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Journal of Bacteriology, June 2009, p. 3726-3735, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01720-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72{triangledown} ,{dagger}

Janina Oetjen and Barbara Reinhold-Hurek*

General Microbiology, Faculty of Biology and Chemistry, University Bremen, D-28359 Bremen, Germany

Received 9 December 2008/ Accepted 29 March 2009

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.

* Corresponding author. Mailing address: General Microbiology, Faculty of Biology and Chemistry, University Bremen, Postfach 33 04 40, D-28334 Bremen, Germany. Phone: 49 (0)421 218 2370. Fax: 49 (0)421 218 9058. E-mail: breinhold{at}uni-bremen.de

{triangledown} Published ahead of print on 3 April 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, June 2009, p. 3726-3735, Vol. 191, No. 11
0021-9193/09/$08.00+0     doi:10.1128/JB.01720-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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