SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A new type of ferredoxin was isolated from Azotobacter vinelandii cells. The protein was able to replace the native chloroplast ferredoxin in the photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) and functioned as a reductant for the Azotobacter nitrogenase.
The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N(2) fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N(2) fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N(2)-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N(2) fixation (0.87 to 4.90 mg of N liter(-1) day(-1)) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.
Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.
Cell extracts of an Escherichia coli strain that overproduces the regulatory protein FIXJ from Rhizobium meliloti promoted transcription of fixK, a known FIXJ-dependent gene, in a coupled transcription-translation assay. Activation by FIXJ was dependent on the sigma 70 holoenzyme form of RNA polymerase.
Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A. vinelandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.
Dinitrogen fixation activity was determined directly on experimental plots in mixtures of grass with red and white clover in the year of sowing as well as in the first year of full utilisation using the method of acetylene reduction (ARA). Furthermore, numbers of bacteria, actinomycetes, fungi and Azotobacter sp. were determined in soils under the experimental mixtures.
The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.
High rates of acetylene (C(2)H(2)) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C(2)H(4) produced per ml per h) and under anaerobic (up to 135 nmol of C(2)H(4) produced per ml per h) conditions. Pasteurized effluent developed C(2)H(2) reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N(2)-fixing Azotobacter and Klebsiella were abundant, and N(2)-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.
Studies on 3H-dihydrostreptomycin accumulation and binding to ribosomes showed that ineffective strain CMts17 carries strB type mutation changing its membrane permeability to the drug. Introduction of high level streptomycin resistance of strA type into strain CMts17 was correlated with acquisition of effectiveness and membrane permeability to the drug. This suggests that changes in membrane permeability, responsible for ineffectiveness of strain CMts17, can be reversed by strA mutation.
DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD(+)-dependent and NADP(+)-dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant.
We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.
Cultured cells of a Rhizobium phaseoli wild-type strain (CE2) possess b-type and c-type cytochromes and two terminal oxidases: cytochromes o and aa3. Cytochrome aa3 was partially expressed when CE2 cells were grown on minimal medium, during symbiosis, and in well-aerated liquid cultures in a complex medium (PY2). Two cytochrome mutants of R. phaseoli were obtained and characterized. A Tn5-mob-induced mutant, CFN4201, expressed diminished amounts of b-type and c-type cytochromes, showed an enhanced expression of cytochrome oxidases, and had reduced levels of N,N,N',N'-tetramethyl-p-phenylenediamine, succinate, and NADH oxidase activities. Nodules formed by this strain had no N2 fixation activity. The other mutant, CFN4205, which was isolated by nitrosoguanidine mutagenesis, had reduced levels of cytochrome o and higher succinate oxidase activity but similar NADH and N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activities when compared with the wild-type strain. Strain CFN4205 expressed a fourfold-higher cytochrome aa3 content when cultured on minimal and complex media and had twofold-higher cytochrome aa3 levels during symbiosis when compared with the wild-type strain. Nodules formed by strain CFN4205 fixed 33% more N2 than did nodules formed by the wild-type strain, as judged by the total nitrogen content found in plants nodulated by these strains. Finally, low-temperature photodissociation spectra of whole cells from strains CE2 and CFN4205 reveal cytochromes o and aa3. Both cytochromes react with O2 at -180 degrees C to give a light-insensitive compound. These experiments identify cytochromes o and aa3 as functional terminal oxidases in R. phaseoli.
Among rhizobia studied, Rhizobium sp. strain ORS571 alone grew unambiguously on N2 as sole N source. In ORS571 , only the glutamine synthetase (GS)-glutamate synthase ( GOGAT ) pathway assimilated ammonium. However, ORS571 exhibited two unique physiological aspects of this pathway: ORS571 had only GS I, whereas all other Rhizobiaceae studied had both GS I and GS II, and both NADPH- and NADH-dependent GOGAT activities were present. ORS571 GS-affected and NADPH- GOGAT -affected mutant strains were defective in both ammonium assimilation (Asm-) and N2 fixation (Nif-) in culture and in planta ; NADH- GOGAT mutants were Asm- but Nif+. "Bacteroid" GS activity was essentially nil, suggesting symbiotic ammonium export. Physiological studies on effects of glutamine, ammonium, methionine sulfoximine, and diazo-oxo-norleucine on nitrogenase induction in culture implied a regulatory role for the intracellular glutamine pool.
A 19-kb DNA region containing genes sharing homology with Rhizobium meliloti fixNOQP and fixGHI was isolated from a genomic library of Azorhizobium caulinodans. Identity of fixG was confirmed by partial nucleotide sequencing. Mutant strains in the fixGHI region were constructed by deletion or Tn5 insertions. In contrast with the situation in R. meliloti, the mutants still displayed a significant nitrogenase activity in symbiosis.
Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.
Steady-state chemostat cultures of Azotobacter vinelandii strain CA11, carrying a deletion of genes encoding the structural polypeptides of nitrogenase nifHDK, were established in a simple defined medium chemically purified to minimize contamination by Mo. The medium contained no utilizable N source. Growth was dependent on N2 (1.1 X 10(8) viable cells X ml-1 at D = 0.176 h-1), and was inhibited by Mo (20 nM). DNA hybridization showed the deletion to be stable during prolonged (55 days) growth in the chemostat (132 doublings). Since batch cultures, using unsupplemented 'spent' chemostat medium, showed good growth (1.9 X 10(8) cells X ml-1), no requirement for subnanomolar concentrations of Mo was found. The biomass yield, as the dilution rate (D) was varied, showed that the N content of the culture, protein and dry wt. increased as D was decreased, indicating that neither N2 nor O2 were limiting growth. The limiting nutrient was not identified. Substantial amounts of H2 were evolved by the chemostat cultures, probably as the result of inhibition of O2-dependent hydrogenase activity by nitrilotriacetic acid present in the medium. Over a range of D values approx. 50% of the electron flux through the alternative system was allocated to H+ reduction. C2H2 was a poor substrate, being reduced at 0.14-0.1 times the rate of N2 fixation, calculated from the N content of the cells. SO4(2-)-limited steady-state continuous cultures of strain UW136 (wild-type for nifHDK) had a 2-fold greater biomass in the presence of MoO4(2-) (1 microM). The significance of this finding for 'Mo-limited' continuous cultures [Eady & Robson (1984) Biochem. J. 224, 853-862] is discussed.
A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.
The nif cistrons indentified by complementation analysis in the preceding paper (Dixon et al., 1977) were mapped with respect to hisD and to each other other by P1 cotransduction and three-factor reciprocal crosses. The order obrained was hisD nifB nifA (nifL) nifF nifE nifK nifD nifH. Analysis of hisD2-nif cotransduction data by the Wu equation (Wu, 1966) suggested that the nif genes are divided into two clusters: a his-proximal cluster comprising nifBA(L)F and a his-distal group of nifEKDH.