Nitrogen Control of Atrazine Utilization in Pseudomonas sp. Strain ADP

ABSTRACT Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas− mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas− mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.

The Saccharomyces cerevisiae YCC5(YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine

ABSTRACT The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.

Characterization of the Aspergillus nidulans nmrA Gene Involved in Nitrogen Metabolite Repression

ABSTRACT The gene nmrA of Aspergillus nidulans has been isolated and found to be a homolog of the Neurospora crassa gene nmr-1, involved in nitrogen metabolite repression. Deletion of nmrA results in partial derepression of activities subject to nitrogen repression similar to phenotypes observed for certain mutations in the positively actingareA gene.

The roles of PUT3, URE2, and GLN3 regulatory proteins in the proline utilization pathway of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as allantoin, urea, γ-aminobutyrate, or proline when preferred nitrogen sources such as asparagine, glutamine, or ammonium ions are unavailable in the environment. To use proline as the sole nitrogen source, cells must activate the expression of the proline transporters and the genes that encode the catabolic enzymes proline oxidase (PUT1) and Δ1-pyrroline-5-carboxylate dehydrogenase (PUT2). Transcriptional activation of the PUT genes requires the PUT3 regulatory protein, proline, and relief from nitrogen repression. PUT3 is a 979 amino acid protein that binds a short DNA sequence in the promoters of PUT1 and PUT2, independent of the presence of proline. The functional domains of PUT3 have been studied by biochemical and molecular tests and analysis of activator-constitutive and activator-defective mutant proteins. Mutations in the URE2 gene relieve nitrogen repression, permitting inducer-independent transcription of the PUT genes in the presence of repressing nitrogen sources. The GLN3 protein that activates the expression of many genes in alternative nitrogen source pathways is not required for the expression of the PUT genes under inducing, derepressing conditions (proline) or noninducing, repressing conditions (ammonia). Although it has been speculated that the URE2 protein antagonizes the action of GLN3 in the regulation of many nitrogen assimilatory pathways, URE2 appears to act independently of GLN3 in the proline-utilization pathway. Key words: Saccharomyces cerevisiae, proline utilization, nitrogen repression.

Roles of URE2 and GLN3 in the proline utilization pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, gamma-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study. Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small. Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product. PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein. The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation.