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Abstract

Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, Tn was constructed. This transposon combines the advantages of mutagenesis of cloned DNA fragments with a conditional read-out promoter from NGR234 (PwA) that reinitiates NifA-dependent transcription downstream of transposition sites. To test the characteristics of the new transposon, the y4vGHIJ operon was mutated using either the Omega interposon or Tn The symbiotic phenotypes on various hosts as well as the transcriptional characteristics of these mutants were analysed in detail and compared with the ineffective (Fix) phenotype of strain NGRΔ, which lacks a functional copy of . transcription from inserted copies of Tn inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in and were Fix on all of the hosts tested, indicating that none of the six genes of the operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.

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2011-10-01
2019-10-19
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vol. , part 10, pp. 2745 - 2758

Mapping of the start points of transcripts produced by PwA. (a) Mapping the +1 site of the y4wAB transcript in NGR234. Top, chromatogram and corresponding nucleotide sequence derived from 5'RACE-PCR products. The nucleotide determined as +1 is shown as a bold capital letter and shaded in grey. Below, the complementary sequence in the context of the PwA promoter region, with the binding sites for NifA (UAS) and RpoN (-12/-24) shown in bold. The +1 and start codon for y4wA are shown in bold red characters. (b) Mapping the +1 site of the y4vG transcript in mutant strain NGR117. Top, chromatogram and corresponding nucleotide sequence derived from 5'RACE-PCR products. Bottom, DNA sequence at the border of Tn in NGR117, with the +1 site and start codon for y4vG shown in bold red characters. The sequence of the PwA promoter embedded in the transposon is shown in green, with the terminal inverted repeat marked in bold capitals. The binding site for σ is shaded. [PDF](740 kb)



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