Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus
| Autor: | Kai-Uwe Riedel, Bernd Masepohl, Petra Dreiskemper, Alice Pawlowski, Holger Bierhoff, Werner Klipp, Silke Gross, Thomas Drepper |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Molybdenum
Rhodobacter biology Bacteria Two-hybrid screening Mutant Structural gene Nitrogenase Nif gene Gene Expression Regulation Bacterial biochemical phenomena metabolism and nutrition biology.organism_classification Microbiology Rhodobacter capsulatus Quaternary Ammonium Compounds Response regulator Biochemistry Azotobacter vinelandii Bacterial Proteins Nitrogen Fixation Two-Hybrid System Techniques bacteria Molecular Biology Signal Transduction |
| Zdroj: | Journal of bacteriology. 185(17) |
| ISSN: | 0021-9193 |
| Popis: | Rhodobacter capsulatus is a nonsulfur phototrophic purple bacterium (α subdivision of the proteobacteria) which is able to fix atmospheric dinitrogen via a nif-encoded molybdenum-containing nitrogenase. The organization and regulation of nif genes have been analyzed genetically and biochemically in great detail (for a review, see references 45 and 46). As in many other bacteria, ammonium is the preferred nitrogen source in R. capsulatus, and consequently, the highly energy-demanding N2 fixation process is regulated by ammonium availability at three levels (12, 45). R. capsulatus measures the cellular nitrogen status by a nitrogen regulation (Ntr) system similar to that of enteric bacteria (see below). Under nitrogen-limiting conditions (in the absence of ammonium), NtrC becomes phosphorylated, and in turn, NtrC-P activates transcription of the nifA1 and nifA2 genes (level 1), which encode two almost identical nif-specific transcriptional activators. Both NifA proteins are able to activate expression of the structural genes for nitrogenase, nifHDK, and all the other nif genes, resulting in synthesis of nitrogenase. In addition to ammonium control of transcription of nifA1 and nifA2, ammonium leads to inhibition of the activity of both NifA1 and NifA2 (level 2) and to inactivation of nitrogenase (level 3). The enteric Ntr system comprises five gene products, GlnD (uridylyltransferase/uridylyl-removing enzyme), two trimeric PII signal transduction proteins, GlnB and GlnK, and the two-component regulatory system NtrB/NtrC (for a review, see reference 52). In response to the cellular glutamine/2-ketoglutarate ratio, GlnB and GlnK are regulated by reversible GlnD-mediated uridylylation. When cells are grown under N-limiting conditions, the PII proteins are uridylylated and thus are unable to interact with the sensor kinase NtrB. Under these conditions, NtrB autophosphorylates and subsequently promotes the phosphorylation of the response regulator NtrC. NtrC-P in turn activates transcription of its target genes. Since R. capsulatus contains genes homologous to glnD, glnB, glnK, ntrB, and ntrC, and based on genetic and biochemical data, regulatory mechanism similar to those of the enteric Ntr system have been proposed (27, 34, 45). In enteric bacteria, NtrB, in concert with GlnB or GlnK, promotes the dephosphorylation of NtrC under conditions of N excess (52), and as a result, expression of NtrC-dependent promoters is switched off. In contrast to the situation in enteric bacteria, in R. capsulatus only GlnB is involved in regulating NtrB activity, whereas GlnK does not seem to play a role in the Ntr signal transduction mechanism (level 1 [12]). Although a glnB mutation results in expression of both nifA1 and nifA2 in the presence of ammonium, NifA-mediated nif gene expression is inhibited under these conditions (level 2 [12]). This posttranslational control of NifA activity is completely abolished in a glnB glnK double mutant but not in single glnB or glnK mutants, suggesting that GlnB and GlnK can substitute for each other in mediating ammonium repression of NifA activity. Similarly, posttranslational ammonium control of nitrogenase activity mediated by the DraT/DraG system (level 3) is relieved in the glnB glnK double mutant. This paper will focus on yeast two-hybrid studies on the interaction of proteins involved in regulation of the synthesis and activity of molybdenum nitrogenase by ammonium. The utility of two-hybrid assays for analysis of the interaction of proteins playing different roles in nitrogen regulation has already been demonstrated for selected protein pairs, including GlnB-NtrB of Escherichia coli and Klebsiella pneumoniae (41, 43), NtrB-NtrC of K. pneumoniae (42), GlnK-NifL of Azotobacter vinelandii (59), and NifL-NifA of A. vinelandii (36). As described above, the PII-like proteins GlnB and GlnK of R. capsulatus play central roles in ammonium regulation of the synthesis and activity of nitrogenase. In the present study, we used a yeast two-hybrid-based approach to analyze whether GlnB and/or GlnK mediates signal transduction by direct interaction with NtrB, NifA1, NifA2, DraT, and DraG, and we examined the ability of GlnB/GlnK and NifA1/NifA2 to form heteromeric structures. In addition, we identified two new putative proteins that interact with GlnB and GlnK by screening an R. capsulatus genomic library constructed for use in yeast two-hybrid studies. (Preliminary results on these yeast two-hybrid studies were presented at the 13th International Congress on Nitrogen Fixation in Canada [44].) |
| Databáze: | OpenAIRE |
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