Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

Abstract

DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class Alphaproteobacteria. Here we present for the first time the DraT/DraG system of a betaproteobacterium, Azoarcus sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one draT ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of draT with two genes encoding hypothetical proteins. Furthermore, draT and draG2 were expressed under all studied conditions, whereas draG1 expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a draG1 mutant and a mutant lacking both draG alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.

PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity

“…In contrast, DraG belongs to the ADP-ribosyl-hydrolase family, and shares a much higher sequence and structural similarity to all members of this family, including orthologues in archaea, bacteria and eukarya (Koch-Nolte et al, 2008). DraG orthologues are widely distributed in prokaryotes, including organisms that do not encode DraT or NifH proteins (Oetjen & Reinhold-Hurek, 2009), suggesting that these orthologues have evolved different functions. The regulation of DraT and DraG activities DraT and DraG activities are subject to opposing regulation in vivo according to the prevailing nitrogen or energy levels (Kanemoto & Ludden, 1984;Liang et al, 1991;Zhang et al, 1993).…”

“…(Oetjen & Reinhold-Hurek, 2009). The genes usually constitute an operon and are presumably co-transcribed.…”

“…However, whilst a draT mutant fails to ADP-ribosylate NifH, it still shows nitrogenase switch-off in response to either ammonium addition or anaerobiosis. Hence, nitrogenase switch-off is independent of ADPribosylation and is presumed to be mediated by a second unknown mechanism (Oetjen & Reinhold-Hurek, 2009). Azoarcus sp.…”

Mass spectrometric characterization of the covalent modification of the nitrogenase Fe‐protein in Azoarcus sp. BH72

“…It is able to express the nif ‐genes in roots of rice [20] and Kallar grass [21], provides fixed nitrogen to its host plant [22], and is thus an interesting candidate for studies of the nitrogenase regulatory mechanism. Phylogenetic analysis indicated that the system for the post‐translational modification of nitrogenase Fe‐protein is probably also present in the δ‐ and γ‐subdivision of the Proteobacteria [17]; however, it has not yet been analyzed in detail outside the α‐subdivision of Proteobacteria .…”

A SIRT4-like auto ADP-ribosyltransferase is essential for the environmental growth of <italic>Mycobacterium smegmatis</italic>

“…One is the well-defined DraT/DraG system, which is involved in the regulation of nitrogenase activity. In response to a negative stimulus such as the presence of ammonium or a decrease in the available cell energy, DraT can ADP-ribosylate Fe protein which in turn dissociates from MoFe protein and ultimately inhibits the nitrogenase activity, while DraG can remove the ADP-ribose moiety from Fe protein and therefore reactivate the nitrogenase [17,18]. In addition to DraT, some toxins secreted by pathogens also possess ADP-ribosylation activity and can disrupt the essential functions of the host cells [13,16,19].…”