandii, named GlnK, and determine its influence on NifA and nitrogen metabolism. The results indicated that glnK is an essential gene and that uridylylation of GlnK is required for activation of glutamine synthetase and NifA. Also presented here is evidence that GlnK interacts with NifL to stimulate its inhibitory properties. These results are consistent with a model in which uridylylation of GlnK in response to nitrogen limitation signals relief of NifL inhibition. In the last section of this dissertation, glnD of Sinorhizobium meliloti was cloned and sequenced because a PII-like protein had been previously implicated in control of nodule development and symbiosis. Unfortunately, S. meliloti glnD mutants could not be isolated unless glnD and flanking genes were provided in trans, indicating that the glnD operon is indispensable. These studies provide new insight into the global mechanisms controlling nitrogen fixation and metabolism and suggest that GlnD and PII-like proteins may regulate other targets, some of which are essential.