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

Abstract

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...

New Rhizobium leguminosarum Flavonoid-Induced Proteins Revealed by Proteome Analysis of Differentially Displayed Proteins

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Molecular cloning of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum

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