title: Stable Fluorescent and Enzymatic Tagging of Bradyrhizobium diazoefficiens to Analyze Host-Plant Infection and Colonization.
abstract: Bradyrhizobium diazoefficiens USDA 110 (formerly named Bradyrhizobium japonicum) can fix dinitrogen when living as an endosymbiont in root nodules of soybean and some other legumes. Formation of a functional symbiosis relies on a defined developmental program mediated by controlled gene expression in both symbiotic partners. In contrast to other well-studied Rhizobium-legume model systems that have been thoroughly examined by means of genetically tagged strains, analysis of B. diazoefficiens host infection has been impaired due to the lack of suitable tagging systems. Here, we describe the construction of B. diazoefficiens strains constitutively expressing single-copy genes for fluorescent proteins (eBFP2, mTurquoise2, GFP+, sYFP2, mCherry, HcRed) and enzymes (GusA, LacZ). For stable inheritance, the constructs were recombined into the chromosome. Effectiveness and versatility of the tagged strains was demonstrated in plant infection assays. (i) The infection process was followed from root-hair attachment to colonization of nodule cells with epifluorescent microscopy. (ii) Monitoring mixed infections with two strains producing different fluorescent proteins allowed rapid analysis of nodule occupancy and revealed that the majority of nodules contained clonal populations. (iii) Microscopic analysis of nodules induced by fluorescent strains provided evidence for host-dependent control of B. diazoefficiens bacteroid morphology in nodules of Aeschynomene afraspera and Arachis hypogaea (peanut), as deduced from their altered morphology compared with bacteroids in soybean nodules.
authors.affiliation: ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Raphael
authors.initials: R
authors.lastname: Ledermann
authors.affiliation: ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Ilka
authors.initials: I
authors.lastname: Bartsch
authors.affiliation: ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Mitja N
authors.initials: MN
authors.lastname: Remus-Emsermann
authors.affiliation: ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
authors.affiliation: ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Fischer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000382:microbiology; D013025:Glycine max / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D004274:DNA, Recombinant; D008164:Luminescent Proteins