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G H Elkan

Publications and source records attributed to G H Elkan.

At least 19 recordsLinked to original sources
PubMed1996

Bradyrhizobium (Arachis) sp. strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants.

The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.

Acyltransferases↗
PubMed1996

Genetic diversity among bradyrhizobium isolates that effectively nodulate peanut (Arachis hypogaea).

Symbiotic gene diversity and other measures of genetic diversity were examined in Bradyrhizobium isolates that form an effective symbiosis with peanut (Arachis hypogaea). Initially, restriction fragment length polymorphism (RFLP) analysis using a nitrogenase (nif) gene probe was performed on 33 isolates along with one Bradyrhizobium elkanii and two Bradyrhizobium japonicum strains. Considerable diversity was observed among the RFLP patterns of many of the isolates, especially those from South America. Some isolates, however, were found to have similar nif and subsequent nod (nodulation) gene RFLP patterns, indicating symbiotic gene relatedness. With some noted exceptions, symbiotic gene relatedness correlated with relatedness based on total DNA homology and ribotyping analyses. Symbiotic gene relatedness also correlated with symbiotic effectiveness. The RFLP and DNA homology analyses indicate that bradyrhizobia effective with peanut are genetically diverse and consist of at least three different species. This diversity, however, was not particularly evident with partial 16S rRNA gene sequencing. Sequences obtained from the isolates were very similar to each other as well as to sequences previously reported for other Bradyrhizobium strains.

Arachis↗
PubMed1991

Oxygen Uptake and Hydrogen-Stimulated Nitrogenase Activity from Azorhizobium caulinodans ORS571 Grown in a Succinate-Limited Chemostat.

Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 muM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 muM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 muM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol min mg of protein and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and beta-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C(2)H(4)h mg (dry weight), and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C(2)H(4) h mg (dry weight). However, when concentration of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in "washout," proving that ORS571 can grow on N(2) and that there was not a nitrogen source in the medium that could substitute.

Journal Article↗
PubMed1990

Growth, Respiration, and Polypeptide Patterns of Bradyrhizobium sp. (Arachis) Strain 3G4b20 from Succinate- or Oxygen-Limited Continuous Cultures.

Succinate- or oxygen-limited continuous cultures were used to study the influences of different concentrations of dissolved oxygen and ammonia on the growth, respiration, and polypeptide patterns of Bradyrhizobium sp. (Arachis) strain 3G4b20. During succinate-limited growth, molar growth yields on succinate (Y(succ)) ranged from 38.9 to 44.4 g (dry weight) of cells mol of succinate and were not greatly influenced by changes in dilution rates or changes in the oxygen concentrations that we tested. Succinate, malate, and fumarate induced the highest rates of oxygen uptake in all of the steady states in which the supply rates of (NH(4))(2)SO(4) ranged between 322 and 976 mumol h. However, the amino acids aspartate, asparagine, and glutamate could also be used as respiratory substrates, especially when the (NH(4))(2)SO(4) supply rate was decreased to 29 mumol h. Glutamine-dependent respiration was seen only when the (NH(4))(2)SO(4) supply rate was 29 mumol h and thus appears to be under tight ammonia control. Nitrogenase activity was detected only when the culture was switched from a succinate-limited steady state to an oxygen-limited steady state. Comparison of major silver-stained proteins from three steady states by two-dimensional gel electrophoresis revealed that nearly 60% were affected by oxygen and 24% were affected by ammonia. These data are consistent with reports that oxygen has a major regulatory role over developmental processes in Rhizobium sp. and Bradyrhizobium sp.

Journal Article↗
PubMed1989

Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in Rhizobium fredii USDA 206.

Rhizobium fredii USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.

Journal Article↗
PubMed1986

Restriction Endonuclease and nif Homology Patterns of Bradyrhizobium japonicum USDA 110 Derivatives With and Without Nitrogen Fixation Competence.

DNAs from Bradyrhizobium japonicum USDA 110 derivatives that differ in nitrogen-fixing ability produced similar electrophoretic patterns with five different restriction enzymes. Our data support the hypothesis of common ancestry for these derivatives. Derivatives I-110 and L1-110 differed as much as 100-fold in acetylene reduction activity when they were tested with several soybean cultivars in both greenhouse and field experiments. While possessing nodulating ability, derivative L1-110 is deficient in symbiotic nitrogen-fixing ability, whereas derivative I-110 is symbiotically competent. Hybridization of nifDK and nifH probes from B. japonicum to Southern blots of restricted DNAs from strain USDA 110 derivatives produced similar patterns. This finding indicates similar structural gene organization for both derivative I-110 and derivative L1-110 and implies that the difference in symbiotic nitrogen fixation is probably not due to structural gene rearrangements. However, our hybridization data do not rule out the possibility of differences in expression of structural nif genes or alterations in the structure or expression of other genes required for symbiotic nitrogen fixation.

Journal Article↗
PubMed1986

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars.

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N(2) than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N(2) than did the USDA 110 during the 20-day period. The superior N(2) fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N(2)-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N(2)-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N(2) in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N(2)-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N(2) fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N(2)-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.

Journal Article↗
PubMed1986

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative.

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N(2)-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K(2)HPO(4)-0.08% MgSO(4) . 7H(2)O-0.02% NaCl-0.001% FeCl(3) . 6H(2)O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.

Journal Article↗
PubMed1986

Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative.

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.

Journal Article↗
PubMed1985

Effect of Sym Plasmid Curing on Symbiotic Effectiveness in Rhizobium fredii.

A mutant, USDA 206C, of Rhizobium fredii USDA 206 was obtained by passage on acridine plates. This mutant was cured of its 197-megadalton Sym plasmid but retained its symbiotic effectiveness. Multiple plasmid and chromosomally borne nif gene copies have previously been shown in R. fredii USDA 206. HindIII and EcoRI restriction enzyme digests of plasmid and total DNA showed that at least two nif gene copies are probably missing in USDA 206C. To compare the symbiotic effectiveness of USDA 206 and USDA 206C, plant tests were carried out. Statistically significant differences were obtained for nodule number, nodule mass, nitrogenase activity per plant, nitrogenase specific activity, and total plant dry weight. There was an apparent correlation between loss of Sym plasmidborne nif gene copies and reduction of overall symbiotic effectiveness. Delayed nodulation by strain USDA 206C relative to strain USDA 206 also indicated an association with the loss of plasmidborne nodulation functions and the reduced symbiotic effectiveness of strain USDA 206C.

Journal Article↗
PubMed1985

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii.

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.

Acridine Orange↗
PubMed1979

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.

Anti-Bacterial Agents↗
PubMed1978

The role of ammonia, L-glutamate, and cyclic adenosine 3',5'-monophosphate in the regulation of ammonia assimilation in Rhizobium japonicum.

The effects of three factors (ammonia, L-glutamate, and cyclic adenosine 3',5'-monophosphate) on the ammonia assimilatory processes in aerobically grown Rhizobium japonicum colony derivatives were examined. Ammonia repressed glutamine synthetase activity and increased the average state of adenylylation of this enzyme. The addition of L-glutamate drastically decreased growth and strongly repressed glutamate synthase levels. Glutamine synthetase repression and adenylylation state were also increased by L-glutamate. The presence of cyclic AMP led to the repression of all three NH+4 assimilatory enzymes.

Ammonia↗
PubMed1978

Method for correcting laboratory model deep-well disposal system data for hydrostatic pressure effects.

A pressure chamber for determining the effect of increased hydrostatic pressure on growth and metabolic activities of groundwater bacteria is described. The chamber was used to show that moderate increases in pressure (to about 100 atmospheres) result in increased growth of mixed cultures of industrial-injection-well bacteria and in the more complete degradation of formate and nitrate by these bacteria, as compared with identical cultures at atmospheric pressure.

Journal Article↗
PubMed1977

Glucose catabolism in two derivatives of a Rhizobium japonicum strain differing in nitrogen-fixing efficiency.

Radiorespirometric and enzymatic analyses reveal that glucose-grown cells of Rhizobium japonicum isolates I-110 and L1-110, both derivatives of R. japonicum strain 3I1b110, possess an active tricarboxylic acid cycle and metabolize glucose by simultaneous operation of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. The hexose cycle may play a minor role in the dissimilation of glucose. Failure to detect the nicotinamide adenine dinucleotide phosphate-dependent decarboxylating 6-phosphogluconate dehydrogenase (EC 1.1.1.44) evidences absence of the pentose phosphate pathway. Transketolase and transaldolase reactions, however, enable R. japonicum to produce the precursors for purine and pyrimidine biosynthesis from fructose-6-phosphate and glyceraldehyde-3-phosphate. A constitutive nicotinamide adenine dinucleotide-linked 6-phosphogluconate dehydrogenase has been detected. The enzyme is stimulated by either mannitol or fuctose and might initiate a new catabolic pathway. R. japonicum isolate I-110, characterized by shorter generation times on glucose and greater nitrogen-fixing efficiency, oxidizes glucose more extensively than type L1-110 and utilizes preferentially the Embden-Meyerhof-Parnas pathway, whereas the Entner-Doudoroff pathway apparently predominates in type L1-110.

Carbohydrate Dehydrogenases↗
PubMed1977

Comparison of colony morphology, salt tolerance, and effectiveness in Rhizobium japonicum.

Four strains of Rhizobium japonicum, two of which produce slimy and non-slimy colony types and two others which produce large and small colony types, were isolated and cloned. All were infective and nodulated Lee soybean host plants. Each colony type was characterized as to its salt sensitivity to Na+ and K+ ions, relative level of symbiotic nitrogen fixation, and relative level of free-living nitrogen fixation. Growth studies performed in the presence of salts demonstrated that the non-slimy or small colony types were sensitive to salt with significantly depressed growth rates and cell yields. Growth rates and cell yields of slimy, large, colony types were relatively unaffected by salt. Both symbiotic and free-living (non-associative) nitrogen fixation analyses (by acetylene reduction) revealed that the non-slimy, small colonies were significantly more effective than slimy, large colonies.

Genetic Variation↗