Title

Functional Analysis of the nifQdctA1y4vGHIJ Operon of Sinorhizobium fredii Strain NGR234 Using a Transposon with a NifA-Dependent Read-Out Promoter

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, Sinorhizobium fredii 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, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro 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 nifQdctA1y4vGHIJ operon was mutated using either the Omega interposon or TnEKm-PwA. 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 NGRΔnifA, which lacks a functional copy of nifA. De novo transcription from inserted copies of TnEKm-PwA inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in dctA1 and nifQ were Fix+ on all of the hosts tested, indicating that none of the six genes of the nifQ operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.

Department(s)

Biological Sciences

Sponsor(s)

University of Geneva
Swiss National Science Foundation
Society of Physics and Natural History of Geneva

Comments

Financial support for this project was provided by the University of Geneva, the Swiss National Science Foundation (grant no. 31003A-116591) and the Society of Physics and Natural History of Geneva.

Keywords and Phrases

Bacterial Strain; Controlled Study; DNA Transposition; Downstream Processing; Gene Mutation; Genetic Transcription; In Vitro Study; Legume; Mutagenesis; nifQdctA1y4vGHIJ Gene; Nitrogen Fixation; Nonhuman; Nucleotide Sequence; Operon; Promoter Region; Rhizobiaceae; Sinorhizobium fredii; Transposon; Bacterial Proteins; Base Sequence; Dicarboxylic Acid Transporters; DNA Transposable Elements; Fabaceae; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutagenesis, Insertional; Promoter Regions, Genetic; Symbiosis; Transcription Factors

International Standard Serial Number (ISSN)

1350-0872;1465-2080

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2011 Microbiology Society, All rights reserved.

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