Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation

Abstract

Reversible ADP-ribosylation of dinitrogenase reductase forms the basis of posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. This report describes the physiological effects of mutations in the genes encoding the enzymes that add and remove the ADP-ribosyl moiety. Mutants lacking a functional draT gene had no dinitrogenase reductase ADP-ribosyltransferase (DRAT, the draT gene product) activity in vitro and were incapable of modifying dinitrogenase reductase with ADP-ribose in vivo. Mutants lacking a functional draG gene had no dinitrogenase reductase-activating glycohydrolase (DRAG, the draG gene product) activity in vitro and were unable to remove ADP-ribose from the modified dinitrogenase reductase in vivo. Strains containing polar mutations in draT had no detectable DRAG activity in vitro, suggesting likely cotranscription of draT and draG. In strains containing draT and lacking a functional draG, dinitrogenase reductase accumulated in the active form under derepressing conditions but was rapidly ADP-ribosylated in response to conditions that cause inactivation. Detection of DRAT in these cells in vitro demonstrated that DRAT is itself subject to posttranslational regulation in vivo. Mutants affected in an open reading frame immediately downstream of draTG showed regulation of dinitrogenase reductase by ADP-ribosylation, although differences in the rates of ADP-ribosylation were apparent.Rhodospirillum rubrum, a purple, nonsulfur, photosynthetic bacterium, has a well-characterized system of posttranslational regulation of its nitrogenase by ADP-ribosylation. The nitrogenase complex consists of two proteins: dinitrogenase, an a232 tetramer of the nifK and nifD gene products, contains the active site of nitrogenase, and dinitrogenase reductase, an a2 dimer of the nifH gene product, supplies the reducing power to dinitrogenase. Regulation of the complex is achieved by reversible mono-ADP-ribosylation of dinitrogenase reductase at Arg-101 (23) in response to light level and a fixed nitrogen concentration (12).Two enzymes have been shown to perform this regulation in vitro. Dinitrogenase reductase ADP-ribosyltransferase (DRAT, the gene product of dra7) transfers an ADP-ribose from NAD to one subunit of dinitrogenase reductase (18,20), thereby inactivating nitrogenase by preventing electron flow within the nitrogenase complex (22). Dinitrogenase reductase-activating glycohydrolase (DRAG, the gene product of draG) removes the ADP-ribosyl group from dinitrogenase reductase, restoring activity to the nitrogenase complex (21, 28-30). The draT and draG genes have been isolated and sequenced (7) and are adjacent to, but transcribed in the opposite direction from, the nifHDK genes (15).The first description of mono-ADP-ribosylation was that of eucaryotic elongation factor 2 by diphtheria toxin (11). Subsequently, a number of other bacterial toxins, including cholera toxin and pertussis toxin, have been shown to act by ADP-ribosylating proteins in eucaryotic cells (reviewed in * Corresponding aut...

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

“…aacC1, encoding gentamicin resistance (Gm r ) from pUCGM (25), was inserted into HindIII site, yielding pUX814, pUX815, pUX816, pUX817, and pUX818. These plasmids were transformed into E. coli S17-1, and then conjugated into R. rubrum glnBJ mutant (UR808) (13) by the method described (26). Sm r Km r Gm r R. rubrum colonies were selected, and whole plasmid was integrated into the chromosome resulting from a single-crossover recombination event.…”

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense

“…The sequence comparison of draTG from these strains showed an extensive similarity, with some conserved regions (17). draTG mutants also were constructed and physiologically characterized in these organisms (15,17,30).In the cases of A. brasilense and R. rubrum, the activities of DRAG and DRAT themselves are subject to some form of posttranslational regulation by an unknown mechanism. Under derepressing (nitrogen-fixing) conditions, DRAG is active and DRAT is inactive (7,15,28,30).…”

“…The sequence comparison of draTG from these strains showed an extensive similarity, with some conserved regions (17). draTG mutants also were constructed and physiologically characterized in these organisms (15,17,30).…”

“…Under derepressing (nitrogen-fixing) conditions, DRAG is active and DRAT is inactive (7,15,28,30). In A. brasilense, for example, shifting a culture from microaerobic to anaerobic conditions eliminates the preferred energy source.…”

“…First, the DRAT/DRAG system responds to different negative stimuli in these bacteria. In the photosynthetic bacteria R. rubrum and R. capsulatus, dinitrogenase reductase is modified in response to addition of fixed nitrogen, such as NH 4 ϩ , or a shift of the culture to darkness, which deprives the cells of energy (10,15,17,22). A. brasilense is a nonphotosynthetic, microaerobic nitrogen-fixing bacterium, and its DRAT/DRAG system negatively regulates nitrogenase activity in response to exogenous NH 4 ϩ or anaerobic conditions, because the latter treatment removes the preferred energy source (8,9,28).…”