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.
authors: University of Geneva, Sciences III, Department of Botany and Plant Biology, Microbiology Unit, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
authors: Coralie
authors: C
authors: Fumeaux
authors: University of Geneva, Sciences III, Department of Botany and Plant Biology, Microbiology Unit, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
authors: Nadia
authors: N
authors: Bakkou
authors: Department of Botany, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, ul. Nowoursynowska 159, 02-776 Warsaw, Poland.
authors: Joanna
authors: J
authors: Kopcińska
authors: Department of Botany, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, ul. Nowoursynowska 159, 02-776 Warsaw, Poland.
authors: Wladyslav
authors: W
authors: Golinowski
authors: Department of Biological Sciences, Missouri University of Science and Technology, 105A Schrenk Hall, 400 West 11th Street, Rolla, 65409-1120 MO, USA.
authors: David J
authors: DJ
authors: Westenberg
authors: Fachbereich Biologie/Zellbiologie, Philipps Universität Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany.
authors: Peter
authors: P
authors: Müller
authors: University of Geneva, Sciences III, Department of Botany and Plant Biology, Microbiology Unit, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
authors: Xavier
authors: X
authors: Perret
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004251:DNA Transposable Elements; D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D007887:Fabaceae / Q000382:microbiology / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon; D011401:Promoter Regions, Genetic; D042181:Sinorhizobium fredii / Q000235:genetics* / Q000502:physiology; D013559:Symbiosis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D029382:Dicarboxylic Acid Transporters; C061338:NifA protein, Bacteria; D014157:Transcription Factors