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.

FIG. 1.

Expression of draT and role of DraT in posttranslational modification of nitrogenase Fe protein and switch-off of nitrogenase activity. (A) Northern blot hybridized with a draT probe and with a 16S rRNA probe as indicated. Band intensities were quantified using the ImageQuant 5.1 program to estimate loading amounts as described below. RNA was extracted from cells grown in SM medium under nitrogen-fixing conditions (−N) or grown aerobically under conditions of nitrogen excess (+N). (B) Western blot analysis of nitrogenase Fe protein of wild-type Azoarcus sp. strain BH72 and BHΔdraT, indicating that DraT is responsible for posttranslational modification of nitrogenase Fe protein of Azoarcus sp. strain BH72. Left panels exhibit the effect of anaerobiosis on nitrogenase Fe protein modification. Cells were taken before (lane 1) and 5 min (lane 2) or 20 min (lane 3) after transfer to anaerobic conditions. Right panels show the effect of addition of 1 mM ammonium chloride to nitrogen-fixing cells. Cells were taken before (lane 1) and 5 min (lane 2) or 15 min (lane 3) after addition of 1 mM ammonium chloride. (C) Western blot analysis of cell extracts from N₂-fixing coculture of Azoarcus sp. strain with fungal strain 2003 7 days after inoculation. wt, wild type. (D) Acetylene reduction assays demonstrating that the switch-off response is not abolished in the draT mutant strain. Diagrams show the ethylene production of wild-type Azoarcus sp. strain BH72 and draT mutant BHΔdraT as indicated without ammonium chloride (filled squares) or with the addition of 1 mM (open squares) or 0.5 mM (filled circles) ammonium chloride at the time indicated by arrows. Error bars were derived from two repetitions.

FIG. 2.

Effect of anaerobiosis shifts on nitrogenase activity and Fe protein modification of nitrogen-fixing cultures of wild-type and draG mutant Azoarcus strains. (A) Acetylene reduction assay representing the percentage of initial nitrogenase activity (nanomoles ethylene/milligrams protein × minute) of nitrogen-fixing cultures of wild-type Azoarcus sp. strain BH72 (open squares), BHΔdraG1 (open triangles), BHΔdraG2 (filled squares), and BHΔdraG1ΔdraG2 (filled triangles) after shifts to anaerobic conditions followed by adjustment to microaerobiosis. Initial activities were for wild-type strain BH72 (147 ± 26), BHΔdraG2 (121 ± 44), BHΔdraG1 (122 ± 12) or BHΔdraG1ΔdraG2 (135 ± 51) (ethylene/milligrams protein × minute), respectively. (B) Western blot analysis with antiserum against nitrogenase Fe protein. Samples were taken at the time points indicated in panel A. The relative amounts of modified Fe protein were determined with a luminescent image analyzer with charge-coupled-device technology and quantified with ImageQuant 5.1. Error bars were derived from two independent experiments with two replicates per strain. mod., modified; unmod., unmodified.

FIG. 3.

Posttranslational modification of nitrogenase under anaerobic conditions in the presence of chloramphenicol (37.5 μg/ml) for inhibition of protein de novo synthesis. Western blot analysis with antiserum against NifH is shown. Strains, as indicated, were transferred to anaerobiosis for 15 min. Then conditions were adjusted to 1% oxygen in the headspace. Samples were taken at the time points indicated. Quantification of the relative amounts of modified Fe protein was as described in legend to Fig. 2. A representative result out of two independent experiments is shown. mod., modified; unmod., unmodified.

FIG. 4.

Effect of ammonium chloride addition (200 μM) on nitrogenase activity and Fe protein modification of nitrogen-fixing cultures of Azoarcus sp. strain BH72 and the draG1 mutant strain. (A) Acetylene reduction (nitrogenase activity in micromoles ethylene/milligram protein) of wild-type Azoarcus strain BH72 (open squares) and draG1 mutant strain BHΔdraG1 (open triangles). Error bars derive from three independent experiments with two replicates each. (B) Western blot analysis with antiserum against nitrogenase Fe protein. Samples were taken at the indicated time points according to the method described for panel A. Quantification of the relative amounts of modified Fe protein as described in legend to Fig. 2. One representative result out of three independent experiments with two replications is shown. mod., modified; unmod., unmodified.