Physiological and proteome analyses to investigate the nitrogen fixation mechanism particularly with respect tothe posttranslational regulation of nitrogenase in Azoarcus sp. BH72

Abstract

Nitrogenase activity control in the grass-endophyte Azoarcus sp. BH72 is subjected to a posttranslational regulation, which is accompanied by covalent modification of its nitrogenase Fe-protein that was characterized here. Nitrogen-fixing cells of the betaproteobacterium Azoarcus sp. BH72 inactivate their nitrogenase after stimulation by addition of an external nitrogen source or energy depletion through ADP-ribosylation of the arginine residue 102 inone subunit of the Fe-protein. The modification was detected in the modified peptide sequence applying high-performance liquid-chromatography electrospray ionization/tandem mass spectrometry and was verified by site-directed mutagenesis of the respective arginineresidue. In addition, analysis of the type of the modification led to the establishment of a MALDI-TOF MS (matrix assisted laser desorption/ionization-time of flight mass spectrometry) based method for the detection of a labile posttranslational modification.The Azoarcus sp. BH72 genome harbors a gene that encodes a protein exhibiting sequence similarity to known dinitrogenase reductase ADP-ribosyltransferases (DraT). It possesses two genes, coding for proteins with sequence similarity to dinitrogenase reductase activatingglycohydrolases (DraG). The expression of draT, which is co-transcribed with two genes encoding a hypothetical protein and a hemerythrin (DcrH1), was neither nitrogen nor oxygen regulated. Likewise, the expression of draG2 was independent on the availability of fixed nitrogen, while transcription of draG1 - probably located in an operon with nifQ - was nitrogen regulated. Physiological analysis of deletion mutants of the genes draT, draG1 and draG2 and of a draG1draG2 double mutant led to the characterization of the DraT/DraGsystem in Azoarcus sp. BH72. DraT was responsible for the inactivation of nitrogenase byADP-ribosylation after ammonium addition or anaerobiosis. However, a DraT-independent, ammonium-mediated nitrogenase "switch-off" was also observed. In addition, physiologicalexperiments of the draG-mutants as well as phylogenetic analysis of DraG amino acid sequences revealed that DraG1 mediates the reversibility of the nitrogenase inactivation by removal of the ADP-ribosylation, while DraG2 may have other functions as it only partially complemented a draG1 deletion mutation. Moreover, a putative role of DcrH1 in nitrogenase protection against oxygen damage under conditions of slightly elevated oxygen concentrations is proposed.Comparative proteomic studies led to the identification of proteins putatively important for the formation and function of intracytoplasmic membrane stacks (diazosomes) and for the interaction of Azoarcus sp. BH72 with a fungal endophyte of Kallar grass.