Fachgebiet Zellbiologie und Angewandte Botanik, Fachbereich Biologie der Philipps-Universität, D-35032 Marburg, Germany
The interaction between soil bacteria of the Rhizobiaceae and their leguminous host plants is a multistep process which finally leads to the formation of new plant organs, the nodules, a very efficient place of biological N2 fixation. Two extremely different partners, the bacteria and a suitable plant, with a completely different organization of their genomes meet, and from the very beginning they will have a strong mutual influence on each other. The free-living bacteria will differentiate to intracellular bacteroids which fix N2 to NH4+, and differentiated cortex cells of the plant regain meristematic activity to build up a shelter for the invading bacteria, where these are protected from competitors in the soil, fed by the plant-derived carbon sources, and exploited by the plant as a source of consumable N compounds. This process is beneficial for both partners, but - as demonstrated by plant or bacterial mutants - the symbiotic relationship might become a pathogenic one when the intimate biochemical dialogue between both representatives is disturbed by a single mutation in any gene related to this communication process, which is controlled by several cascades of transcriptional regulation systems.
The four papers of this session contribute to various aspects of the differentiation processes of the bacteria and the plants. Daniel Kahn and co-workers report on "The FixJ transcriptional activator: from structure to genome" (this volume). Yi-Ping Wang and co-workers study "CRP-cAMP-mediated repression effect on the glnAp2 promoter in E. coir (this volume). Furthermore, the "Functional analysis of regulatory genes involved in M. truncatula nodule organogenesis" is presented by Martin Crespi and co-workers (this volume). Finally Pawel Strözycki presents new data on "Iron proteins in legume plant development and nodulation - Ferritin" (this volume). The combination of these four contributions nicely represents the great diversity of aspects which play important roles in the formation and functional operation of nitrogen-fixing nodules.
With the establishment of the genomes of Sinorhizobium meliloti and Mesorhizobium loti, and the partial genomes of several others, e.g. the symbiotic plasmid of Rhizobium spp. NGR234 and the symbiotic region of Bradyrhizobium japonicum, the molecular analysis of the legume/Rhizobium symbiosis has now shifted from the specific level of single genes to whole sets of genes which are expressed under defined conditions (e.g. absence or presence of plant-derived inducers, availability of C compounds, oxygen concentration, etc.). The combination of transcriptomics and proteomics, based on the availability of bacterial genomes, is a powerful tool for elucidating the concerted events which are going on in the regulation and developmental processes in and between the symbiotic partners. Nevertheless, these experimental approaches cannot replace a detailed analysis of single genes in order to define the real functional importance of their gene products in the symbiotic differentiation process. It is necessary to keep in mind this particular aspect, because there are numerous examples for the existence of duplicate or even multiple allelic genes in the rhizobial genomes which might functionally substitute for each other.
In our laboratory, we have focused on the analysis of genes encoding extracytoplasmic proteins, because they might be required for the symbiotic relationships with the legume host plant. Surface components can play important roles in the exchange of metabolites and/or signals between the partners. As a rule, many of these proteins have N-terminal signal peptides and therefore are supposed to be translocated by the general secretory pathway (Pugsley 1993). To detect genes in Bradyrhizobium japonicum encoding extracytoplasmic proteins involved in symbiotic traits we previously used a delivery system for TnphoA (Miiller et al. 1995), because the promoterless reporter gene, 'phoA, is a suitable means to identify putative secreted proteins, if a translational fusion has occurred by a proper insertion of the genetic element (right orientation and in frame fusion within a gene region encoding a periplasmic domain of the protein). Based on our experience with B. japonicum symbiotic mutants defective in two different signal peptidases, sipS (Miiller et al. 1995) and sipF (Bairl et al. 1998), the construction of mutants, the subsequent screening of their symbiotic phenotypes and the detailed genetic analysis of the mutations turned out to be a successful, but very time consuming procedure. Thus, based on an in vitro transposon mutagenesis system (Reznikoff et Goryshin, 1999) and a 'phoA-aphll cassette (Rodriguez-Quinones et al. 1994), TnKPK2 was constructed and used to establish an alternative and rapid strategy which enabled the identification of new B. japonicum loci required in symbiosis. The following experimental scheme should be generally applicable in many other systems:
electroporation into E. coli in vitro transposition
nodulation electroporation into E. coli in vitro transposition
DNA sequencing / transformation in E. coli S17.1
nodulation selection and plant inoculation
DNA sequencing / transformation in E. coli S17.1
selection and plant inoculation
Bairl A, Müller P (1998) Mol. Gen. Genet. 260, 346-356
Manoil C, Beckwith J (1985) Proc. Natl. Acad. Sei. USA 8129-8133
Miiller P et al. (1995) Mol. Microbiol. 18, 831-840
Miiller P et al. (1995) Planta 197, 163-175
Pugsley AP (1993) Microbiol. Rev. 57, 50-108
Rezinkoff W, Goryshin I (1999) Epicentre Forum 6, 5-7
Rodriguez-Quinones et al. (1994) Gene 151, 125-130
THE FixJ TRANSCRIPTIONAL ACTIVATOR: FROM STRUCTURE TO GENOME
L. Ferrières, J. Schumacher, B. Ton-Hoang, J. Fourment, P. Roche, S. Rouillé, D. Kahn
Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes INRA / CNRS, BP 27, 31326 Castanet-Tolosan Cedex, France
In the alfalfa symbiont Sinorhizobium meliloti, nitrogen fixation genes are controlled by the oxygen-regulated FixLJ 'two-component' regulatory system. Under microoxic conditions as provided by root nodules, the FixL histidine kinase phosphorylates FixJ, turning it into a transcriptional activator of the nifA and fixK promoters. As a consequence a genetic cascade is switched on, allowing expression of the nitrogen fixation apparatus during symbiosis (for review see Jf JFixJN Fischer 1994). Like other response regulators, FixJ is a modular protein, which has been dissected into an N-terminal phosphorylatable 'receiver' domain FixJN and a C-terminal transcriptional activator domain FixJC. In its non-phosphorylated form, the FixJN receiver domain inhibits the latent activity of FixJC at Figure 1. General scheme for FixJ activation, the nifA promoter (Kahn, Ditta 1991; Da Re et al. 1994). Phosphorylation simultaneously relieves this inhibition and triggers the dimerization of the protein via the FixJN receiver domain (Figure 1), resulting in a FixJ~P dimer which is the active form of FixJ (Da Re et al. 1999).
The structure of FixJN (Gouet et al. 1999) shares basic features found in other receiver domains, with a parallel 2-1-3-4-5 p-sheet sandwiched between helices al/a5 on one side and helices a2/a3/a4 on the other side. The phosphorylated Asp-54 residue participates in an acidic pocket located at the C-terminal edge of the central parallel p-sheet. The conformational change resulting from the phosphorylation of FixJN has recently been characterized at atomic resolution, showing how phosphorylation reshapes the ot4-|35 face into a dimerization interface (Birck et al. 1999).
Here we used alanine-scanning mutagenesis in order to map functional interfaces of the FixJN receiver domain. Two interfaces were identified, one interacting with the FixJC output domain, the other required for transcriptional activation at the fixK promoter. In addition we present the outcome of a systematic search of FixJ targets in the S. meliloti genome.
2. The 'Aromatic Switch' Hypothesis for 2-Component Signal Transduction
A systematic alanine-scanning mutagenesis of the FixJN receiver domain was undertaken in order to map the interface with the FixJC transcriptional activator domain. Effects of mutations were tested in vivo in nifA-lacZ and fixK-lacZ reporter strains. Mutated proteins were purified from GST fusion proteins and assayed for acetyl-phosphate dependent phosphorylation, dimerization and DNA-binding ability. Mutations affecting the interaction between FixJN and FixJC were expected to confer a strong 'up' phenotype for nifA activation in vivo. Among 26 mutations tested, only one mutation (F101A) exhibited a strong 'up' effect both on in vivo activity and on DNA binding. This 'up' phenotype suggests that the mutated FixJ protein adopts an open conformation liberating the activity of the C-terminal domain. In addition, the F101A Fix J protein could not be phosphorylated with acetyl-phosphate, suggesting a propagating conformational change between Phe-101 and the phosphorylation site. This dual phenotype of the F101A mutation indicates that Phe-101 interacts both with the phosphorylation site and the C-terminal domain. Thus Phe-101 lies at the heart of signal transduction between the phosphorylation site and the output domain.
Conversely crystallographic analysis of the phosphorylated receiver domain showed that phosphorylation of Asp-54 causes a marked change in the conformation of Phe-101, which switches from an outward to an inward oriented conformation (Bircke^a/. 1999; Figure 2). This conformational change is associated with a large displacement of the (34-a4 loop and the rotation of Thr-82 towards the phosphorylation site. We used a molecular dynamics approach to simulate this conformational change and found that the conformation of Phe-101 appears exquisitely sensitive to the conformation of the p4-a4 loop.
Because position 101 is usually conserved as an aromatic residue in receiver domains, we propose that the conformation of this aromatic residue plays a key role in 2-component signal transduction. This 'aromatic switch' hypothesis states that the inward or outward conformation of the aromatic ring determines the on/off state of the switch. The switch is triggered by a concerted displacement of the (34-a4 loop following phosphorylation of Asp-54, opening up a hydrophobic cavity filled by the aromatic residue. In the case of FixJ, this change results in two functionally important consequences: (i) liberating the FixJC output domain and (ii) reshaping the a4-(35 face into a dimerization interface.
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