Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.     

Role of TnrA in nitrogen source-dependent repression of Bacillus subtilis glutamate synthase gene expression

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis, the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the gltAB promoter. As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.     

Modulation of activity of Bacillus subtilis regulatory proteins GltC and TnrA by glutamate dehydrogenase

The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA. The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity has remained elusive. GltC-dependent gltAB expression was drastically reduced when cells were grown in media containing arginine or ornithine or proline, all of which are inducers and substrates of the Roc catabolic pathway. Analysis of gltAB expression in mutants with various defects in the Roc pathway indicated that rocG-encoded glutamate dehydrogenase was required for such repression, suggesting that the substrates or products of this enzyme are the real effectors of GltC. Given that RocG is an enzyme of glutamate catabolism, the main regulatory role of GltC may be prevention of a futile cycle of glutamate synthesis and degradation in the presence of arginine-related amino acids or proline. In addition, high activity of glutamate dehydrogenase was incompatible with activity of TnrA.     

Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.     

trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.     

Role of glutamine synthetase activity in the uptake and metabolism of arginine and proline in the cyanobacterium Anabaena cycadeae

Abstract The uptake of arginine and proline and their assimilation as nitrogen source have been studied in the cyanobacterium Anabaena cycadeae and its glutamine auxotropic mutant lacking glutamine synthetase activity. The uptake pattern of arginine and proline was found to be biphasic in both wild-type and mutant strains, consisting of an initial fast phase lasting up to 60 s followed by a slower second phase. The uptake activities of both the amino acids were also found to be similar in both the strains. The wild-type strain, having normal glutamine synthetase activity, utilized arginine and proline as sole nitrogen source, whereas the mutant strain lacking glutamine synthetase activity could not do so. These results suggest that: (1) glutamine synthetase activity is necessarily required for the assimilation of arginine and proline as nitrogen source, but it is not required for the uptake of these amino acids; and (2) glutamine synthetase serves as the sole ammonia-assimilating enzyme as well as glutamine-forming route in heterocystous cyanobacteria.