Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nod− mutants

Long, Sharon R.; Buikema, William J.; Ausubel, Frederick M.

doi:10.1038/298485a0

Cited by 233 publications

(164 citation statements)

References 15 publications

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“…For the Rhizobium meliloti/alfalfa symbiosis, we have isolated rhizobial mutants and categorized them into three classes according to the point at which they impede the formation of effective nodules (Müller, 1987;Müller et al, 1988). lnto the first class fall nodulation mutants (Nod-) that cannot proceed beyond the early steps of nodulation (Long, Buikema, and Ausubel, 1982;Meade et al, 1982;Debelle et al, 1986;Horvath et al, 1986;Müller, 1987). The nodulation and host specificity genes have been identified and located on megaplasmid 1, the symbiotic plasmid of R. meliloti (Banfalvi et al, 1981;Rosenberg et al, 1981).…”

Section: Introductionmentioning

confidence: 99%

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules.

et al. 1990

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Alfalfa plants co-inoculated with Rhizobium meliloti nodulation (Nod-) and infection mutants deficient in exopolysaccharide production (Inf-EPS) formed mixed infected nodules that were capable of fixing atmospheric nitrogen. The formation of infected nodules was dependent on close contact between the inoculation partners. When the partners were separated by a filter, empt) Fix-nodules were formed, suggesting that infection thread formation in alfalfa is dependent on signals from the nodulation and infection genes. In rnixed infected nodules, both nodulation and infection mutants colonized the plant cells and differentiated into bacteroids. The formation of bacteroids was not dependent on cell-to-cell contact between the mutants. Immunogold/silver staining revealed that the ratio of the two mutants varied considerably in colonized plant cells following mixed inoculation. The introduction of an additional niflfix mutation into one of the inoculation partners did not abolish nitrogen fixation in mixed infected nodules. The expression of nif D::lacZ fusions additionally demonstrated that mutations in the nodulation and infection genes did not prevent the nif genes from being expressed in the mutant bacteroids.

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Section: Introductionmentioning

confidence: 99%

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules.

et al. 1990

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“…Four weeks after inoculation of roots with the conjugation mixture, nodules were removed and surface-sterilized as described by Long et al (26). The nodules were squashed and the macerates were spread on mannitol-yeast agar plates (18).…”

Section: Methodsmentioning

confidence: 99%

Nodule formation by clover-Rhizobium carrying chromosomal nod genes.

Uchiumi

Higashi

Abe

1995

J. Gen. Appl. Microbiol.

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Plasmid pCINod l carrying attP of a lysogenic rhizobiophage and the nod genes was constructed. Inoculation of a conjugation mixture of Escherichia coli harbouring pCINod l and a nodulation-defective Rhizobium leguminosarum biovar trifolii strain resulted in formation of root nodules on white clover. Rhizobium transconjugant strain CNH 1 isolated from these nodules had integrated pCINod 1 into its chromosome. The light and electron microscopic observation of the nodulation process revealed that the nod genes on the chromosome of strain CNH 1 were completely functional.In fast-growing Rhizobium spp. including Rhizobium leguminosarum biovars, the nodulation (nod) genes and nitrogen fixation (nif) genes are located on a large symbiotic (Sym) plasmid (3,14,15,21). Many genes have been introduced into Rhizobium using wide host range cloning vectors (24). However, often the cloned genes and/or the vector were eliminated during the nodulation process ( 7,16,22,24). Incompatibility between the vector and the endogeneous Sym plasmid will also occur (5). To overcome these problems, cloning vectors that integrate into the chromosome of Rhizobium have been developed using transposable elements (4,19,20), repeat sequence (1) and the attachment site (attP) of a lysogenic phage (10).In recent work, we developed the chromosomal integrative vector system using the attP site of a lysogenic phage ~5U (33). Here, we report on the integration of nod genes into the chromosome of Rhizobium using the chromosomal integrative vector system and the nodulation phenotype of the Rhizobium transconjugant strain.

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“…The nodulation genes, described so far, are located on a large plasmid and are closely linked to nif genes [I -51. Nodulation genes are arranged into two clusters: early nodulation functions, which are conserved in many Rhizobium species, form one cluster ('common' nod genes) [4,6, 71 were reported. To explain the lack or decreased level of protein synthesis from some heterologous DNA templates in the E. coli system, differences in sequence signals involved in transcription and translation processes were invoked.…”

mentioning

confidence: 99%

“…Nodulation genes are arranged into two clusters: early nodulation functions, which are conserved in many Rhizobium species, form one cluster ('common' nod genes) [4,6,71, while genes determining host specificity are located in another cluster [6]. Protein-coding regions of 'common' nod genes (nodABC) were mapped [8].…”

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confidence: 99%

A cell‐free system from Rhizobium meliloti to study the specific expression of nodulation genes

Dusha

Schröder

Putnoky

et al. 1986

European Journal of Biochemistry

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An in vitro transcription-translation system was developed using cell-free extracts from the symbiotic nitrogenfixing bacterium Rhizobium meliloti strain 41. Conditions for preparation of the 30000 x g supernatant extract and for measurement of protein-synthesizing activity were determined and compared to the activity of an Eschericha coli cell-free system. Genes expressed in the free-living or in the symbiotic state were studied. The product of a recA-like gene (41-kDa protein) was synthesized both in R. meliloti and E. coli extracts, although less efficiently in the heterologous system. In agreement with earlier results obtained in E. coli minicells, three proteins (44, 28.5 and 23 kDa) were synthesized from a cloned 3.3 x 103-base DNA region carrying genes for nodulation (nod). However, differences in the transcription-translation of nod and host specificity (hsn) genes were observed when protein expression was compared in R. meliloti and E. coli cell-free extracts, and the possible explanations of these findings are discussed.Rhizobium meliloti, a member of the family Rhizobiaceae, is able to establish a symbiotic relationship with leguminous plants. As a result of the interaction of plant and bacterial genes, root nodules are formed which are able to fix atmospheric nitrogen.Bacterial genes required for nodule formation have been identified in R . meliloti. The nodulation genes, described so far, are located on a large plasmid and are closely linked to nif genes [I -51. Nodulation genes are arranged into two clusters: early nodulation functions, which are conserved in many Rhizobium species, form one cluster ('common' nod genes) [4,6, 71 were reported. To explain the lack or decreased level of protein synthesis from some heterologous DNA templates in the E. coli system, differences in sequence signals involved in transcription and translation processes were invoked. Recently we published the observation that SmR of transposon Tn5 is expressed in vivo in R. meliloti although it is not detectable in E. coli [20]. This finding also supports the idea that differences either on the transcriptional or translational level or both between the two species may exist. To circumvent these difficulties our aim was to establish an in vitro proteinsynthesizing system from R. meliloti, which would enable us to study specific expression of Rhizobium genes. MATERIALS AND METHODS Preparation of R. meliloti cellqree extractStrain AK631, a prototrophic, symbiotically effective derivative of R. meliloti 41 with compact colony morphology, was used for preparation of the 30000 x g supernatant (S-30) extract. Cells were grown with shaking at 170 rpm at 32°C in TY medium [21] to A600 = 0.7-1.0. Cultures were cooled rapidly in an ice/water bath and cells were collected by centrifugation at 6000 rpm for 20 min at 4°C. The bacterial pellet was washed twice with buffer A containing 20 mM Trislacetate (pH 8.2), 10 mM magnesium acetate, 40 mM potassium acetate, 1 mM dithiothreitol. Bacteria were stored at -20°C until used. For the prepara...

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Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nod− mutants

Cited by 233 publications

References 15 publications

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules.

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules.

Nodule formation by clover-Rhizobium carrying chromosomal nod genes.

A cell‐free system from Rhizobium meliloti to study the specific expression of nodulation genes

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