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R H Burris

Publications and source records attributed to R H Burris.

At least 19 recordsLinked to original sources
PubMed1992

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.

ADP Ribose Transferases↗
PubMed1991

Nitrogenases.

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Cyanobacteria↗
PubMed1991

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

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.

Ammonia↗
PubMed1990

Citrate substitutes for homocitrate in nitrogenase of a nifV mutant of Klebsiella pneumoniae.

An organic acid extracted from purified dinitrogenase isolated from a nifV mutant of Klebsiella pneumoniae has been identified as citric acid. H2 evolution by the citrate-containing dinitrogenase is partially inhibited by CO, and by some substrates for nitrogenase. The response of maximum velocities to changes in pH for both the wild-type and the NifV- dinitrogenase was compared. No substantial differences between the enzymes were observed, but there are minor differences. Both enzymes are stable in the pH range 4.8-10, but the enzyme activities dropped dramatically below pH 6.2.

Citrates↗
PubMed1990

Cloning and expression of draTG genes from Azospirillum lipoferum.

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.

ADP Ribose Transferases↗
PubMed1990

Reversible ADP-ribosylation is demonstrated to be a regulatory mechanism in prokaryotes by heterologous expression.

The primary product of biological nitrogen fixation, ammonia, reversibly regulates nitrogenase activity in a variety of diazotrophs by a process called "NH4(+)-switch-off/on." Strong correlative evidence from work in Azospirillum lipoferum and Rhodospirillum rubrum indicates that this regulation involves both the inactivation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase and the reactivation by dinitrogenase reductase activating glycohydrolase. The genes encoding these two enzymes, draT and draG, have been cloned from these two organisms, so that direct genetic evidence can be marshaled to test this model in vivo. The draT/G system has been transferred to and monitored in the enteric nitrogen-fixing bacterium Klebsiella pneumoniae, an organism normally devoid of such a regulatory mechanism. The expressed draT and draG genes allowed K. pneumoniae to respond to NH4Cl with a reversible regulation of nitrogenase activity that was correlated with the reversible ADP-ribosylation of dinitrogenase reductase in vivo. Thus, the expression of draT and draG genes in K. pneumoniae is necessary and sufficient to support NH4(+)-switch-off/on, and ADP-ribosylation serves as a reversible regulatory mechanism for controlling nitrogenase activity in prokaryotes.

ADP Ribose Transferases↗
PubMed1989

Functional expression of a Rhodospirillum rubrum gene encoding dinitrogenase reductase ADP-ribosyltransferase in enteric bacteria.

The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.

ADP Ribose Transferases↗
PubMed1989

Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae.

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N2-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O2 level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversibly inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N2-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was not significantly disturbed when cultures were treated with ammonium in vivo. Possible mechanisms for inhibition by ammonium of whole-cell nitrogenase activity in H. seropedicae are discussed.

Adenosine Diphosphate Ribose↗
PubMed1989

N2O reduction and HD formation by nitrogenase from a nifV mutant of Klebsiella pneumoniae.

Dinitrogenase from a nifV mutant of Klebsiella pneumoniae contains an altered form of iron-molybdenum cofactor (FeMoco) that lacks a biologically active homocitric acid molecule. Change in the composition of FeMoco led to substantial variation in the kinetics of nitrogenase action. The KmS of the mutant enzyme for N2 and N2O were 0.244 and 0.175 atm (24,714 and 17,726 kPa), respectively. The km for N2 was higher and the Km for N2O was lower than that for the wild-type enzyme. The mutant enzyme was ineffective in N2 fixation, in N2O reduction, and in HD formation, as indicated by the low Vmax of these reactions with saturating levels of substrate and under conditions of saturating electron flux. These observations provide further support for the concept that N2, N2O, and D2 interact with the same form of dinitrogenase. H2 evolution by the mutant enzyme is only partially inhibited by CO. Observation that different numbers of electrons are stored in CO-inhibited than in noninhibited dinitrogenase before H2 is released suggests that the mutant enzyme has more sites responsible for H2 evolution than the wild-type enzyme, whose H2 evolution is not inhibited by CO.

Ammonia↗
PubMed1989

Posttranslational regulatory system for nitrogenase activity in Azospirillum spp.

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.

ADP Ribose Transferases↗
PubMed1988

Hydrogen burst associated with nitrogenase-catalyzed reactions.

We have used a membrane-leak mass spectrometer to follow the time courses of H2 evolution and substrate reduction by nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. In the absence of added substrates, dinitrogenase passes all of its electrons to protons to form H2, but when a reducible substrate is added the electrons from dinitrogenase are shared between protons and the added substrate so that the steady-state rate of H2 production is decreased. If a reducible substrate is added before the nitrogenase reaction is initiated, a pre-steady-state burst of H2 is evident upon initiation of the reaction. This burst is associated with all the substrates of nitrogenase examined--i.e., N2, N2O, C2H2, NaN3, and NaCN. The H2 burst is stoichiometric with dinitrogenase, but not with dinitrogenase reductase. In the H2 burst phase, 1 H2 is evolved per dinitrogenase molybdenum. Although a change in the ratio of nitrogenase components changed the initial rate of the H2 burst, the stoichiometry was not affected. Production of H2 by the burst in the presence of a high concentration of substrate is terminated after production of 1 H2 per dinitrogenase molybdenum, and a steady-state rate of H2 production is established. This response suggests that the H2 burst is not a catalytic event but a result of a once-only activation process.

Hydrogen↗
PubMed1988

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1987

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.

Amino Acids↗
PubMed1986

N2O as a substrate and as a competitive inhibitor of nitrogenase.

We have investigated the inhibitory effect of N2O on NH3 formation by purified component proteins from Klebsiella pneumoniae and have confirmed that the inhibition is competitive with respect to N2 and that N2O is reduced to N2, which in turn is further reduced to NH3. In addition, we have shown that N2O is unable to support HD formation from D2 and H2O. N2-supported HD formation from D2 and H2O was found to be inhibited by N2O. In contrast to N2, N2O was found to suppress nitrogenase-mediated H2 evolution completely at infinitely high pN2O. H2 was found to inhibit N2O-supported NH3 production but not N2O-supported N2 production. The steady-state kinetics of N2O reduction showed a good fit to Michaelis-Menten kinetics with a Km for N2O of 5 mM at 30 degrees C, corresponding to 24 kPa of N2O. A model is proposed that fits the observed results.

Ammonia↗
PubMed1986

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants.

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O2. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2 M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H2 had about half the nitrogen-fixation activity expected on the basis of intact filaments. H2 did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0 degree C under N2.

Acetylene↗