Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum

Summary Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.     

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

A novel potassium deficiency-induced stimulon in<Emphasis Type="Italic">Anabaena torulosa</Emphasis>

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacteriumAnabaena torulosa and of nine proteins inEscherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K⁺ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase ofA. torulosa and the glycine betaine-binding protein ofE. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria.     

Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum

The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity. We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme. DRAT activity is stabilized by NaCl and ADP. The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase. NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R. rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors. No other proteins or small molecules, including water, have been found to be effective as acceptors. Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product. DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.     

Interactions between PII proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense

In Azospirillum brasilense ADP-ribosylation of dinitrogenase reductase (NifH) occurs in response to addition of ammonium to the extracellular medium and is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG). The P(II) proteins GlnB and GlnZ have been implicated in regulation of DraT and DraG by an as yet unknown mechanism. Using pull-down experiments with His-tagged versions of DraT and DraG we have now shown that DraT binds to GlnB, but only to the deuridylylated form, and that DraG binds to both the uridylylated and deuridylylated forms of GlnZ. The demonstration of these specific protein complexes, together with our recent report of the ability of deuridylylated GlnZ to be sequestered to the cell membrane by the ammonia channel protein AmtB, offers new insights into the control of NifH ADP-ribosylation.     

Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-(32)P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-(14)C]NAD individually upon UV irradiation, but most (14)C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-(14)C]NAD suggested that Arg 101 is not absolutely required for NAD binding.     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.     

ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.     

The early formation of the photosynthetic apparatus in Rhodospirillum rubrum

The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c ₂do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-¹⁴C-proteinhydrolyzate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism.Abbreviations Bchl bacteriochlorophyll - LH light harvesting - RC reaction center - R. Rhodospirillum - R. Rhodopseudomonas     

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins

The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years.Clostridium botulinum C2 toxin,Clostridium perfringens iota toxin andClostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization.Clostridium botulium exoenzyme C3 andClostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.     

Wachstum und Ausbildungsformen des Gynoeceums vonRochelia (Boraginaceae)

The ontogeny of the gynoecium ofRochelia disperma has been investigated by LM and SEM. From the floral apex only one carpel primordium arises abaxially and eventually shifts into a subterminal position. Neither an initial stadium of a second carpel nor an adaxial vascular strand in gynobase and style could be observed. InR. stylaris two vascular strands run through the style and two undifferentiated lobes in adaxial position may be regarded as rudimentary mericarps. Only from comparison with related taxa the conclusion can be drawn thatRochelia is really pseudomonomerous, more so inR. disperma than inR. stylaris. The primary gynoecial bulge splits up into three parts inR. disperma: style/stigma, nutlets with gynobase, and disc. While the mericarps originate in mostBoraginoideae from the symplicate region and the ascidiate one is restricted to the very basal parts, inR. disperma the ascidiate part extends and forms the nutlets. The hood-shaped mouth of the carpel (the plicate zone) is closed to a triangular slit in lateral position, the stigma.The nutlets are triangular with broad base and do not surround the adaxial part of the gynobase inR. disperma, R. persica, R. bungei, R. stylaris, andR. macrocalyx. In contrast,R. peduncularis, R. cardiosepala, andR. cancellata have nutlets clasping the gynobase; they may be more closely related than was assumed up to now. The glochids ofRochelia are fascicled unicellular hairs (with different shapes) and not emergences as in theCynoglosseae. There is an evolutionary trend towards fruit formation with only one mericarp, especially inR. disperma.

     

Effect of nucleotides on the activity of dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum

The mechanism by which MgADP stimulates the activity of dinitrogenase reductase ADP-ribosyltransferase (DRAT) has been examined by using dinitrogenase reductases from Rhodospirillum rubrum, Klebsiella pneumoniae, and Azotobacter vinelandii as acceptor substrates. In the presence of 0.2 mM NAD, maximal rates of ADP-ribosylation of all three acceptors were observed at an ADP concentration of 150 microM; in the absence of added ADP, DRAT activity with the dinitrogenase reductases from R. rubrum and K. pneumoniae was less than 5% of the maximal rate, but the A. vinelandii protein was ADP-ribosylated at 40% of the maximal rate. Of eight dinucleotides tested, only ADP, 2'-deoxy-ADP, and ADP-beta S served as activators of the DRAT reaction; ADP, 2'-deoxy-ADP, and ADP-beta S were also the only dinucleotides found which inhibited acetylene reduction activity by dinitrogenase reductase. The dinucleotide specificities for both DRAT activation and acetylene reduction inhibition were the same for all three dinitrogenase reductases. In the DRAT reaction with the dinitrogenase reductases from K. pneumoniae and A. vinelandii, the Km for NAD was 30-fold higher in the absence of ADP than in its presence; the Km for NAD with the R. rubrum acceptor was not measurable. In the presence of saturating ADP, ADP-ribosylation of dinitrogenase reductase from R. rubrum was inhibited 63% by 1.5 mM ATP. It is concluded that MgADP stimulates DRAT activity by lowering the Km for NAD and that MgADP exerts its effect by binding to dinitrogenase reductase. MgATP inhibits DRAT activity by competing with MgADP for binding to dinitrogenase reductase.     

Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

rbcL sequences support exclusion ofRetzia,Desfontainia, andNicodemia from theGentianales

The taxonomic positions ofRetzia, Desfontainia, andNicodemia have been much discussed, and all three genera have been included inLoganiaceae (Gentianales). We have made a cladistic analysis ofrbcL gene sequences to determine the relationships of these taxa toGentianales. Four newrbcL sequences are presented; i.e., ofRetzia, Desfontainia, Diervilla (Caprifoliaceae), andEuthystachys (Stilbaceae). Our results show thatRetzia, Desfontainia, andNicodemia are not closely related toLoganiaceae or theGentianales. Retzia is most closely related toEuthystachys and is better included inStilbaceae. The positions ofDesfontainia andNicodemia are not settled, butDesfontainia shows affinity for theDipsacales s.l. andNicodemia for theLamiales s.l.     

NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum.

Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state.     

Zur systematischen Stellung der GattungProckia: Karyologie und Epidermisskulptur im Vergleich zuFlacourtia (Flacourtiaceae),Grewia (Tiliaceae) und verwandten Gattungen

Detailed analyses of karyology and leaf morphology do not support relationships betweenFlacourtiaceae andTiliaceae. In spite of different chromosome numbers,Prockia (2n = 18),Flacourtia (2n = 22) andRawsonia (2n = 22) are very similar in karyomorphology, indicating a certain karyological uniformity withinFlacourtiaceae. Lacistema (2n = ca. 62) appears more isolated. On the other hand, theTiliaceae Grewia (2n = 18) andLuhea (2n = 36) have much in common and differ remarkably from the Flacourtiaceous genera. The salicoid leaf-teeth ofProckia are also found inIdesia, but never inTiliaceae. Epidermis ultrastructure reveals certain relationships betweenProckia andFlacourtia in contrast to the strongly differingGrewia. Idesia has a rare und unique epidermis sculpture. — Basic chromosome numbers and chromosomal evolution within theFlacourtiaceae are discussed.