Derivation and molecular characterization of symbiotically deficient mutants of Rhizobium meliloti

A collection of symbiotically defective mutants of Rhizobium meliloti JJ1c10 was derived by Tn5 mutagenesis using the suicide vector pGS9. They include two Nod− and about 250 Fix− mutants. The mutants were found to be heterogenous in acetylene reduction activity and in the morphology and ultrastructure of the nodules which they induced. Over 90% were found to contain bona fide Tn5 insertions in a variety of DNA restriction fragments. When Tn5-carrying DNA segments cloned from 24 of the mutants were introduced into the equivalent location in the genome of the wild-type strain by recombination-mediated replacement, only eight produced a symbiotically defective phenotype similar to that of the original mutant. This result indicated that many of the symbiosis mutations were not directly caused by Tn5 insertion. DNA segments apparently containing mutated fix genes but not containing Tn5 were found in eight mutants by identifying cosmids carrying wild-type DNA which complemented their symbiosis defects. Probing of the DNA of these mutants with their complementing cosmids revealed no detectable physical alteration of the homologous DNA. A segment of DNA including the hsn and nifHDK genes was favoured for these non-Tn5 mutations. Three regions of the genome in which Tn5 caused fix mutations were identified. One of these was the known megaplasmid nod-nif region. The other two regions, designated fix-e5 and fix-h21, were found to be chromosomal. Mutants in one of these chromosomal regions fluoresced more intensely on calcofluor plates than the wild type.

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Expression of the nodulation and nitrogen fixation genes in Rhizobium meliloti during development

Genome ◽

10.1139/g89-054 ◽

1989 ◽

Vol 31 (1) ◽

pp. 354-360 ◽

Cited By ~ 2

Author(s):

San Chiun Shen ◽

Shui Ping Wang ◽

Guan Qiao Yu ◽

Jia Bi Zhu

Keyword(s):

Nitrogen Fixation ◽

Rhizobium Meliloti ◽

Abnormal Development ◽

Wild Type ◽

Free Living ◽

Nod Genes ◽

Nodule Initiation ◽

Fix Genes ◽

Nitrogen Fixation Genes

Genes that specify nodulation (nod genes) are only active in the free-living rhizobia or in the nodule initiation state of rhizobia. As soon as the repression of nod genes occurs in the bacteroids of the nodule, nifA is induced, while ntrC is inactivated and thus the nifA-mediated nif/fix genes are turned on. Limitation of available oxygen brings about the induction of nifA, which reflects the actual status of nif/fix gene activities in symbiotic state of rhizobia. Oxygen thus appears to be a major symbiotic signal to the expression of bacteroid nif/fix genes. Mutation of nifA or shortage of nifA product in wild-type rhizobia caused by the inhibition of multicopy nifH/fixA promoters leads to an abnormal development of nodules and premature degradation of bacteroids in nodules.Key words: nitrogen fixation, nodulation, nif/fix regulation, nifA mutant.

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Genetic characterization of Rhizobium sp. strain TAL1145 that nodulates tree legumes

Canadian Journal of Microbiology ◽

10.1139/m94-034 ◽

1994 ◽

Vol 40 (3) ◽

pp. 208-215 ◽

Cited By ~ 16

Author(s):

M. L. C. George ◽

J. P. W. Young ◽

D. Borthakur

Keyword(s):

Rhizobium Leguminosarum ◽

Genetic Characterization ◽

Rhizobium Meliloti ◽

Wild Type ◽

Tree Legumes ◽

Wide Range ◽

The Common ◽

Rrna Sequences ◽

Rhizobium Sp

Rhizobium sp. strain TALI 145 nodulates Leucaena ieucocephaia and Phaseolus vulgaris, in addition to a wide range of tropical tree legumes. Six overlapping clones that complemented nodulation defects in leucaena and bean rhizobia were isolated and a 40-kb map of the symbiosis region was constructed. The common nod and nifA genes were situated approximately 17 kb apart, with the nodlJ genes in between. These clones enabled a derivative of TAL1145 carrying a partially deleted pSym to form ineffective nodules on both leucaena and bean, and a similar derivative of Rhizobium etli TAL182 to form ineffective nodules on bean. When two representative clones, pUHR9 and pUHR114, were each transferred to wild-type rhizobial strains, they allowed ineffective nodulation by Rhizobium meliloti on both leucaena and bean and by Rhizobium leguminosarum bv. viciae on bean. Transconjugants of R. leguminosarum bv. trifolii formed effective nodules on leucaena and ineffective nodules on bean. Tn5 mutagenesis of the symbiosis region resulted in a variety of nodulation and fixation phenotypes on leucaena and bean. On the basis of 16S rRNA sequences, TAL1145 was found to be distinct from both R. tropici and NGR234, the two groups of leucaena symbionts that were previously described.Key words: Rhizobium, Leucaena leucocephala, nodulation, nitrogen fixation.

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Isolation and characterization of rhizobiophages specific for Bradyrhizobium japonicum USDA 117

Canadian Journal of Microbiology ◽

10.1139/m86-064 ◽

1986 ◽

Vol 32 (4) ◽

pp. 326-329 ◽

Cited By ~ 12

Author(s):

F. M. Hashem ◽

J. S. Angle ◽

P. A. Ristiano

Keyword(s):

Bradyrhizobium Japonicum ◽

Generation Time ◽

Burst Size ◽

Maximum Size ◽

Acetylene Reduction Activity ◽

Reduction Activity ◽

Isolation And Characterization ◽

Mutant Phage ◽

Shoot Weight

The isolation and characterization of two phages specific for Bradyrhizobium japonicum USDA 117 are reported. The original phage was isolated from a Chester silt loam soil cropped to soybeans. A mutant of this isolate was obtained during long-term incubation in soil of the original isolate. Both phages were specific for B. japonicum USDA 117. The primary distinction between the two phages was the plaque size produced on the host. The original isolate produced plaques with a maximum size of 2.2 mm2. The mutant phage produced plaques with a maximum size of 11.4 mm2. Both phages exhibited similar morphologies. The head was hexagonal in shape with a diameter of 60 nm. An adsorption rate experiment revealed that the mutant phage was adsorbed faster to the host than the original isolate. Maximum adsorption of the original isolate to the host occurred after 10 min, while the mutant phage required 7 min. Characterization of the original isolate in a one-step growth experiment revealed that the burst size, rise period, and generation time were 100 plaque-forming units/cell, 12 min, and 80 min, respectively. A similar experiment for the mutant phage demonstrated a burst size, rise period, and generation time of 210 plaque-forming units/cell, 6 min, and 70 min, respectively. The ecological competitiveness of the mutant phage appeared to have been altered. In a greenhouse soil incubation experiment, the original isolate reduced nodule number, nodule weight, shoot weight, and acetylene reduction activity to a significantly greater extent than the mutant phage. These results indicate that, while the overall growth rate of the mutant phage was faster, it was less efficient in its parasitism of its host.

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Azospirillum effects on susceptibility to Rhizobium nodulation and on nitrogen fixation of several forage legumes

Canadian Journal of Microbiology ◽

10.1139/m87-085 ◽

1987 ◽

Vol 33 (6) ◽

pp. 510-514 ◽

Cited By ~ 38

Author(s):

Eli Yahalom ◽

Yaacov Okon ◽

Amos Dovrat

Keyword(s):

Rhizobium Meliloti ◽

Root Hairs ◽

Acetylene Reduction Activity ◽

Nodule Formation ◽

Medicago Polymorpha ◽

Reduction Activity ◽

Colony Forming Units ◽

Macroptilium Atropurpureum ◽

Rhizobium Nodulation ◽

Increased Susceptibility

Azospirillum brasilense Cd cell concentration of 105–107 colony-forming units (cfu)/mL applied 24 h before Rhizobium (106 cfu/mL), increased nodule formation in the non root hair zone, more than twofold, in pouch-grown Medicagopolymorpha and Macroptilium atropurpureum seedlings, compared with Rhizobium alone. The increase in nodule formation in pouch-grown Trifolium alexandrinum following preinoculation with Azospirillum was 20%. The percentage of nodulated seedlings rose from 0 to 25% when Medicago polymorpha was preinoculated with Azospirillum followed by the application of 10 cfu/mL Rhizobium meliloti, a level which by itself was not sufficient to initiate nodule formation. Acetylene reduction activity in Medicago polymorpha and Macroptilium atropurpureum seedlings after inoculation with Azospirillum–Rhizobium was markedly increased. A possible reason for the increased susceptibility to Rhizobium infection may be that Azospirillum stimulates the formation of a larger number of epidermal cells that differentiate into infectable root hairs.

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Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.383 ◽

1995 ◽

Vol 128 (3) ◽

pp. 383-392 ◽

Cited By ~ 92

Author(s):

B Drees ◽

C Brown ◽

B G Barrell ◽

A Bretscher

Keyword(s):

Sequence Analysis ◽

Wild Type ◽

Purification And Characterization ◽

Reading Frame ◽

Over Expression ◽

Sequence Identity ◽

Bona Fide ◽

Essential Function

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

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Cloning and characterization of the nifA gene from Herbaspirillum seropedicae strain Z78

Canadian Journal of Microbiology ◽

10.1139/m91-069 ◽

1991 ◽

Vol 37 (6) ◽

pp. 425-429 ◽

Cited By ~ 10

Author(s):

Emanuel M. Souza ◽

Shigehiro Funayama ◽

Liu U. Rigo ◽

Fábio O. Pedrosa

Keyword(s):

Nitrogen Fixation ◽

Azospirillum Brasilense ◽

Genomic Library ◽

Acetylene Reduction Activity ◽

Nif Genes ◽

Herbaspirillum Seropedicae ◽

Reduction Activity ◽

Nitrogen Fixation Gene ◽

Nifa Gene

A genomic library of Herbaspirillum seropedicae was constructed and screened for the nifA gene by complementation of a nifA mutant of Azospirillum brasilense (FP10). A recombinant plasmid, pEMS1, capable of restoring acetylene reduction activity in the mutant FP10, was isolated and found to hybridize to the nifA gene of Klebsiella pneumoniae. The results suggest that nifA is involved in the regulation of nif genes in H. seropedicae. Key words: diazotroph, nitrogen fixation gene, nif regulation, complementation cloning, Herbaspirillum seropedicae.

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Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii

Journal of Bacteriology ◽

10.1128/jb.152.1.72-80.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 72-80

Author(s):

G D Riddle ◽

J G Simonson ◽

B J Hales ◽

H D Braymer

Keyword(s):

Gel Electrophoresis ◽

Azotobacter Vinelandii ◽

Acetylene Reduction Activity ◽

Wild Type ◽

Two Dimensional Gel Electrophoresis ◽

Whole Cells ◽

Reduction Activity ◽

Spin Resonance ◽

Resistant Mutants ◽

Fixation System

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo.

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Preparation and characterization of highly radioactive in vitro labeled adenovirus DNA and DNA restriction fragments

Biochemistry ◽

10.1021/bi00639a023 ◽

1977 ◽

Vol 16 (20) ◽

pp. 4478-4483 ◽

Cited By ~ 66

Author(s):

Jesse K. Mackey ◽

Karl H. Brackmann ◽

Michael R. Green ◽

Maurice Green

Keyword(s):

Restriction Fragments ◽

Dna Restriction Fragments ◽

Dna Restriction

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CLONING AND PHYSICAL MAPPING OF AN EARLY REGION OF THE BACTERIOPHAGE T4 GENOME

Genetics ◽

10.1093/genetics/106.1.1 ◽

1984 ◽

Vol 106 (1) ◽

pp. 1-16

Author(s):

Paul M Macdonald ◽

Gisela Mosig

Keyword(s):

Physical Mapping ◽

Bacteriophage T4 ◽

Restriction Endonucleases ◽

Restriction Map ◽

Wild Type ◽

End Points ◽

Restriction Fragments ◽

Early Region ◽

Dna Restriction Fragments ◽

Dna Restriction

ABSTRACT We have cloned DNA restriction fragments from the largely nonessential region of bacteriophage T4 located between genes 39 and 56. The cloned DNA fragments were used to construct a precise map of the sites in this region recognized by eight restriction endonucleases. This restriction map allowed us to compare the cytosine-containing T4 DNA used for cloning with the hydroxymethylcytosine-containing DNA of wild-type T4; there were no detectable rearrangements in the region tested. We were also able to determine the physical locations of several deletion end points and of several genes.

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